Professor Brett Neilan

This article discusses the economic implications of toxin production by aquatic cyanobacteria, predominantly within the context of freshwater reservoirs. It presents an overview of the various cyanobacterial bloom compositions and toxin profiles typically encountered in these ecosystems. The chapter provides a brief overview of the major cyanotoxin toxocological subgroups, including the hepatotoxins, the neurotoxins, the cytotoxins, and the dermatoxins. The economic costs associated with toxic cyanobacterial blooms are complex and difficult to fully evaluate. If successful, a monitoring program can drastically reduce the economic costs associated with cyanobacterial blooms and their toxins by allowing for early intervention and prevention of exposure of individuals.

Cyanobacteria produce a wealth of secondary metabolites that are very diverse in chemical structure and in biological activity. A number of neurotoxins have been identified in strains of cyanobacteria including anatoxin-a and its higher homologue homoanatoxin-a, anatoxin-a(S), saxitoxins, and related paralytic shellfish toxins. This chapter focuses on these neurotoxic alkaloids from cyanobacteria, with emphasis on the chemical, biochemical, toxicological, and biosynthetic aspects. Anatoxin-a and homoanatoxin-a are two deadly potent agonists of the nicotinic acetylcholine receptor at the neuromuscular junction, provoking the rapid death of animals after respiratory failure. The chemistry and the pharmacology of these neurotoxins have been well studied, while their biosynthesis was recently deciphered. Our knowledge concerning anatoxin-a(S), an irreversible inhibitor of acetylcholine esterase, lies far behind and future work will be needed. Saxitoxin and its analogues, the paralytic shellfish toxins, have been associated with massive intoxication episodes in marine environment. They act as blockers of voltage-gated sodium channels provoking death in animals at low doses. The chemistry, pharmacology, and biosynthesis of saxitoxins have been well described. The genetic origin of the biosynthetic genes of this ancient toxin has been studied by detailed phylogenetic analysis.

The transition metal ions including cobalt, copper, iron, manganese, nickel, and zinc are essential for the viability of cyanobacteria as well as many other organisms. Cellular trace elements are required for the structure and function of many molecules essential for life including proteins and nucleic acids [1]. Metal ions are involved in a range of essential processes in particular enzyme functions where they are required for catalysis or may act as catalytic cofactors, for example, in reversible oxidation-reduction and hydrolytic reactions [2,3]. Individual metal ions may perform multiple biochemical roles or may be limited to just one role.

The transition metal ions including cobalt, copper, iron, manganese, nickel, and zinc are essential for the viability of cyanobacteria as well as many other organisms. Cellular trace elements are required for the structure and function of many molecules essential for life including proteins and nucleic acids [1]. Metal ions are involved in a range of essential processes in particular enzyme functions where they are required for catalysis or may act as catalytic cofactors, for example, in reversible oxidation-reduction and hydrolytic reactions [2,3]. Individual metal ions may perform multiple biochemical roles or may be limited to just one role.

Prochloron is an oxygenic photosynthetic prokaryotes that possess not only chlorophyll a but also b and lacks any phycobilins. This cyanobacterium lives in obligate symbiosis with colonial ascidians inhabiting tropical/subtropical waters and free-living Prochloron cells have never been recorded so far. There are about 30 species of host ascidians that are all belong to four genera of the family Didemnidae. Asicidiancyanobacteria symbiosis has attracted considerable attention as a source of biomedicals: many bioactive compounds were isolated from photosymbiotic ascidians and many of them are supposed to be originated from the photosymbionts. Since the stable in vitro culture of Prochloron has never been established, there are many unsolved question about the biology of Prochloron. Recent genetic, physiological, biochemical, and morphological studies are partly disclosing various aspects of its enigmatic life, e.g., photophysiology, metabolite synthesis, symbiosis, and evolution. Here, we tried to draw a rough sketch of the life of Prochloron and some related cyanobacteria. © 2009 Nova Science Publishers, Inc. All rights reserved.

Cyanobacterial harmful algal blooms (CHABs) are a global environmental concern that encompasses public health issues, water availability, and water quality owing to the production of various secondary metabolites (SMs), including cyanotoxins in freshwater, brackish water, and marine ecosystems. The frequency, extent, magnitude, and duration of CHABs are increasing globally. Cyanobacterial species traits and changing environmental conditions, including anthropogenic pressure, eutrophication, and global climate change, together allow cyanobacteria to thrive. The cyanotoxins include a diverse range of low molecular weight compounds with varying biochemical properties and modes of action. With the application of modern molecular biology techniques, many important aspects of cyanobacteria are being elucidated, including aspects of their diversity, gene-environment interactions, and genes that express cyanotoxins. The toxicological, environmental, and economic impacts of CHABs strongly advocate the need for continuing, extensive efforts to monitor cyanobacterial growth and to understand the mechanisms regulating species composition and cyanotoxin biosynthesis. In this review, we critically examined the genomic organization of some cyanobacterial species that lead to the production of cyanotoxins and their characteristic properties discovered to date.

This study was aimed to produce zinc oxide nanoparticles (ZnO-NPs) using the supernatant of Weissella confusa UPM22MT04 and assess their effectiveness in inhibiting methicillinresistant Staphylococcus aureus (MRSA). An isolate of Weissella confusa UPM22MT04 was isolated from a wastewater treatment plant in Johor, Malaysia, and was utilized to synthesize ZnO-NPs. The synthesized ZnO-NPs were characterized through several techniques, including UV-visible spectroscopy, Fourier-transform infrared spectroscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering. Monodisperse spherical ZnO-NPs of 1.7-7.9 nm were obtained with 0.1 M zinc nitrate at 80°C. The biosynthesized ZnO-NPs exhibited vigorous inhibitory activity against MRSA. Results found that ZnO-NPs inhibited MRSA at a minimum concentration of 0.625 mg/mL and were bactericidal at a minimum concentration of 1.25 mg/mL. In MTT assays, ZnO-NPs showed no toxicity to HS-27 fibroblasts. The supernatant of Weissella confusa UPM22MT04 could be used to synthesize ZnO-NPs, which are an antibacterial agent, eco-friendly and nontoxic.

Siderophores are low molecular weight iron-chelating molecules that many organisms secrete to scavenge ferric iron from the environment. While cyanobacteria inhabit a wide range of environments with poor iron availability, only two siderophore families have been characterized from this phylum. Herein, we sought to investigate siderophore production in the marine genus, Leptolyngbya. A 12 open reading frame (14.5 kb) putative nonribosomal peptide synthetase-independent siderophore biosynthesis gene cluster, identified in the genome of Leptolyngbya sp. PCC 7376, was cloned and heterologously expressed in Escherichia coli. Under iron-limiting conditions, expression strains harboring the first seven genes (lidA to lidF), produced a potent siderophore, which was subsequently identified via UPLC-MS/MS and NMR as schizokinen. The enzymes encoded by the remaining genes (lidG1 to lidG5) did not appear to be active in E. coli, therefore their function could not be determined. Bioinformatic analysis revealed gene clusters with high homology to lidA to lidF in phylogenetically and biogeographically diverse cyanobacteria, suggesting that schizokinen-based siderophore production is widespread in this phylum. Siderophore yields in E. coli expression strains were significantly higher than those achieved by Leptolyngbya, highlighting the potential of this platform for producing siderophores of industrial value.

The soil surface of drylands can typically be colonized by cyanobacteria and other microbes, forming biological soil crusts or ¿biocrusts¿. Biocrusts provide critical benefits to ecosystems and are a common component of the largely arid and semi-arid Australian continent. Yet, their distribution and the parameters that shape their microbial composition have not been investigated. We present here the first detailed description of Australia's biocrust microbiome assessed from 15 sites across the continent using 16S rRNA sequencing. The most abundant bacterial phyla from all sites were Cyanobacteria, Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Cyanobacterial communities from northern regions were more diverse and unclassified cyanobacteria were a noticeable feature of northern biocrusts. Segregation between northern and southern regions was largely due to the differential abundance of Microcoleus spp., with M. paludosus dominating in the north and M. vaginatus dominating in the south. The geographical shifts in bacterial composition and diversity were correlated to seasonal temperatures and summer rainfall. Our findings provide an initial reference for sampling strategies to maximize access to bacterial genetic diversity. As hubs for essential ecosystem services, further investigation into biocrusts in arid and semi-arid regions may yield discoveries of genetic mechanisms that combat increases in warming due to climate change.

Microbialites are sedimentary rocks created in association with benthic microorganisms. While they harbour complex microbial communities, Cyanobacteria perform critical roles in sediment stabilisation and accretion. Microbialites have been described from permanent and ephemeral saline lakes in South Australia; however, the microbial communities that generate and inhabit these biogeological structures have not been studied in detail. To address this knowledge gap, we investigated the composition, diversity and metabolic potential of bacterial communities from different microbialite-forming mats and surrounding sediments in five South Australian saline coastal lakes using 16S rRNA gene sequencing and predictive metagenome analyses. While Proteobacteria and Bacteroidetes were the dominant phyla recovered from the mats and sediments, Cyanobacteria were significantly more abundant in the mat samples. Interestingly, at lower taxonomic levels, the mat communities were vastly different across the five lakes. Comparative analysis of putative mat and sediment metagenomes via PICRUSt2 revealed important metabolic pathways driving the process of carbonate precipitation, including cyanobacterial oxygenic photosynthesis, ureolysis and nitrogen fixation. These pathways were highly conserved across the five examined lakes, although they appeared to be performed by distinct groups of bacterial taxa found in each lake. Stress response, quorum sensing and circadian clock were other important pathways predicted by the in silico metagenome analysis. The enrichment of CRISPR/Cas and phage shock associated genes in these cyanobacteria-rich communities suggests that they may be under selective pressure from viral infection. Together, these results highlight that a very stable ecosystem function is maintained by distinctly different communities in microbialite-forming mats in the five South Australian lakes and reinforce the concept that ¿who¿ is in the community is not as critical as their net metabolic capacity.

Municipal wastewater treatment facilities (WWTFs) are prone to the proliferation of cyanobacterial species which thrive in stable, nutrient-rich environments. Dense cyanobacterial blooms frequently disrupt treatment processes and the supply of recycled water due to their production of extracellular polymeric substances, which hinder microfiltration, and toxins, which pose a health risk to end-users. A variety of methods are employed by water utilities for the identification and monitoring of cyanobacteria and their toxins in WWTFs, including microscopy, flow cytometry, ELISA, chemoanalytical methods, and more recently, molecular methods. Here we review the literature on the occurrence and significance of cyanobacterial blooms in WWTFs and discuss the pros and cons of the various strategies for monitoring these potentially hazardous events. Particular focus is directed towards next-generation metagenomic sequencing technologies for the development of site-specific cyanobacterial bloom management strategies. Long-term multi-omic observations will enable the identification of indicator species and the development of site-specific bloom dynamics models for the mitigation and management of cyanobacterial blooms in WWTFs. While emerging metagenomic tools could potentially provide deep insight into the diversity and flux of problematic cyanobacterial species in these systems, they should be considered a complement to, rather than a replacement of, quantitative chemoanalytical approaches.

The contamination of water catchments by nonpoint source faecal pollution is a major issue affecting the microbial quality of receiving waters and is associated with the occurrence of a range of enteric illnesses in humans. The potential sources of faecal pollution in surface waters are diverse, including urban sewage leaks, surface runoff and wildlife contamination originating from a range of hosts. The major contributing hosts require identification to allow targeted management of this public health concern. In this study, two high-performing Microbial Source Tracking (MST) assays, HF183/Bac242 and BacCan-UCDmodif, were used for their ability to detect host-specific Bacteroides 16Sr RNA markers for faecal pollution in a 12-month study on an urban coastal lagoon in Sydney, Australia. The lagoon was found to contain year-round high numbers of human and canine faecal markers, as well as faecal indicator bacteria counts, suggesting considerable human and animal faecal pollution. The high sensitivity and specificity of the HF183/Bac242 and BacCan-UCDmodif assays, together with the manageable levels of PCR inhibition and high level DNA extraction efficiency obtained from lagoon water samples make these markers candidates for inclusion in an MST ¿toolbox¿ for investigating host origins of faecal pollution in urban surface waters.

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15N2] arginine and [1,2-13C2] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.

Two cyanobacterial strains, one collected from an epidermal mat present on a dead bottlenose dolphin and the other as a bioaerosol 457 m (1500¿ft) above the river, were recently analysed from the St. Johns River, Jacksonville, Florida, USA. Both samples had major phenotypic plasticity which confused morphological identification. Amplicon sequencing of the 16S rRNA gene from the isolates revealed that both samples were closely aligned (branch bootstrap support¿=¿100%) with the recently erected genus Komarekiella. Whole genome sequencing and phylogenetic construction also supported the isolation of a new species of cyanobacteria branching from the Nostoc clade. A total evidence approach of molecular, genetic, and ecological examination of these strains supported the erection of a new species, Komarekiella delphini-convector. A prior study determined that the dolphin with the epidermal mat had low levels of microcystins/nodularins (MCs/NODs) in the hepatic tissue. To investigate whether these toxins originated from the epidermal mat, immunoassay (ELISA) and 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) techniques were conducted on the original mat and subsequent culture samples. The results from both analyses were not conclusive. Genome mining was conducted and revealed diverse biosynthetic capabilities of this species but could not support toxin-producing potential. Further analytical work is required to determine the pathogenic capacity of this epizoic species.

Marine microalgae produce a variety of specialised metabolites that have toxic effects on humans, farmed fish, and marine wildlife. Alarmingly, many of these compounds bioaccumulate in the tissues of shellfish and higher trophic organisms, including species consumed by humans. Molecular methods are emerging as a potential alternative and complement to the conventional microscopic diagnosis of toxic or otherwise harmful microalgal species. Quantitative PCR (qPCR) in particular, has gained popularity over the past decade as a sensitive, rapid, and cost-effective method for monitoring harmful microalgae. Assays targeting taxonomic marker genes provide the opportunity to identify and quantify (or semi-quantify) microalgal species and importantly to pre-empt bloom events. Moreover, the discovery of paralytic shellfish toxin biosynthesis genes in dinoflagellates has enabled researchers to directly monitor toxigenic species in coastal waters and fisheries. This review summarises the recent developments in qPCR detection methods for harmful microalgae, with emphasis on emerging toxin gene monitoring technologies.

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2¿µM added Co, 0.5¿µM added Cu, 500¿µM Mn, 1¿µM Ni, or 18¿µM Zn. For cells treated with 60¿µM H2O2, no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60¿µM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60¿µM H2O2 and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582- cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582- cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that a-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated a-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.

A novel light-to-bioenergy system produced 3.5 times the baseline methane output using a co-culture of cyanobacteria (Oscillatoria sp.) and a methanogenic microbial community. Analysis of micronutrients in the system during the growth phase indicated that cobalt, iron, nickel and zinc were not appreciably consumed. The stable consumption and return of macronutrients calcium and magnesium were also observed. Essential macronutrients nitrogen, in the form of nitrate, and phosphorus showed no cycling during the growth phase and were depleted at rates of 0.35¿mg/L/day and 0.40¿µg/L/day, respectively. Biofilm formation increased the resilience of biomass to bacterial degradation in an anaerobic digester, as shown by viability assays of cyanobacterial biofilms in the co-culture.

Blooms of cyanobacteria are an increasingly frequent phenomenon in freshwater ecosystems worldwide as a result of eutrophication. Many species can produce hepatotoxins that cause severe health hazards to humans. The aim of this study was to identify the bloom forming cyanobacteria species by molecular methods and to amplify genes responsible for hepatotoxin biosynthesis from the environmental samples and isolated strains of cyanobacteria from Küçükçekmece Lagoon, Sapanca, Iznik, Manyas and Taskisi Lakes. A total of 10 bloom samples and 11 isolated strains were examined and Microcystis spp., Planktothrix spp., Nodularia spumigena, Anabaenopsis elenkinii, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis raciborskii were identified. Hepatotoxin genes were detected in 60% of the bloom samples and 45% of the strains. Two Microcystis strains were obtained from Küçükçekmece Lagoon. While the strain assigned to Microcystis flosaquae was non-toxic, Microcystis aeruginosa strain produced microcystin. According to PCR results, the M. aeruginosa and Planktothrix agardhii bloom samples of Küçükçekmece Lagoon contained the microcystin synthetase gene E (mcyE) indicative of microcystin production, however, no microcystin was detected by HPLC. The mcyE gene was also found in Microcystis wesenbergii isolated from Taskisi Lake, and in all Planktothrix rubescens bloom samples from Sapanca Lake. To our knowledge, this is the first detailed study for identifiying different toxic cyanobacteria species and their hepatotoxin production from several waterbodies in Turkey using molecular methods.

Historically microbial natural product biosynthesis pathways were elucidated mainly by isotope labelled precursor directed feeding studies. Now the genetics underpinning the assembly of microbial natural products biosynthesis is so well understood that some pathways and their products can be predicted from DNA sequences alone. The association between microbial natural products and their biosynthesis gene clusters is now driving the field of 'genetics guided natural product discovery'. This account overviews our research into cyanotoxin biosynthesis before the genome sequencing era through to some recent discoveries resulting from the mining of Australian biota for natural product biosynthesis pathways.

The effects of mammalian ecosystem engineers on soil microbial communities and ecosystem functions in terrestrial ecosystems are poorly known. Disturbance from livestock has been widely reported to reduce soil function, but disturbance by animals that forage in the soil may partially offset these negative effects of livestock, directly and/or indirectly by shifting the composition and diversity of soil microbial communities. Understanding the role of disturbance from livestock and ecosystem engineers in driving soil microbes and functions is essential for formulating sustainable ecosystem management and conservation policies. We compared soil bacterial community composition and enzyme concentrations within four microsites: foraging pits of two vertebrates, the indigenous short-beaked echidna (Tachyglossus aculeatus) and the exotic European rabbit (Oryctolagus cuniculus), and surface and subsurface soils along a gradient in grazing-induced disturbance in an arid woodland. Microbial community composition varied little across the disturbance gradient, but there were substantial differences among the four microsites. Echidna pits supported a lower relative abundance of Acidobacteria and Cyanobacteria, but a higher relative abundance of Proteobacteria than rabbit pits and surface microsites. Moreover, these microsite differences varied with disturbance. Rabbit pits had a similar profile to the subsoil or the surface soils under moderate and high, but not low disturbance. Overall, echidna foraging pits had the greatest positive effect on function, assessed as mean enzyme concentrations, but rabbits had the least. The positive effects of echidna foraging on function were indirectly driven via microbial community composition. In particular, increasing activity was positively associated with increasing relative abundance of Proteobacteria, but decreasing Acidobacteria. Our study suggests that soil disturbance by animals may offset, to some degree, the oft-reported negative effects of grazing-induced disturbance on soil function. Further, our results suggest that most of this effect will be derived from echidnas, with little positive effects due to rabbits. Activities that enhance the habitat for echidnas or reduce rabbit populations are likely to have a positive effect on soil function in these systems.

Bleomycin (BLM) is used clinically in combination with a number of other agents for the treatment of several types of tumours. Members of the BLM family of drugs include zorbamycin (ZBM), phleomycin D1, BLM A2 and BLM B2. By manipulating the BLM biosynthetic machinery, we have produced two new BLM analogues, BLM Z and 6'-deoxy-BLM Z, with the latter exhibiting significantly improved DNA cleavage activity. Here we determined the DNA sequence specificity of BLM Z, 6'-deoxy-BLM Z and ZBM, in comparison with BLM, with high precision using purified plasmid DNA and our recently developed technique. It was found that ZBM had a different DNA sequence specificity compared with BLM and the BLM analogues. While BLM and the BLM analogues showed a similar DNA sequence specificity, with TGTA sequences as the main site of cleavage, ZBM exhibited a distinct DNA sequence specificity, with both TGTA and TGTG as the predominant cleavage sites. These differences in DNA sequence specificity are discussed in relation to the structures of ZBM, BLM and the BLM analogues. Our findings support the strategy of manipulating the BLM biosynthetic machinery for the production of novel BLM analogues, difficult to prepare by total synthesis; some of which could have beneficial cancer chemotherapeutic properties.

Aim: To initiate a genetic and bioactivity-based screening programme of culturable endophytes to identify micro-organisms capable of producing bioactive polyketides and peptides. Methods and Results: Fungal endophytes were isolated from flowers, leaves and roots of Rhoeo spathacea, revealing a community consisting of Colletotrichum sp., Fusarium sp., Guignardia sp., Phomopsis sp., Phoma sp. and Microdochium sp. Genetic screening showed that all isolates had polyketide synthase (PKS) genes and most had nonribosomal peptide synthetase (NRPS) genes. Ethyl acetate extracts of the fungal isolates exhibited antiproliferative activity against at least one of the seven bacterial and mycobacterial test strains. Nuclear Magnetic Resonance -guided fractionation of the crude extract from a Fusarium sp. strain which exhibited strong antiproliferative activity against Mycobacterium tuberculosis resulted in the isolation of the polyketide javanicin. This compound was active against Myco. tuberculosis (MIC = 25 µg ml-1) and Mycobacterium phlei (MIC = 50 µg ml-1). Conclusions: The medicinal plant R. spathacea hosts a variety of fungal endophytes capable of producing antibacterial and antimycobacterial compounds. There is a positive correlation between the presence of PKS and/or NRPS encoding genes in endophytes and the bioactivity of their respective organic extracts. Significance and Impact of the Study: This is the first report on the fungal endophytic diversity of R. spathacea, and the isolation of an antimycobacterial compound from the plant which has been traditionally used for the treatment of tuberculosis symptoms.

In Australia, saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model saxitoxin-producing A. circinalisAWQC131C and non-toxic A. circinalisAWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria.

The bloom-forming cyanobacteria species Microcystis aeruginosa includes toxic and non-toxic (microcystin-producing) strains. Certain stress conditions stimulate synthesis of microcystin (MCYST) and enhance the binding of the MCYST molecule to proteins. In this quantitative proteomic study, we compared the response of a wild-type toxic strain PCC 7806, an mcyH- knockout non-toxic strain, and a naturally occurring non-toxic strain, PCC 7005, after 8 days in low iron (Fe) and nitrogen (N) starvation in order to assess the benefit of MCYST synthesis in non-optimal conditions. Fe limitation increased MCYST synthesis and caused an accumulation of phycobilisome proteins and the ferric iron transporter FutA only in the toxic PCC 7806 but not the non-toxic strains. In N starvation, photosynthetic, C and N metabolism proteins were more abundant in the non-toxic strains, as were chaperones and proteases. Significant interaction between nutrient availability and toxicity existed for thioredoxin peroxidase and several thioredoxin-regulated proteins. We propose a competition of MCYST for binding sites in thioredoxin-regulated proteins during oxidative stress (low Fe) but not in growth-limiting conditions (low N). This then leads to differences in the regulation of C:N metabolism in toxic and non-toxic M.aeruginosa in nutrient-replete and nutrient-limited conditions.

The cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. It is of particular concern because strains in some geographic areas are capable of producing toxins with implications for human and animal health. Studies of this species have increased rapidly in the last two decades, especially in the southern hemisphere where toxic strains are prevalent. A clearer picture is emerging of the strategies adopted by this species to bloom and out-compete other species. This species has a high level of flexibility with respect to light and nutrients, with higher temperatures and carbon dioxide also promoting growth. There are two types of toxins produced by C. raciborskii: cylindrospermopsins (CYNs) and saxitoxins (STXs). The toxins CYNs are constitutively produced irrespective of environmental conditions and the ecological or physiological role is unclear, while STXs appear to serve as protection against high salinity and/or water hardness. It is also apparent that strains of this species can vary substantially in their physiological responses to environmental conditions, including CYNs production, and this may explain discrepancies in findings from studies in different geographical areas. The combination of a flexible strategy with respect to environmental conditions, and variability in strain response makes it a challenging species to manage. Our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology of this species.

Modern microbial mats are potential analogues of some of Earth's earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.

The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn's disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn's disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn's disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC.We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC.

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19-39.60µg/L and 15.22-128.14µg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18¿µM of zinc for 1¿h, NpunR4017 mRNA levels increased by up to 1300¿% above basal levels. The accumulation and retention of radiolabelled 65Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24¿% greater than the wild type, while cells overexpressing NpunR4107 accumulated 22¿% less than the wild type. Accumulation of 65Zn in ZntA-Escherichia coli overexpressing NpunR4017 was reduced by up to 21¿%, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Aims: To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. Methods and Results: Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N. punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3 h. Interestingly, Cdf31- and Cdf33- mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31- mutant had a more profound effect on cdf33 expression than Cdf33- did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA-- and ZitB--deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E. coli and the cyanobacterial transporters. Conclusions: The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N. punctiforme. Significance and Impact of the Study: This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.

Halococcus hamelinensis was the first archaeon isolated from stromatolites. These geomicrobial ecosystems are thought to be some of the earliest known on Earth, yet, despite their evolutionary significance, the role of Archaea in these systems is still not well understood. Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites. The genome of H. hamelinensis consisted of 3,133,046 base pairs with an average G+C content of 60.08% and contained 3,150 predicted coding sequences or ORFs, 2,196 (68.67%) of which were protein-coding genes with functional assignments and 954 (29.83%) of which were of unknown function. Codon usage of the H. hamelinensis genome was consistent with a highly acidic proteome, a major adaptive mechanism towards high salinity. Amino acid transport andmetabolism, inorganic ion transport andmetabolism, energy production and conversion, ribosomal structure, and unknown function COG genes were overrepresented. The genome of H. hamelinensis also revealed characteristics reflecting its survival in its extreme environment, including putative genes/pathways involved in osmoprotection, oxidative stress response, and UV damage repair. Finally, genome analyses indicated the presence of putative transposases as well as positive matches of genes of H. hamelinensis against various genomes of Bacteria, Archaea, and viruses, suggesting the potential for horizontal gene transfer.

Animals that modify their physical environment by foraging in the soil can have dramatic effects on ecosystem functions and processes. We compared bacterial and fungal communities in the foraging pits created by bilbies and burrowing bettongs with undisturbed surface soils dominated by biocrusts. Bacterial communities were characterized by Actinobacteria and Alphaproteobacteria, and fungal communities by Lecanoromycetes and Archaeosporomycetes. The composition of bacterial or fungal communities was not observed to vary between loamy or sandy soils. There were no differences in richness of either bacterial or fungal operational taxonomic units (OTUs) in the soil of young or old foraging pits, or undisturbed soils. Although the bacterial assemblage did not vary among the three microsites, the composition of fungi in undisturbed soils was significantly different from that in old or young foraging pits. Network analysis indicated that a greater number of correlations between bacterial OTUs occurred in undisturbed soils and old pits, whereas a greater number of correlations between fungal OTUs occurred in undisturbed soils. Our study suggests that digging by soil-disturbing animals is likely to create successional shifts in soil microbial and fungal communities, leading to functional shifts associated with the decomposition of organic matter and the fixation of nitrogen. Given the primacy of organic matter decomposition in arid and semi-arid environments, the loss of native soil-foraging animals is likely to impair the ability of these systems to maintain key ecosystem processes such as the mineralization of nitrogen and the breakdown of organic matter, and to recover from disturbance.

Cyanobacteria produce a vast array of natural products, some of which are toxic to human health, while others possess potential pharmaceutical activities. Genome mining enables the identification and characterisation of natural product gene clusters; however, the current number of cyanobacterial genomes remains low compared to other phyla. There has been a recent effort to rectify this issue by increasing the number of sequenced cyanobacterial genomes. This has enabled the identification of biosynthetic gene clusters for structurally diverse metabolites, including non-ribosomal peptides, polyketides, ribosomal peptides, UV-absorbing compounds, alkaloids, terpenes and fatty acids. While some of the identified biosynthetic gene clusters correlate with known metabolites, genome mining also highlights the number and diversity of clusters for which the product is unknown (referred to as orphan gene clusters). A number of bioinformatic tools have recently been developed in order to predict the products of orphan gene clusters; however, in some cases the complexity of the cyanobacterial pathways makes the prediction problematic. This can be overcome by the use of mass spectrometry-guided natural product genome mining, or heterologous expression. Application of these techniques to cyanobacterial natural product gene clusters will be explored.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions. © 2014 Federation of European Microbiological Societies.

In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes. © 2014 American Chemical Society.

Species of the PST producing planktonic marine dinoflagellate genus Alexandrium have been intensively scrutinised, and it is therefore surprising that new taxa can still be found. Here we report a new species, Alexandrium diversaporum nov. sp., isolated from spherical cysts found at two sites in Tasmania, Australia. This species differs in its morphology from all previously reported Alexandrium species, possessing a unique combination of morphological features: the presence of 2 size classes of thecal pores on the cell surface, a medium cell size, the size and shape of the 6¿, 1', 2¿¿ and Sp plates, the lack of a ventral pore, a lack of anterior and posterior connecting pores, and a lack of chain formation. We determined the relationship of the two strains to other species of Alexandrium based on an alignment of concatenated SSU-ITS1, 5.8S, ITS2 and partial LSU ribosomal RNA sequences, and found A. diversaporum to be a sister group to Alexandrium leei with high support. A. leei shares several morphological features, including the relative size and shapes of the 6¿, 1', 2¿¿ and Sp plates and the fact that some strains of A. leei have two size classes of thecal pores. We examined A. diversaporum strains for saxitoxin production and found them to be non-toxic. The species lacked sequences for the domain A4 of sxtA, as has been previously found for non-saxitoxin producing species of Alexandrium. © 2013 Elsevier B.V.

The phylum candidatus Saccharibacteria formerly known as Candidate Division TM7 is a highly ubiquitous phylum with 16S rRNA gene sequences reported in soils, sediments, wastewater and animals, as well as a host of clinical environments. Here, the application of two taxon-specific primers on environmental and human-associated samples using bar-coded tag pyrosequencing revealed two new clades for this phylum to exist and we propose that the division consists of 2 monophyletic and 2 polyphyletic clades. Investigation into TM7 ecology revealed that a high proportion (58%) of phylotypes were sample specific, few were widely distributed and of those most widely distributed all belonged to subdivision 3. Additionally, 50% of the most relatively abundant phylotypes observed were also subdivision 3 members. Community analysis showed that despite the presence of a high proportion of unique phylotypes, specific groups of samples still harbor similar TM7 communities with samples clustering together. The lack of relatively abundant phylotypes from subdivisions 1, 2 and 4 and the presence of very few cosmopolitan members' highlights not only the site specific nature of this phylum but provides insight into why the majority of studies into TM7 have been biased towards subdivision 3.

Background and aims: This study investigated the effect of cyanobacterial inoculants on salt tolerance in wheat. Methods: Unicyanobacterial crusts of Nostoc, Leptolyngbya and Microcoleus were established in sand pots. Salt stress was targeted at 6 and 13 dS m-1, corresponding to the wheat salt tolerance and 50 % yield reduction thresholds, respectively. Germinated wheat seeds were planted and grown for 14 (0 and 6 dS m-1) and 21 (13 dS m-1) days by which time seedlings had five emergent leaves. The effects of cyanobacterial inoculation and salinity on wheat growth were quantified using chlorophyll fluorescence, inductively coupled plasma-optical emission spectrometry and biomass measurements. Results: Chlorophyll fluorescence was negatively affected by soil salinity and no change was observed in inoculated wheat. Effective photochemical efficiency correlated with a large range of plant nutrient concentrations primarily in plant roots. Inoculation negatively affected wheat biomass and nutrient concentrations at all salinities, though the effects were fewer as salinity increased. Conclusions: The most likely explanation of these results is the sorption of nutrients to cyanobacterial extracellular polymeric substances, making them unavailable for plant uptake. These results suggest that cyanobacterial inoculation may not be appropriate for establishing wheat in saline soils but that cyanobacteria could be very useful for stabilising soils. © 2013 Springer Science+Business Media Dordrecht.

Cyanobacteria produce an unparalleled variety of toxins that can cause severe health problems or even death in humans, and wild or domestic animals. In the last decade, biosynthetic pathways have been assigned to the majority of the known toxin families. This review summarizes current knowledge about the enzymatic basis for the production of the hepatotoxins microcystin and nodularin, the cytotoxin cylindrospermopsin, the neurotoxins anatoxin and saxitoxin, and the dermatotoxin lyngbyatoxin. Elucidation of the biosynthetic pathways of the toxins has paved the way for the development of molecular techniques for the detection and quantification of the producing cyanobacteria in different environments. Phylogenetic analyses of related clusters from a large number of strains has also allowed for the reconstruction of the evolutionary scenarios that have led to the emergence, diversification, and loss of such gene clusters in different strains and genera of cyanobacteria. Advances in the understanding of toxin biosynthesis and evolution have provided new methods for drinking-water quality control and may inspire the development of techniques for the management of bloom formation in the future. We report the biosynthetic pathways of cyanobacterial toxins and describe the evolutionary scenarios that have led to the emergence, diversification and loss of such gene clusters. © 2012 Federation of European Microbiological Societies.

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn2+-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn2+-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08-, znuA18-, znuB- and znuC- mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn2+-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA- mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur- mutant. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The ZIP family of metal transporters is involved in the transport of Zn2+ and other metal cations from the extracellular environment and/or organelles into the cytoplasm of prokaryotes, eukaryotes and archaeotes. In the present study, we identified twin ZIP transporters, Zip11 (Npun-F3111) and Zip63 (Npun-F2202) encoded within the genome of the filamentous cyanobacterium, Nostoc punctiforme PCC73120. Sequence-based analyses and structural predictions confirmed that these cyanobacterial transporters belong to the SLC39 subfamily of metal transporters. Quantitative real-time (QRT)-PCR analyses suggested that the enzymes encoded by zip11 and zip63 have a broad allocrite range that includes zinc as well as cadmium, cobalt, copper, manganese and nickel. Inactivation of either zip11 or zip63 via insertional mutagenesis in N. punctiforme resulted in reduced expression of both genes, highlighting a possible co-regulation mechanism. Uptake experiments using 65Zn demonstrated that both zip mutants had diminished zinc uptake capacity, with the deletion of zip11 resulting in the greatest overall reduction in 65Zn uptake. Over-expression of Zip11 and Zip63 in an E. coli mutant strain (ZupT736::kan) restored divalent metal cation uptake, providing further evidence that these transporters are involved in Zn uptake in N. punctiforme. Our findings show the functional role of these twin metal uptake transporters in N. punctiforme, which are independently expressed in the presence of an array of metals. Both Zip11 and Zip63 are required for the maintenance of homeostatic levels of intracellular zinc N. punctiforme, although Zip11 appears to be the primary zinc transporter in this cyanobacterium, both ZIP's may be part of a larger metal uptake system with shared regulatory elements. © 2013 Springer-Verlag Berlin Heidelberg.

The presence of Helicobacter spp. was examined in the liver and in different regions of the gastrointestinal tract (GIT) including the stomach, 3. cm above ileum, ileum, caecum, colon and rectum of 10 ringtail possums (RTPs) and 3 koalas using a combination of microscopy, culture and PCR. Helicobacter was detected in the distal end of the GIT of 7 of 10 RTPs by direct PCR and in all (10/10) RTPs by nested PCR. Five 'S' shaped isolates with bipolar sheathed flagella were isolated from the lower bowel of 3 of the 10 RTPs. 16S rRNA sequence analysis of these 5 isolates confirmed them as potentially novel Helicobacter species. No Helicobacter species were cultured from the koalas, however Helicobacter DNA was detected, in the majority of liver and/or stomach samples of the three koalas and in the colonic region of one koala, using nested PCR. The 16S rRNA gene was sequenced directly from DNA extracted from the homogenised livers and mucus scrapings of the stomach from koala 1 and were confirmed to be Helicobacter species. Based on histopathological examination of sections from the liver and intestine no evidence of infection could be related to the presence of helicobacters in either the RTP or koala. Based on our results, it is possible that diet may influence the detection of Helicobacter species; however this required further investigation. © 2013 Elsevier B.V.

Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [. i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of =97.7% for each assay. © 2013 Elsevier Ltd.

Families of closely related chemical compounds, which are relatively resistant to degradation, are often used as biomarkers to help trace the evolutionary history of early groups of organisms and the environments in which they lived. Biomarkers derived from hopanoid variations are particularly useful in determining bacterial community compositions. 2-Methylhopananoids have been thought to be diagnostic for cyanobacteria, and 2-methylhopanes in the geological record are taken as evidence for the presence of cyanobacteria-containing communities at the time of sediment deposition. Recently, however, doubt has been cast on the validity of 2-methylhopanes as cyanobacterial biomarkers, since non-cyanobacterial species have been shown to produce significant amounts of 2-methylhopanoids. This study examines the diversity of hpnP, the hopanoid biosynthesis gene coding for the enzyme that methylates hopanoids at the C2 position. Genomic DNA isolated from stromatolite-associated pustular and smooth microbial mat samples from Shark Bay, Western Australia, was analysed for bacterial diversity, and used to construct an hpnP clone library. A total of 117 partial hpnP clones were sequenced, representing 12 operational taxonomic units (OTUs). Phylogenetic analysis showed that 11 of these OTUs, representing 115 sequences, cluster within the cyanobacterial clade. We conclude that the dominant types of microorganisms with the detected capability of producing 2-methylhopanoids within pustular and smooth microbial mats in Shark Bay are cyanobacteria. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Major cyanobacterial blooms (biovolume>4mm3L-1) occurred in the main water reservoirs on the upper Murray River, Australia during February and March 2010. Cyanobacterial-infested water was released and contaminated rivers downstream. River flow velocities were sufficiently high that in-stream bloom development was unlikely. The location has a temperate climate but experienced drought in 2010, causing river flows that were well below the long-term median values. This coupled with very low bed gradients meant turbulence was insufficient to destroy the cyanobacteria in-stream. Blooms in the upper 500km of the Murray and Edward Rivers persisted for 5 weeks, but in the mid and lower Murray blooms were confined to a small package of water that moved progressively downstream for another 650km. Anabaena circinalis was the dominant species present, confirmed by 16S rRNA gene sequencing, but other potentially toxic species were also present in smaller amounts. Saxitoxin (sxtA), microcystin (mcyE) and cylindrospermopsin (aoaA) biosynthesis genes were also detected, although water sample analysis rarely detected these toxins. River water temperature and nutrient concentrations were optimal for bloom survival. The operational design of weirs and retention times within weir pools, as well as tributary inflows to and diversions from the Murray River all influenced the distribution and persistence of the blooms. Similar flow, water quality and river regulation factors were underlying causes of another bloom in these rivers in 2009. Global climate change is likely to promote future blooms in this and other lowland rivers. © 2013 Elsevier B.V.

The title compound (systematic name: 3,7-dihydroxy-9-methoxy-1-methyl-6H- benzo[c]chromen-6-one dimethyl sulfoxide monosolvate), C15H 12O5·C2H6OS, was isolated from an unidentified endophytic fungus (belonging to class Ascomycetes) of Taxus sp. In the crystal, both the alternariol 9-O-methyl ether (AME) and the dimethyl sulfoxide (DMSO) molecules exhibit crystallographic mirror symmetry. One of the hydroxy groups makes bifurcated hydrogen bonds, viz. an intramolecular bond with the carbonyl group and an intermolecular bond with the carbonyl group in an inversion-related AME molecule. In the crystal, the AME molecules are organized into stacks parallel with the b axis by p-p interactions between centrosymmetrically related molecules [the distance between the centroid of the central ring and the centroid of the methoxy-substituted benzene ring in the next molecule of the stack is 3.6184 (5) Å]. Pairs of DMSO molecules, linked via centrosymmetric C - H¿O contacts, are inserted into the voids created by the AME molecules, making O - H¿O and C - H¿O contacts with the hosts. © Dasari et al. 2013.

A tropical planktic filamentous cyanobacterium, tentatively identified as Lyngbya robusta, recently increased in abundance in Lake Atitlán, Guatemala, and since 2008 annual water-blooms occurred. This was one from the first known cases of L. robusta water-blooms worldwide. A polyphasic evaluation of L. robusta using 16S rRNA gene sequencing, cytomorphological markers, and ecological characteristics was made. This species had several unique features. It produced aerotopes that were irregularly spaced in cells; cyanotoxins were not found, and it fixed nitrogen in spite of the lack of heterocytes. It contained a high amount of carotenoids, which caused an unusual brown color of the macroscopic scum on the water level. Molecular phylogenetic analyses using the 16S rRNA gene showed that L. robusta, together with few other planktic species, formed a clade, separated from typical Lyngbya species. The main diacritical markers of this clade were the planktic type of life and formation of gas vesicles in cells. Based upon molecular, morphological and ecological data, a new genus Limnoraphis was proposed with four species. © Czech Phycological Society (2013).

The amino acid variant ß-methyl-amino-L-alanine (BMAA) has long been associated with the increased incidence and progression of the amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). Previous studies have indicated that BMAA damages neurons via excitotoxic mechanisms. We have challenged rat olfactory ensheathing cells (OECs) with exogenous BMAA and found it to be cytotoxic. BMAA also induces a significant increase in Ca2+ influx, enhanced production of reactive oxygen species (ROS), and disrupts mitochondrial activity in OECs. This is the first study investigating BMAA toxicity using pure glial cells. These findings align BMAA with the three proposed mechanisms of degeneration in ALS, those being non-cell autonomous death, excitotoxicity and mitochondrial dysfunction.

The toxicity of the cyanobacterial modified amino acid, BMAA, has been described in rat, mouse and leech neurons. Particular emphasis has been placed on the potential ability of BMAA to induce neuronal damage via excitotoxic mechanisms. Here we present data indicating that the effects observed on lower organisms are also evident in a human model. Our data indicates that BMAA induces increased intracellular Ca2+ influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of reactive oxygen species (ROS). The amelioration of LDH release in the presence of the N-methyl-d-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Additionally, we have shown that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors. © 2012.

The bloom-forming, toxic cyanobacterium, Cylindrospermopsis raciborskii exhibits global distribution. In recent years both the occurrence and dominance of this species, particularly in temperate regions, has increased. Whilst this may be due to increased sensitivity of analytical detection methods or more rigorous sampling routines, it is possible that this expansion has been assisted by a number of changing conditions in these environments. The geographical expansion of both the organism and toxin production can be attributed to phenomena such as eutrophication and climate change. In this review, we discuss the occurrence of C. raciborskii with respect to current literature against the backdrop of increasing global temperatures. Critically, we identify a concerning trend between the geographical spread of this organism and global climate change. © 2011.

The bioactive compounds of medicinal plants are products of the plant itself or of endophytes living inside the plant. Endophytes isolated from eight different anticancer plants collected in Yunnan, China, were characterized by diverse 16S and 18S rRNA gene phylogenies. A functional gene-based molecular screening strategy was used to target nonribosomal peptide synthetase (NRPS) and type I polyketide synthase (PKS) genes in endophytes. Bioinformatic analysis of these biosynthetic pathways facilitated inference of the potential bioactivity of endophyte natural products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. All of the endophyte culture broth extracts demonstrated antiproliferative effects in at least one test assay, either cytotoxic, antibacterial or antifungal. From the perspective of natural product discovery, this study confirms the potential for endophytes from medicinal plants to produce anticancer, antibacterial and antifungal compounds. In addition, PKS and NRPS gene screening is a valuable method for screening isolates of biosynthetic potential. © 2012 Springer Science+Business Media, LLC.

A cyanobacterial bloom impacted over 1,100 km of the Murray River, Australia, and its tributaries in 2009. Physicochemical conditions in the river were optimal to support a bloom at the time. The data suggest that at least three blooms occurred concurrently in different sections of the river, with each having a different community composition and associated cyanotoxin profile. Microscopic and genetic analyses suggested the presence of potentially toxic Anabaena circinalis, Microcystis flos-aquae, and Cylindrospermopsis raciborskii at many locations. Low concentrations of saxitoxins and cylindrospermopsin were detected in Anabaena and Cylindrospermopsis populations. A multiplex quantitative PCR was used, employing novel oligonucleotide primers and fluorescent TaqMan probes, to examine bloom toxigenicity. This single reaction method identified the presence of the major cyanotoxin-producing species present in these environmental samples and also quantified the various toxin biosynthesis genes. A large number of cells present throughout the bloom were not potential toxin producers or were present in numbers below the limit of detection of the assay and therefore not an immediate health risk. Potential toxin-producing cells, possessing the cylindrospermopsin biosynthesis gene (cyrA), predominated early in the bloom, while those possessing the saxitoxin biosynthesis gene (sxtA) were more common toward its decline. In this study, the concentrations of cyanotoxins measured via enzymelinked immunosorbent assay (ELISA) correlated positively with the respective toxin gene copy numbers, indicating that the molecular method may be used as a proxy for bloom risk assessment. © 2012, American Society for Microbiology.

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths > 10 cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Lichens, algae and cyanobacteria have been detected growing endolithically in natural rock and in stone buildings in various countries of Australasia, Europe and Latin America. Previously these organisms had mainly been described in natural carbonaceous rocks in aquatic environments, with some reports in siliceous rocks, principally from extremophilic regions. Using various culture and microscopy methods, we have detected endoliths in siliceous stone, both natural and cut, in humid temperate and subtropical climates. Such endolithic growth leads to degradation of the stone structure, not only by mechanical means, but also by metabolites liberated by the cells. Using in vitro culture, transmission, optical and fluorescence microscopy, and confocal laser scanning microscopy, both coccoid and filamentous cyanobacteria and algae, including Cyanidiales, have been identified growing endolithically in the facades of historic buildings built from limestone, sandstone, granite, basalt and soapstone, as well as in some natural rocks. Numerically, the most abundant are small, single-celled, colonial cyanobacteria. These small phototrophs are difficult to detect by standard microscope techniques and some of these species have not been previously reported within stone. © 2012 Springer Science+Business Media, LLC.

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co2+, Cu2+, Mn2+, Ni2+,and Zn 2+ and the nonessential divalent cation Cd2¿ in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5 µM Cd2+, 2 lM Co2+, 0.5 lM Cu2+, 500 lMMn2+, 1 lM Ni2+, and 18 lM Zn2+.Cells exposed to these non-toxic concentrations with combinations of Zn2+ and Cd2+, Zn2+ and Co2+, Zn2+ and Cu2+ or Zn2+ and Ni2+, had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500 lMMn2+ showed similar growth compared to the untreated controls. Metal levels were measured after one and 72 h for whole cells and absorbed (EDTAresistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using 65Zn showed that after 72 h of exposure Zn2+ uptake was reduced by most metals particularly 0.5 lM Cd 2+, while 2 lM Co2+ increased Zn2+ uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals. © Springer Science+Business Media, LLC. 2012.

The debris cones known as Amorphous Glacier and Boulder Clay are located in an ice-free region in Northern Victoria Land, Antarctica, and differ in their isotopic composition, mechanisms of ice distribution, geological formation and age. However, to date it is not known if bacterial community profiles within ice and permafrost can be established for these environments, and then whether glaciological differences between the two areas would be reflected in the bacterial community composition. In order to gather first evidence for the bacterial communities in these glacial zones, we carried out terminal-restriction fragment length polymorphism (T-RFLP) analysis on the 16S rRNA gene using a universal bacterial amplification protocol on two permafrost cores. The DNA yields from ice-core samples ranged from 0.29ng µL -1 in Amorphous Glacier to 88ng µL -1 in Boulder Clay. Bray-Curtis cluster analysis suggested Boulder Clay bacterial profiles were similar to each other, but cluster separately from the Amorphous Glacier bacterial profile. © 2012 John Wiley & Sons, Ltd.

Toxic bloom-forming cyanobacteria are a global health hazard. These photosynthetic microorganisms produce a suite of secondary metabolite toxins including hepatotoxins such as microcystin, nodularin and cylindrospermopsin and neurotoxins such as saxitoxin. These toxins can threaten the safety of drinking water supplies and in the case of saxitoxin, can accumulate to dangerous levels in shellfish, affecting the seafood industry. Several molecular methods have been described for the detection and quantification of toxigenic cyanobacteria, however, to date there is no method for the simultaneous detection and quantification of hepatotoxin and neurotoxin producing genera. This paper describes the development and validation of a quadruplex quantitative-PCR (qPCR) assay capable of detecting and quantifying toxin genes from the microcystin, nodularin, cylindrospermopsin and saxitoxin biosynthesis pathways. The primers and probes employed in this assay were designed from conserved regions within toxin biosynthesis genes from most of the representative cyanobacterial genera. The qPCR assay was optimized to reliably determine the copy number of cyanotoxin biosynthesis genes, as well as an internal cyanobacteria 16S rDNA control, in a single reaction. Amplification efficiency and reproducibility were similar among the cyanotoxin genes, while the sensitivity of the reaction for the toxin genes ranged from 10 2 to 10 6 gene copies per reaction. This multiplex qPCR assay is a powerful tool for detecting and quantifying potentially toxic cyanobacteria in laboratory and field samples. Such technology will enable water quality and food safety authorities to better forecast, evaluate and reduce the impact of future harmful algal bloom events. © 2011.

The discovery of novel natural products for drug development relies heavily upon a rich biodiversity, of which the marine environment is an obvious example. Marine natural product research has spawned several drugs and many other candidates, some of which are the focus of current clinical trials. The sponge megadiversity of Papua New Guinea is a rich but underexplored source of bioactive natural products. Here, we review some of the many natural products derived from PNG sponges with an emphasis on those with interesting biological activity and, therefore, drug potential. Many bioactive natural products discussed here appear to be derived from non-ribosomal peptide and polyketide biosynthesis pathways, strongly suggesting a microbial origin of these compounds. With this in mind, we also explore the notion of sponge-symbiont biosynthesis of these bioactive compounds and present examples to support the working hypothesis. © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

A novel prokaryotic l-arginine:glycine amidinotransferase (CyrA;) is involved in the biosynthesis of the polyketide-derived cytotoxin cylindrospermopsin in the cyanobacterium Cylindrospermopsis raciborskii AWT250, and was previously characterized with regard to kinetic mechanism and substrate specificity [Muenchhoff J et al. (2010) FEBS J277, 3844-3860]. In order to elucidate the structure-function-stability relationship of this enzyme, two residues in its active site were replaced with the residues that occur in the human l-arginine:glycine amidinotransferase (h-AGAT) at the corresponding positions (F245N and S247M), and a double variant carrying both substitutions was also created. In h-AGAT, both of these residues are critical for the function of this enzyme with regard to substrate binding, ligand-induced structural changes, and stability of the active site. In this study, we demonstrated that both single residue replacements resulted in a dramatic broadening of substrate specificity, but did not affect the kinetic mechanism. Experiments with substrate analogues indicate that donor substrates require a carboxylate group for binding. Evidence from initial velocity studies suggests that CyrA undergoes ligand-induced structural changes that involve Phe245. Stability parameters (T opt and T max) of the CyrA variants differed from those of wild-type CyrA. Structural flexibilities of the wild type and all three variants were comparable on the basis of dynamic fluorescence quenching, indicating that changes in T opt are most likely attributable to localized effects within the active site. Overall, the results indicated that these two residues are essential for both stringent substrate specificity and the active site stability and flexibility of this unique cyanobacterial enzyme. In order to elucidate the structure-function-stability relationship of the novel prokaryotic l-arginine:glycine amidinotransferase CyrA, two amino acid residues in its active site were replaced with the residues occurring in the human l-arginine:glycine amidinotransferase at the corresponding position. We demonstrate that either amino acid replacement affected the structural stability of the enzyme and dramatically broadened its substrate specificity. © 2011 FEBS.

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. Macrozamia riedlei 65.1 and Nostoc sp. Macrozamia serpentina 73.1 both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg1 chlorophyll a in Nostoc sp. Macrozamia riedlei 65.1 and 12.5±8.4 ng µg1 Chl a in Nostoc sp. Macrozamia serpentina 73.1 extracts. Further scans indicated the presence of the rare isoform L-Har 2 nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml 1 (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of L-Har 2 nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and L-Har 2 nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta. © 2012 International Society for Microbial Ecology. All rights reserved.

The title compound (AME; systematic name: 3,7-dihydroxy-9-methoxy-1-methyl- 6H-benzo[c]chromen-6-one), C15H12O5, was isolated from an endophytic fungi Alternaria sp., from Catharanthus roseus (common name: Madagascar periwinkle). There is an intramolecular O - H¿O hydrogen bond in the essentially planar molecule (r.m.s. deviation 0.02 Å). In the crystal, the molecule forms an O - H¿O hydrogen bond with its centrosymmetric counterpart with four bridging inter-actions (two O - H¿O and two C - H¿O). The almost planar sheets of the dimeric units thus formed are stacked along b axis via C - H¿p and p-p contacts [with C¿C short contacts between aromatic moieties of 3.324 (3), 3.296 (3) and 3.374 (3) Å]. © Dasari et al. 2012.

Traditional Chinese medicine encompasses a rich empirical knowledge of the use of plants for the treatment of disease. In addition, the microorganisms associated with medicinal plants are also of interest as the producers of the compounds responsible for the observed plant bioactivity. The present study has pioneered the use of genetic screening to assess the potential of endophytes to synthesize bioactive compounds, as indicated by the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. The total DNA extracts of 30 traditional Chinese herbs, were screened for functional genes involved in the biosynthesis of bioactive compounds. The four PCR screens were successful in targeting four bacterial PKS, six bacterial NRPS, ten fungal PKS and three fungal NRPS gene fragments. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes in medicinal herbs. This investigation describes a rapid method for the initial screening of medicinal herbs and has highlighted a subset of those plants that host endophytes with biosynthetic potential. These selected plants can be the focus of more comprehensive endophyte isolation and natural product studies. © 2012 Miller et al.

Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml-1. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality. © 2011 Elsevier Ltd.

The presence of Helicobacter species in Australian marsupials was examined systematically using microscopy, culture, and PCR in different regions of the gastrointestinal tract (GIT) and in the liver of brushtail possums (BTPs) (Trichosurus vulpecula), a common Australian marsupial that feeds on eucalyptus leaves. The spatial distribution of Helicobacter species in the GIT sections also was examined microscopically in silverstained sections and by fluorescent in situ hybridization (FISH) using a Helicobacter genus-specific probe. Helicobacter species were found colonizing the lower bowel of all BTPs studied. Good agreement was observed between the detection of Helicobacter species using culture and PCR, which was supported by the microscopic examination of silver-stained sections and FISH. The lower bowel of BTPs were colonized by one to three morphologically different (a comma-shaped species with no apparent flagella, a fusiform-shaped species entwined with periplasmic fibers and a bipolar sheathed flagella, and an S-shaped species with bipolar sheathed flagella) and potentially novel Helicobacter species, as well as in one case with a potentially novel Campylobacter species, which was a tightly coiled rod with bipolar unsheathed flagella. The isolation and characterization of these Helicobacter species in BTPs provides important information regarding the specific natural niche of these bacteria and their corelationship within their host, and it increases our understanding of the ecology of Helicobacter species. © 2011, American Society for Microbiology.

The potent neurotoxin tetrodotoxin (TTX) has been identified from taxonomically diverse marine organisms. TTX possesses a unique cage-like structure, however, its biosynthesis has yet to be elucidated. Biosynthetic studies in the TTX-producing newt Taricha torosa, and in bacterial genera, including Vibrio, have proven inconclusive. Indeed, very few studies have been performed that address the cellular production of TTX. Here we review the sources of TTX described to date and provide evidence for the biosynthesis of TTX by symbiotic microorganisms in higher taxa. Chemical and genetic based biosynthesis studies of TTX undertaken thus far are discussed and we outline approaches which may be useful for expanding upon the current body of knowledge. The complex biosynthesis of structurally similar toxins, that reveal clues into the biosynthetic pathway of TTX, is also presented. © 2011 Elsevier B.V.

To better understand the DNA restriction-modification (R-M) systems for more amenable strain development of the alternative industrial ethanologen, Zymomonas mobilis, three gene knockout mutants were constructed. The gene knockout mutants were tested for their DNA restriction activities by the determination of transformation efficiency using methylated and unmethylated foreign plasmid DNAs. Inactivation of a putative mrr gene encoded by ZMO0028 (zmrr) resulted in a 60-fold increase in the transformation efficiency when unmethylated plasmid DNA was used. This indicated that the putative mrr gene may serve as a type IV restriction-modification system in Z. mobilis ZM4. To assign the function of a putative type I DNA methyltransferase encoded by ZMO1933 (putative S subunit) and ZMO1934 (putative M subunit), the putative S subunit was inactivated. The gene inactivation of ZMO1933 resulted in a 30-fold increase in the transformation efficiency when methylated plasmid DNA was introduced, indicating that the putative S subunit possibly serves as a part of functional type I R-M system(s). Growth studies performed on the mutant strains indicate inactivation of the type I S subunit resulted in a lower maximum specific glucose consumption rate and biomass yield, while inactivation of the type IV Zmrr had the opposite effect, with an increase in the maximum specific growth rate and biomass yield. © 2010 Springer-Verlag.

The recent determination of the genetic basis for the biosynthesis of the neurotoxin, saxitoxin, produced by cyanobacteria, has revealed a highly complex sequence of reactions, involving over 30 biosynthetic steps encoded by up to 26 genes clustered at one genomic locus, sxt. Insights into evolutionary-ecological processes have been found through the study of such secondary metabolites because they consist of a measurable phenotype with clear ecological consequences, synthesized by known genes in a small number of species. However, the processes involved in and timing of the divergence of prokaryotic secondary metabolites have been difficult to determine due to their antiquity and the possible frequency of horizontal gene transfer and homologous recombination. Through analyses of gene synteny, phylogenies of individual genes, and analyses of recombination and selection, we identified the evolutionary processes of this cluster in five species of cyanobacteria. Here, we provide evidence that the sxt cluster appears to have been largely vertically inherited and was therefore likely present early in the divergence of the Nostocales, at least 2,100 Ma, the earliest reliably dated appearance of a secondary metabolite. The sxt cluster has been extraordinarily conserved through stabilizing selection. Genes have been lost and rearranged, have undergone intra- and interspecific recombination, and have been subject to duplication followed by positive selection along the duplicated lineage, with likely consequences for the toxin analogues produced. Several hypotheses exist as to the ecophysiological role of saxitoxin: as a method of chemical defense, cellular nitrogen storage, DNA metabolism, or chemical signaling. The antiquity of this gene cluster indicates that potassium channels, not sodium channels, may have been the original targets of this compound. The extraordinary conservation of the machinery for saxitoxin synthesis, under radically changing environmental conditions, shows that it has continued to play an important adaptive role in some cyanobacteria. © The Author 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

The halophilic archaeon Halococcus hamelinensis was isolated from living stromatolites in Shark Bay, Western Australia, that are known to be exposed to extreme conditions of salinity, desiccation, and UV radiation. Modern stromatolites are considered analogues of very early life on Earth and thus inhabitants of modern stromatolites, and Hcc. hamelinensis in particular, are excellent candidates to examine responses to high UV radiation. This organism was exposed to high dosages (up to 500 J/m2) of standard germicidal UVC (254 nm) radiation and overall responses such as survival, thymine-thymine cyclobutane pyrimidine dimer formation, and DNA repair have been assessed. Results show that Hcc. hamelinensis is able to survive high UVC radiation dosages and that intact cells give an increased level of DNA protection over purified DNA. The organism was screened for the bacterial-like nucleotide excision repair (NER) genes uvrA, uvrB, uvrC, as well as for the photolyase phr2 gene. All four genes were discovered and changes in the expression levels of those genes during repair in either light or dark were investigated by means of quantitative Real-Time (qRT) PCR. The data obtained and presented in this study show that the uvrA, uvrB, and uvrC genes were up-regulated during both repair conditions. The photolyase phr2 was not induced during dark repair, yet showed a 20-fold increase during repair in light conditions. The data presented is the first molecular study of different repair mechanisms in the genus Halococcus following exposure to high UVC radiation levels. © 2010 Elsevier B.V.

Cyanobacterial strains of the genus Spirulina have recently been identified as an excellent source of sulfolipids, some of which possess anti-HIV properties. Thus, to investigate the distribution of sufolipid biosynthesis pathways in Spirulina, a genetic screening/phylogentic study was performed. Five different strains of Spirulina [Spirulina (Jiangmen), Spirulina sp., S. platensis, S. maxima, and Spirulina seawater] sourced from different locations were initially classified via 16S rDNA sequencing, and then screened for the presence of the sulfolipid biosynthesis genes sqdB and sqdX via a PCR. To assess the suitability of these strains for human consumption and safe therapeutic use, the strains were also screened for the presence of genes encoding nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs), which are often associated with toxin pathways in cyanobacteria. The results of the 16S rDNA analysis and phylogenetic study indicated that Spirulina sp. is closely related to Halospirulina, whereas the other four Spirulina strains are closely related to Arthrospira. Homologs of sqdB and sqdX were identified in Spirulina (Jiangmen), Spirulina sp., S. platensis, and the Spirulina seawater. None of the Spirulina strains screened in this study tested positive for NRPS or PKS genes, suggesting that these strains do not produce NRP or PK toxins.

This paper reports an investigation of salinity-induced glycolate metabolism in the cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena PCC 7120). Quantitative analysis of transcripts for the photosynthesis-associated genes encoding ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), phosphoribulokinase and transketolase, as well as those involved in glycolate metabolism (phosphoglycolate phosphatase, glycolate oxidase, alanine-glyoxylate aminotransferase and serine hydroxymethyltransferase) was performed. The expression of all investigated photosynthesis-associated genes except Rubisco was downregulated after 24 h NaCl treatment. However, under the same conditions, the transcripts encoding enzymes involved in glycolate metabolism were overexpressed. This was further confirmed by the quantitative analysis of the intermediates involved in glycolate metabolism. The intracellular levels of organic acids (glyceric, glycolic and glyoxylic acids) and amino acids (glycine and serine) were elevated in salt-treated cells as compared to those in the control cells. Transcriptional inhibition of photosynthesis-associated genes, and upregulation of genes and enhanced synthesis of intermediates associated with glycolate metabolism, indicate the occurrence of this photorespiratory metabolic pathway metabolism in Anabaena PCC 7120 under salt stress. © 2011 SGM.

Toxin production during cyanobacterial blooms poses a significant public health threat in water bodies globally and requires the development of effective bloom management strategies. Previously, synthesis of the hepatotoxin microcystin has been proposed to be regulated by iron availability, but the contribution of the toxin to the adaptation of cyanobacteria to environmental stresses, such as changing light intensity and nutrient limitation, remains unclear. The aim of this study was to compare the iron stress response in toxic and non-toxic strains of Microcystis aeruginosa subjected to moderate and severe iron limitation. The transcription of a number of genes involved in iron uptake, oxidative stress response, toxin synthesis and transcriptional control of these processes was accessed by quantitative real-time PCR (qRT-PCR). The process of adaptation of M. aeruginosa to iron stress was found to be highly dynamic and strain-specific. Toxin production in PCC 7806 increased in an iron-dependent manner and appeared to be regulated by FurA. The inability to produce microcystin, either due to natural mutations in the mcy gene cluster or due to insertional inactivation of mcyH, affected the remodelling of the photosynthetic machinery in iron-stressed cells, the transport of Fe(II) and transcription of the Fur family of transcriptional regulators. The presence of the toxin appears to give an advantage to microcystin-producing cyanobacteria in the early stages of exposure to severe iron stress and may protect the cell from reactive oxygen species-induced damage. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Biogenic stromatolites are sources of significant information on the evolution of microbial life. Despite their evolutionary significance, little is known about the mechanisms of osmoadaptation by microorganisms that comprise living stromatolites thriving in hypersaline environments. Osmoadaptive strategies for Halococcus hamelinensis, a novel halophilic archaeon recently isolated from living stromatolites in the hypersaline reaches of Shark Bay, were thus a particular interest in this study. To investigate the possibility of "salt-in-cytoplasm"-associated osmoadaptation for this archaeon, flame photometry studies were performed. From the results, it was evident that this halophilic archaeon did not accumulate intracellular K + ions when cells were exposed to either osmotic shock or conditions with gradual increments in salinity. These results were further supported by polymerase chain reaction (PCR) analyses where there was no evidence for the existence of homologous genes to an ATP-driven, high-affinity potassium uptake system in Halococcus hamelinensis. To identify an alternative salt adaptation mechanism associated with accumulation of compatible solutes for this archaeon, 1H nuclear magnetic resonance (NMR) spectroscopy experiments were carried out. Results indicate that glycine betaine, trehalose, and glutamate are solutes likely to be involved in osmoregulation in this archeaon. Subsequent 1H NMR analysis of cell extracts from this microorganism grown under various NaCl concentrations revealed that intracellular levels of glycine betaine increased with increasing concentrations of NaCl. This behavior of increasing glycine betaine concentration with increasing external NaCl is consistent with its identity as an osmolyte. In contrast, intracellular levels of trehalose were decreased in high concentrations of NaCl. This provides evidence that compatible solute accumulation appears to be the preferential salt regulation mechanism for this haloarchaeon, in contrast to the salt-in-cytoplasm strategy employed by many other halophilic archaea. © Copyright 2011, Mary Ann Liebert, Inc.

Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the labelfree normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds. © 2011 Mihali et al.

Chlorophylls are essential for light-harvesting and energy transduction in photosynthesis. Four chemically distinct varieties have been known for the past 60 years. Here we report isolation of a fifth, which we designate chlorophyll f. Its in vitro absorption (706 nanometers) and fluorescence (722 nanometers) maxima are red-shifted compared to all other chlorophylls from oxygenic phototrophs. On the basis of the optical, mass, and nuclear magnetic resonance spectra, we propose that chlorophyll f is [2-formyl]-chlorophyll a (C 55H70O6N4Mg). This finding suggests that oxygenic photosynthesis can be extended further into the infrared region and may open associated bioenergy applications.

Outbreaks of human illness caused by the consumption of contaminated seafood, continues to be a major problem particularly for the shellfish industry. Toxins from marine, brackish and freshwater environments, which are often produced as a result of harmful algal blooms, have been implicated as the causative agents of these poisonings. Commonly, poisoning events have been grouped into one of six classes, Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Ciguatera Fish Poisoning (CFP), Azaspiracid Shellfish Poisoning (AZP), and Amnesiac Shellfish Poisoning (ASP). The causative agents of these specific poisonings along with their biosyntheses are discussed in this review. The highly unusual and complex structures of most common seafood toxins have made them interesting targets for biosynthetic studies. Many of the toxins presented are biosynthesized via complex pathways that have been elucidated either through isotope labelled precursor feeding studies and/or characterization of the genes encoding the producing organism's biosynthetic machinery. Feeding studies key to our understanding of a particular toxin's biosynthesis, such as the incorporation of unusual precursors, as well as unique biosynthetic pathways and rare chemical mechanisms involved in the assembly process are highlighted. More recently, however, modern genomics-based techniques have been used for the elucidation of biosynthetic pathways and these are presented in the context of polyketide, non-ribosomal peptide, and hybrid pathway derived, toxin assembly. © 2009 Elsevier Ltd.

NtcA is a transcription factor that has been found in a diverse range of cyanobacteria. This nitrogencontrolled factor was focused on as a key component in the yet-to-be-deciphered regulatory network controlling microcystin production. Adaptor-mediated PCR was utilized to isolate the ntcA gene from Microcystis aeruginosa PCC 7806. This gene was cloned, and the recombinant (His-tagged) protein was overexpressed and purified for use in mobility shift assays to analyze NtcA binding to putative sites identified in the microcystin mcyA/D promoter region. Autoregulation of NtcA in M. aeruginosa was shown via NtcA binding in the upstream ntcA promoter region. The observation of binding of NtcA to the mcyA/D promoter region has direct relevance for the regulation of microcystin biosynthesis, as transcription of the mcyABCDEFGHIJ gene cluster appears to be under direct control of nitrogen. Copyright © 2010, American Microbiology, All Rights Reserved.

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria. Copyright © 2010, American Society for Microbiology.

Comprehensive lipid biomarker profiles were determined for extant intertidal columnar stromatolites and non-lithified smooth and pustular microbial mats from Hamelin Pool, Shark Bay, Western Australia. Hydrocarbons, alkyl (wax) esters, sterols, fatty acids, triterpenoids and ether-linked hydrocarbons were analysed using gas chromatography-mass spectrometry (GC-MS) and triterpenoids were analysed using high temperature GC-MS. Cyanobacterial markers were abundant in each sample and lipids diagnostic of heterotrophic bacteria, sulfate-reducing bacteria, anoxygenic phototropic bacteria and archaea were also detected. Limited input from higher plants and diatoms was observed. For the first time, 2-methylhopanoids were detected in Hamelin Pool microbial communities. The overall lipid profiles of the three sediment types were similar, suggesting that extant non-lithified microbial mats and stromatolites can comprise similar microbial communities. © 2010 Elsevier Ltd.

Cyanobacteria are able to survive in various extreme environments via the production of organic compounds known as compatible solutes. In particular, cyanobacteria are capable of inhabiting hypersaline environments such as those found in intertidal regions. Cyanobacteria in these environments must possess regulatory mechanisms for surviving the changing osmotic pressure as a result of desiccation, rainfall and tidal fluxes. The objective of this study was to determine the compatible solutes that are accumulated by cyanobacteria from hypersaline regions, and specifically, the stromatolite ecosystems of Shark Bay, Western Australia. Previously, the cyanobacterial populations associated with these stromatolites were characterized in two separate studies. Compatible solutes were extracted from isolated cyanobacteria here and identified by nuclear magnetic resonance. As the media of isolation contained no complex carbon source, the solutes accumulated were likely synthesized by the cyanobacteria. The data indicate that from this one habitat taxonomically distinct cyanobacteria exposed to varying salinities accumulate a range of known compatible solutes. In addition, taxonomically similar cyanobacteria do not necessarily accumulate the same compatible solutes. Glucosylglycerol, a compatible solute unique to marine cyanobacteria was not detected; however, various saccharides, glycine betaine, and trimethylamine-N-oxide were identified as the predominant solutes. We conclude that the cyanobacterial communities from these hypersaline stromatolites are likely to possess more complex mechanisms of adaptation to osmotic stress than previously thought. The characterization of osmoregulatory properties of stromatolite microorganisms provides further insight into how life can thrive in such extreme environments. © 2010 Springer-Verlag.

N2-fixation is an important mechanism in microbial mats of the McMurdo Ice Shelf as nitrogen sources are limited. Here we applied molecular analyses of the N2-fixing diversity in cyanobacterial dominated microbial mats in a meltwater pond, known as Orange Pond, on the McMurdo Ice Shelf. Phylogenetic analyses of nifH genes and nifH gene transcripts were performed in association with acetylene reduction assay measurements. Eighteen phylotypes with the highest similarities to cyanobacteria, firmicutes, beta-, gamma- and deltaproteobacteria, spirochaetes and verrumicrobia were identified. All cyanobacterial nifH phylotypes grouped solely in the genus Nostoc spp. Clone-library analysis of nifH gene transcripts only identified sequences with a highest match to Nostoc spp. and acetylene reduction activity was identified in the presence of light and absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. These molecular results indicate that a variety of bacterial phyla possess the ability to fix nitrogen. However, under the tested conditions the only organisms actively transcribing nifH genes were Nostoc spp. This underlines the importance of Nostoc for the nitrogen budget on the McMurdo Ice Shelf. © 2009 Antarctic Science Ltd.

We report the first characterization of an l-arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well-characterized human l-arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an l-arginine:glycine amidinotransferase by 1H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis-Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (Tmax of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic l-arginine:glycine amidinotransferases. © 2010 FEBS.

The cyanobacteria or "blue-green algae", as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites. © 2010 by the authors; licensee MDPI.

Background. Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results. We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion. The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.

Cyanobacteria are recognised globally as a human health threat due to their proliferation into toxic blooms. Of particular concern are strains that produce the hepatotoxins, microcystin and nodularin. Research over the past decade has revealed the biochemical and molecular mechanisms behind hepatotoxin production. However, there is still much to learn regarding the regulation of these biologically active metabolites. This review provides an overview of cyanobacterial hepatotoxin research to date and additionally, elaborates on the putative involvement of nitrogen and iron homeostatic mechanisms in cyanotoxin regulation.

The lipid biomarker composition of microbial mat communities from three meltwater ponds from the McMurdo Ice Shelf, Antarctica, was investigated for the first time. Hydrocarbons, ether-linked components, fatty acids (FAs), wax esters, hopanols and sterols from Fresh, Orange and Salt Ponds were analysed. The dominance of cyanobacteria and the presence of bacterial sulfate reducers were confirmed using signature FAs in all three mats. Wax ester analysis suggested the presence of Chloroflexus spp. The dominance of short chain hydrocarbons, wax esters and FAs indicated that microorganisms are the major source of organic matter in these meltwater ponds. A variety of sterols were present in different relative abundances. The greatest diversity of sterols was in Salt Pond, followed by Fresh Pond, which was attributed to differences in the present eukaryotic diversity. Lipid profiles of the three communities were similar despite the presence of a salinity gradient. Analysis of lipid biomarkers allowed the creation of profiles for these unique Antarctic cryo-ecosystems. This will assist in the comparison of present and past microbial communities and in the monitoring of Antarctic biodiversity in response to global climate change and other environmental perturbations. Crown Copyright © 2008.

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics. © 2009 Springer-Verlag.

This review describes genome mining of cyanobacteria for natural product discovery and biosynthesis pathways. Also presented is an overview of the genetic basis of natural product biosynthesis in cyanobacteria. It includes 143 references. © 2009 The Royal Society of Chemistry.

Revealing the geological history of microbial life is very challenging. Microbes rarely are preserved with morphological fidelity, and even when they are, morphology is a poor guide to phylogeny and metabolism. Biological studies of environments considered analogous to those of paleobiological interest on the ancient Earth can inform interpretations and suggest new approaches. This paper reviews recent advances in our understanding of the biological diversity of two environments relevant to Archean paleobiology: those of extreme acidity and temperature (the Mt. Hood and White Island volcanoes), and high salinity (living stromatolites in Shark Bay). The combination of traditional microbial isolation with the use of modern molecular techniques has revealed that the microbial communities in these environments are much more diverse than originally thought. Through the extraction of whole microbial community DNA, enzymatic amplification of evolutionarily conserved genes, and cloning and sequencing of these genes, more specific and informed inferences concerning functional complexity in these extreme environments have now been made. Studies of the modern stromatolites have demonstrated that they have a very diverse range of micoorganisms, and contrary to previous interpretations, cyanobacteria are not the most abundant microbes present. In addition, many of the microorganisms are unique with no known close relatives, and these microorganisms may also possess novel physiologies vital to the integrity and persistence of stromatolites through space and time. Microbes in the volcanoes studied are present ubiquitously and include geochemically significant sulfur- and iron-cycling taxa. The findings from the studies reviewed here suggest that the Archean biota may have been functionally diverse and much more complex than has yet been revealed. The importance of studying modern analogues is stressed in that the biogeochemical processes occurring in these communities leave morphological, mineralogical, lipid and isotopic signals that could be sought in the rock record. © 2009 Elsevier B.V. All rights reserved.

The cyanobacterium Synechocystis sp. PCC6803 harbours one phosphopantetheinyl transferase (PPTase), Sppt. Protein modelling supported previous bioinformatics analyses, which suggested that Sppt is a Sfp-type PPTase with the potential to phosphopantetheinylate a broad range of carrier proteins from both primary and secondary metabolism. However, no natural products are synthesised by this species, which raises interesting evolutionary and functional questions. Phosphopantetheinylation assays and kinetic data demonstrate that Sppt was able to activate its cognate fatty acid synthesis carrier protein, SACP, but was unable to effectively activate various cyanobacterial carrier proteins from secondary metabolism or glycolipid biosynthesis pathways. To our knowledge, this is the first example of a PPTase with a Sfp-type structure, but with activity more closely resembling AcpS-type enzymes. The broad-range PPTase from Nodularia spumigena NSOR10 was introduced into Synechocystis sp. PCC6803 and was shown to activate a noncognate carrier protein, in vivo. This engineered strain could provide a future biotechnological platform for the heterologous expression of cyanobacterial biosynthetic gene clusters. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool. © 2009 The Authors.

Responses to changes in external salinity were examined in Halobacterium salinarum NRC-1. H. salinarum NRC-1 grows optimally at 4.3 M NaCl and is capable of growth between 2.6 and 5.1 M NaCl. Physiological changes following incubation at 2.6 M NaCl were investigated with respect to growth behavior and proteomic changes. Initial observations indicated delayed growth at low NaCl concentrations (2.6 M NaCl), and supplementation with different sugars, amino acids, or KCl to increase external osmotic pressure did not reverse these growth perturbations. To gain a more detailed insight into the adaptive responses of H. salinarum NRC-1 to changes in salinity, the proteome was characterized using iTRAQ (amine specific isobaric tagging reagents). Three hundred and nine differentially expressed proteins were shown to be associated with changes in the external sodium chloride concentration, with proteins associated with metabolism revealing the greatest response. © 2009 American Chemical Society. .

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were a- and -proteobacteria (58), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology. © 2009 International Society for Microbial Ecology All rights reserved.

Background. Salinity is known to affect almost half of the world's irrigated lands, especially rice fields. Furthermore, cyanobacteria, one of the critical inhabitants of rice fields have been characterized at molecular level from many different geographical locations. This study, for the first time, has examined the molecular diversity of cyanobacteria inhabiting Indian rice fields which experience various levels of salinity. Results. Ten physicochemical parameters were analyzed for samples collected from twenty experimental sites. Electrical conductivity data were used to classify the soils and to investigate relationship between soil salinity and cyanobacterial diversity. The cyanobacterial communities were analyzed using semi-nested 16S rRNA gene PCR and denaturing gradient gel electrophoresis. Out of 51 DGGE bands selected for sequencing only 31 which showed difference in sequences were subjected to further analysis. BLAST analysis revealed highest similarity for twenty nine of the sequences with cyanobacteria, and the other two to plant plastids. Clusters obtained based on morphological and molecular attributes of cyanobacteria were correlated to soil salinity. Among six different clades, clades 1, 2, 4 and 6 contained cyanobacteria inhabiting normal or low saline (having EC < 4.0 ds m-1) to (high) saline soils (having EC > 4.0 ds m-1), however, clade 5 represented the cyanobacteria inhabiting only saline soils. Whilst, clade 3 contained cyanobacteria from normal soils. The presence of DGGE band corresponding to Aulosira strains were present in large number of soil indicating its wide distribution over a range of salinities, as were Nostoc, Anabaena, and Hapalosiphon although to a lesser extent in the sites studied. Conclusion. Low salinity favored the presence of heterocystous cyanobacteria, while very high salinity mainly supported the growth of non-heterocystous genera. High nitrogen content in the low salt soils is proposed to be a result of reduced ammonia volatilization compared to the high salt soils. Although many environmental factors could potentially determine the microbial community present in these multidimensional ecosystems, changes in the diversity of cyanobacteria in rice fields was correlated to salinity.

The paralytic shellfish poisoning (PSP) toxins, saxitoxin, and its derivatives, are produced by a complex and unique biosynthetic pathway. It involves reactions that are rare in other metabolic pathways, however, distantly related organisms, such as dinoflagellates and cyanobacteria, produce these toxins by an identical pathway. Speculative explanations for the unusual phylogenetic distribution of this metabolic pathway have been proposed, including a polyphyletic origin, the involvement of symbiotic bacteria, and horizontal gene transfer. This study describes for the first time the identity of one gene, sxt1, that is involved in the biosynthesis of saxitoxin in cyanobacteria. It encoded an O-carbamoyltransferase (OCTASE) that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in PSP-toxic strains of cyanobacteria and had a high sequence similarity to each other. L. wollei had a naturally mutated sxt1 gene that encoded an inactive enzyme, and was incapable of producing carbamoylated PSP-toxin analogues, supporting the proposed function of Sxt1. Phylogenetic analysis revealed that OCATSE genes were present exclusively in prokaryotic organisms and were characterized by a high rate of horizontal gene transfer. OCTASE has most likely evolved from an ancestral O-sialoglycoprotein endopeptidase from proteobacteria, whereas the most likely phylogenetic origin of sxt1 was an ancestral a-proteobacterium. The phylogeny of sxt1 suggested that the entire set of genes required for saxitoxin biosynthesis may spread by horizontal gene transfer. © 2008 Springer Science+Business Media, LLC.

Antibiotics are commonly detected in the environment as contaminants. Exposure to antibiotics may induce antimicrobial-resistance, as well as the horizontal transfer of resistance genes in bacterial populations. We selected the resistance gene marA, mediating resistance to multiple antibiotics, and explored its distribution in sediment and water samples from surface and sewage treatment waters. Ciprofloxacin and ofloxacin (fluoroquinolones), sulphamethoxazole (sulphonamide), erythromycin, clarythromycin, and spiramycin (macrolides), lincomycin (lincosamide), and oxytetracycline (tetracycline) were measured in the same samples to determine antibiotic contamination. Bacterial populations from environmental samples were challenged with antibiotics to identify resistant isolates. The gene marA was found in almost all environmental samples and was confirmed by PCR amplification in antibiotic-resistant colonies. 16S rDNA sequencing revealed that the majority of resistant isolates belonged to the Gram-positive genus Bacillus, not previously known to possess the regulator marA. We assayed the incidence of marA in environmental bacterial populations of Escherichia coli and Bacillus by quantitative real-time PCR in correlation with the levels of antibiotics. Phylogenetic analysis indicated the possible lateral acquisition of marA by Bacillus from Gram-negative Enterobacteriaceae revealing a novel marA homolog in Bacillus. Quantitative PCR assays indicate that the frequency of this gene in antropised environments seems to be related to bacterial exposure to water-borne antibiotics. © 2008 Elsevier Ltd. All rights reserved.

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5a (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(±0.3) U mg-1 protein, compared to 0.1(±0.01) U mg-1 protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases. © 2008 Elsevier B.V. All rights reserved.

Toxic cyanobacteria pose a significant hazard to human health and the environment. The recent characterisation of cyanotoxin synthetase gene clusters has resulted in an explosion of molecular detection methods for these organisms and their toxins. Conventional polymerase chain reaction (PCR) tests targeting cyanotoxin biosynthesis genes provide a rapid and sensitive means for detecting potentially toxic populations of cyanobacteria in water supplies. The adaptation of these simple PCR tests into quantitative methods has additionally enabled the monitoring of dynamic bloom populations and the identification of particularly problematic species. More recently, DNA microarray technology has been applied to cyanobacterial diagnostics offering a high-throughput option for detecting and differentiating toxic genotypes in complex samples. Together, these molecular methods are proving increasingly important for monitoring water quality. Crown Copyright © 2008.

Over the last 10 years, we have witnessed major advances in our understanding of natural product biosynthesis, including the genetic basis for toxin production by numerous groups of cyanobacteria. Cyanobacteria produce an unparalleled array of bioactive secondary metabolites, including alkaloids, polyketides and non-ribosomal peptides, some of which are potent toxins. This review addresses the molecular genetics underlying the production of hepatotoxins, microcystin and nodularin in fresh and brackish water. These toxins pose a serious threat to human health and their occurrence in water supplies is increasing, because of the prevalence of toxic algal blooms worldwide. Toxin biosynthesis gene-cluster-associated transposition and the natural transformability of certain species suggest a broader distribution of toxic cyanobacterial taxa. The information gained from the discovery of these toxin biosynthetic pathways has enabled the genetic screening of various environments for drinking-water quality management. Understanding the role of cyanotoxins in the producing microorganisms and the environmental regulation of their biosynthesis genes may also suggest the means of controlling toxic-bloom events. Copyright © 2008 Informa UK Ltd.

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis. © 2007 Springer.

Extremely halophilic archaea were cultivated from smooth and pustular microbial mats collected from Hamelin Pool, Shark Bay, Western Australia. On the basis of morphology, two phenotypes were present and 16S rRNA gene sequence analysis indicated that all strains were most closely related to members of the genus Haloferax (98.1-99.4% similarity). One representative strain from each phenotype was selected for further taxonomic characterization. Strain SA5T, isolated from the smooth mat, formed small (~1 mm diameter), red, translucent colonies on agar medium and strain PA12T, isolated from the pustular mat, formed large (3-5 mm diameter), pink, mucoid, domed colonies. Both strains grew in media with 1.7-5.1 M NaCl, required at least 0.2 M Mg2+ for growth and had pH optima of 7.4. The 16S rRNA gene similarity between strains SA5T and PA12T was 97.1 %. Physiological properties, G+C content and polar lipid composition supported placement of both strains in the genus Haloferax. Phenotypic analysis indicated that the two strains were distinct from each other and from all other members of the genus. This was confirmed by the low DNA-DNA relatedness between strains SA5T and PA12T (18-30 %) and between both strains and all other recognized Haloferax species. Two novel species of the genus Haloferax are proposed to accommodate these novel isolates, Haloferax elongans sp. nov. (type strain SA5T=JCM 14791T=ATCC BAA-1513T5UNSW 104100T) and Haloferax mucosum sp. nov. (type strain PA12T=JCM 14792T=ATCC BAA-1512T=UNSW 104200T). © 2008 IUMS.

Taxol is a powerful and complex anti-cancer compound that was first isolated from the bark of the Pacific yew Taxus brevifolia. Although it offered huge potential as an anti-cancer agent, it experienced a long development period, attributed to by its low availability from its traditional source. Research into alternate sources and methods of production for Taxol have been crucial in meeting with demand for the drug. Three main avenues of research have resulted. Firstly, chemical syntheses of this complex diterpene consist of multiple steps and are not economically feasible due to their low yield. Developments have therefore concentrated on enhancing production in vivo. Efforts have been made to understand the enzymatic steps involved in the synthesis within the yew and innovations to produce Taxol and Taxol-like substances in high yield from cell cultures of Taxus species. An alternative stream of research focuses on endophytes as the producer of Taxol. Endophytes can be isolated from the yew tree and produce Taxol in culture. Encouraging findings with endophytes resulted in much interest in the prospect of using endophytes as the producer of Taxol and Taxol-like substances. This review also discusses patents and the future prospects of each of the main streams of production. © 2008 Bentham Science Publishers Ltd.

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 ± 1.8 nmol Pi min-1 mg protein -1, with a requirement for Mg2+. Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thiotemplate function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of D-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce a-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Saxitoxin (STX) and its analogs are voltage-gated sodium-channel blockers that cause paralytic shellfish poisoning (PSP) and negatively affect human health and seafood industries worldwide. Little is known about the molecular biology of PSP-toxin synthesis. Saxitoxin precursors were identified 25 years ago, and a hypothetical biosynthesis pathway was proposed; however, the correct sequence of reactions and enzymes involved in their catalysis remains to be identified. This study describes the optimization of in vitro biosynthesis of PSP toxins by cellular lysates of the toxic cyanobacterium Cylindrospermopsis raciborskii (Wolosz.) Seenaya et Subbaraju T3 and the characterization of its biochemical requirements. Enzymes involved in PSP-toxin synthesis are located in the cytosol. The molecular components of in vitro biosynthesis reactions could not be completely defined because of the requirement of an unknown cofactor. Evidence is presented that supports the previous suggestion that STX biosynthesis involves a Claisen condensation between arginine and acetate. In addition, carbamoyl phosphate was identified as a likely precursor for carbamated PSP toxins. Predictions have been made regarding the enzymes that may be involved in the biosynthesis of PSP toxins. These included class II aminotransferase; nonheme iron oxygenase, containing flavin, and possibly ferredoxin, as the prosthetic groups; and an O-carbamoyltransferase. On the other hand, the involvement of cytochrome P450 monooxygenase was excluded. © 2007 Phycological Society of America.

Cyanobacteria that produce the toxin microcystin have been isolated from many parts of the world. Most of these organisms are planktonic; however, we report on several microcystin-producing benthic filamentous cyanobacterial isolates from four drinking-water reservoirs in southern California (USA): Lake Mathews, Lake Skinner, Diamond Valley Lake (DVL), and Lake Perris. Some samples of benthic material from these reservoirs tested positive for microcystin by an ELISA tube assay, and all the positive samples had in common a green filamentous cyanobacterium 10-15 µm in diameter. Seventeen unialgal strains of the organism were isolated and tested positive by ELISA, and 11 cultures of these strains were found to contain high concentrations of microcystin-LR (90-432 µg L-1). The cultures were analyzed by protein phosphatase inhibition assay (PPIA) and HPLC with photodiode array detector (PDA) or liquid chromatography/mass spectrometry (LC/MS). Microcystin per unit carbon was determined for six cultures and ranged from 1.15 to 4.15 µg mg-1 C. Phylogenetic analysis of four cultures from Lake Skinner and DVL using cyanobacterial-specific PCR and sequencing of the partial 16S rRNA gene suggested the highest similarity to an unidentified cyanobacterium in the oscillatoriales, and to a Phormidium sp. Morphologically, some of the isolates were similar to Oscillatoria, and others resembled Lyngbya. The significance of these organisms lies in the relative scarcity of known toxin producers among freshwater benthic cyanobacteria, and also as a source of cell-bound microcystin in these reservoirs. © 2006 Elsevier Ltd. All rights reserved.

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA-PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment. © 2006 Springer.

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm-1. The bands at 1505 cm-1 and 1152 cm-1 are due to in-phase C = C (v1) and C-C stretching (v2) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH3 groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm-1 region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C = C stretches (1600-1500 cm-1), the CCH in-plane rocks (1400-1250 cm-1), the C-C stretches (1250-1100 cm-1), and the hydrogen out-of-plane wags (1000-700 cm-1). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry. © Mary Ann Liebert, Inc.

Rhodococcus sp. 33 can tolerate and efficiently degrade various concentrations of benzene, one of the most toxic and prevailing environmental pollutants. This strain produces a large quantity of extracellular polysaccharide (33 EPS), which plays an important role in the benzene tolerance in Rhodococcus sp. 33, especially by helping the cells to survive an initial challenge with benzene. This EPS has been reported to be composed of d-galactose, d-glucose, d-mannose, d-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1. To understand the protective effect of 33 EPS, we determined its chemical structure by using 1H and 13C NMR spectroscopy including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (Equation presented). © 2006 Elsevier Ltd. All rights reserved.

The alkaloid cylindrospermopsin is the most recently discovered cyanotoxin and has caused epidemic outbreaks of human poisoning. Cylindrospermopsin producing cyanobacteria have in recent times appeared in countries all over the world where they had not been observed previously and, thus, represent a global public health concern. Three putative cylindrospermopsin biosynthesis genes, encoding an amidinotransferase (aoaA), a nonribosomal peptide synthetase (aoaB), and a polyketide synthase (aoaC), have been described. Most cyanotoxins are the product of nonribosomal peptide and polyketide synthesis, but the involvement of an amidinotransferase is novel. In the present study, functional modeling was carried out to gain insight into the mechanism of precursor recruitment in cylindrospermopsin biosynthesis. In addition, the molecular phylogenies of putative cylindrospermopsin biosynthesis genes and producer organisms were determined. The model indicated that AoaA may catalyze the formation of guanidino acetate from glycine and arginine. The catalytic site of the AoaB adenylation domain provided two aspartate residues, instead of the usual one, which may be involved in the binding of the guanidino moiety of guanidino acetate. Molecular phylogenetic analysis grouped cylindrospermopsin producing cyanobacteria into two divergent groups. Although the phylogeny of the cylindrospermopsin biosynthesis genes followed that of the producer organisms, they were less divergent, which may indicate the recent horizontal transfer of these genes. © Springer Science+Business Media, Inc. 2006.

Recently there has been recognition of the importance of reductive processes in the acquisition of iron by microorganisms in marine environments with Fe(III) reduction induced by either membrane-bound reductases or by superoxide, a powerful Fe(III) reducing agent generated either by photochemical or biological means. We have measured the relative rates of iron uptake achieved by the cyanobacterium L. majuscula in the presence of a variety of model- and naturally-derived organic ligands exhibiting a broad range of conditional ferric and ferrous stability constants. Additionally, we have investigated the effect upon iron uptake rate of varying the concentration of both iron and the iron-binding ligands. We have reconciled this data with previous work in which we measured rates of reduction by exogenous superoxide of Fe(III) bound to these same complexes. We show that the rate of formation of ferrous iron and the rate of uptake of iron by Lyngbya majuscula are each independent of the concentration of Fe' and demonstrate that this is consistent with our previous finding that this organism acquires iron via nondissociative reduction of ferric complexes. We also show that the rate of reoxidation of organically complexed Fe(II) is a critical determinant of the subsequent bioavailability of iron, a feature not previously addressed in the literature. In view of the central importance of the complexation and redox behavior of the iron-organic complexes to iron uptake rate, we propose a new kinetic model of iron acquisition, termed the FeL model, that is consistent with presented and previously published data and which describes processes both in natural and artificial conditions. © 2006, by the American Society of Limnology and Oceanography, Inc.

Cyanobacteria are well known for their production of non-ribosomal cyclic peptide toxins, including microcystin, in temperate and tropical regions, however, the production of these compounds in extremely cold environments is still largely unexplored. Therefore, we investigated the production of protein phosphatase inhibiting microcystins by Antarctic cyanobacteria. We have identified microcystin-LR and for the first time [d-Asp3] microcystin-LR by mass spectrometric analysis in Antarctic cyanobacteria. The microcystins were extracted from a benthic microbial community that was sampled from a meltwater pond (Fresh Pond, McMurdo Ice Shelf, Antarctica). The extracted cyanobacterial cyclic peptides were equivalent to 11.4 ng MC-LR per mg dry weight by semi-quantitative analyses using HPLC-DAD and the protein phosphatase inhibition assay. Furthermore, we were able to identify the presence of cyanobacterial non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes in total DNA extracts from the mat community. © 2005 Elsevier Ltd. All rights reserved.

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis, The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Endolithic photosynthetic communities in geothermal siliceous rocks in the area of Rotorua, New Zealand, were analysed using traditional microbiological and molecular biology techniques. Rock surface temperatures varied between 40°C and 60°C. Major endoliths included cyanobacteria of subsections I, II and V. Few subsection IV organisms were found and subsection III (filamentous, non-heterocystous) cyanobacteria were present only as epiliths or chasmoendoliths. Therefore, the endolithic cyanobacterial communities in these sites resembled assemblages as reported for carbonate rocks in other geothermal regions. Cells of the rhodophyte family Cyanidiaceae were detected within rock at various sites. Some of these phototrophic organisms were associated with mineral (presumably silica) deposits and could be important geological agents in siliceous rock deposition.

Blooms of the freshwater cyanobacterium Anabaena circinalis are recognised as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives, also known as paralytic shellfish poisoning (PSP) toxins. In this study the transcriptional profile of PSP toxin-producing and non-toxic strains of A. circinalis was investigated by means of a DNA microarray approach. Additionally, gene expression was studied after exposure of toxic A. circinalis cultures to lidocaine hydrochloride at 1¿µM for 2¿h. Under standard growth conditions, a limited number of putative toxic-strain distinctive DNA fragments, identified in previous studies, were preferentially expressed in toxic versus non-toxic strains. The same genes did not significantly change their expression after exposure to 1¿µM lidocaine, conditions previously shown to induce STX production in the cyanobacterium Cylindrospermopsis raciborskii T3. Lidocaine supplementation, however, enhanced the transcription of genes involved in physiological adaptive responses and bloom formation in cyanobacteria, such as the gas vesicle structural protein A and phycocyanin. The heat shock protein HSP-70 and the chlorophyll-a binding protein isiA were significantly repressed by lidocaine exposure. Stress response proteins and genes implicated in secondary metabolism were repressed, including phosphopantetheinyl transferases. The BGGM1 DNA microarray, used in this study, was shown to be suitable for gene expression studies in cultured toxic cyanobacteria and allowed the analysis of gene transcripts associated with surface scum formation by toxic A. circinalis. © 2006 Elsevier Ltd. All rights reserved.

The cyanobacterial hepatotoxins, microcystin and nodularin, are produced by a wide range of cyanobacteria. Microcystin production has been reported in the four cyanobacterial orders: Oscillatoriales, Chroococcales, Stigonematales, and Nostocales. The production of nodularin is a distinct characteristic of the Nostocales genus Nodularia. A single rapid method is needed to reliably detect cyanobacteria that are potentially capable of producing these hepatotoxins. To this end, a PCR was designed to detect all potential microcystin and nodularin-producing cyanobacteria from laboratory cultures as well as in harmful algal blooms. The aminotransferase (AMT) domain, which is located on the modules mcyE and ndaF of the microcystin and nodularin synthetase enzyme complexes, respectively, was chosen as the target sequence because of its essential function in the synthesis of all microcystins as well as nodularins. Using the described PCR, it was possible to amplify a 472 bp PCR product from the AMT domains of all tested hepatotoxic species and bloom samples. Sequence data provided further insight into the evolution of the microcystin and nodularin synthetases through bioinformatic analyses of the AMT in microcystin and nodularin synthetases, with congruence between the evolution of 16S rRNA and the AMT domain. © Springer-Verlag 2006.

There are few modern analyses of the cyanobacterial communities in biofilms on external building surfaces. As the classification of cyanobacteria is rapidly changing, we aimed to identify them on historic buildings in Brazil using both established and molecular techniques. In mature biofilms, cyanobacteria of subsections I and II were generally the major biomass; occasionally filamentous genera of the Scytonemataceae, Microchaetaceae and Rivularaceae were dominant. Filamentous organisms of subsections III and IV were more frequently isolated in culture. PCR products using cyanobacteria-specific 16S rDNA primers were sequenced from morphologically identified organisms. Homologies with deposited sequences were generally low. Phylogenetic analysis showed that many isolates were distant from their nearest neighbours, even though they grouped with their appropriate taxa. The majority of cyanobacterial DNA sequences deposited in data banks are aquatic; our results indicate that cyanobacteria from external walls are an ecologically isolated group. © 2006.

The gene slr0388 was previously annotated to encode a hypothetical protein in Synechocystis sp. strain PCC 6803. When a positively phototactic strain of this cyanobacterium was insertionally inactivated at slr0388, the mutants were not transformable, and appeared to aggregate as a result of increased bundling of type IV pili. Also, these mutants were rendered non-phototactic compared to the wild-type. Quantitative real-time PCR revealed a 3·5-fold increase in pilA1 transcript levels in the mutant over wild-type cells, while there were no changes in the level of pilT 1 and comA transcripts. Supernatant from mutant liquid culture contained more PilA1 protein, confirmed by mass spectrometric analysis, compared to the wild-type cells, which corresponded to the increase in pilA1 transcripts. The increase in PilA1 subunits may contribute to the bundling morphology of pili that was observed, which in turn may act to retard DNA uptake by hindering the retraction of pili. This gene is therefore proposed to be designated comF, as it possesses a phosphoribosyltransferase domain, a distinguishing feature of other ComF proteins of naturally transformable heterotrophic bacteria. This report is the second of a competence-related gene from Synechocystis sp. strain PCC 6803, the product of which does not show homology to other well-studied type IV pili proteins. © 2006 SGM.

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50-80°C and pH 6.0-8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA-DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures. © Springer-Verlag 2006.

Several halophilic archaea belonging to the genus Halococcus were isolated from stromatolites from Hamelin Pool, Shark Bay, Western Australia, collected during field trips in 1996 and 2002. This is the first incidence of halophilic archaea being isolated from this environment. Stromatolites are biosedimentary structures that have been formed throughout the earth's evolutionary history and have been preserved in the geological record for over 3 billion years. The stromatolites from Hamelin Pool, Western Australia, are the only known example of extant stromatolites forming in hypersaline coastal environments. Based on their 16S rRNA gene sequences and morphology, the isolates belong to the genus Halococcus. Strain 100NA1, isolated from stromatolites collected in 2002, was closely related to strain 100A6T that was isolated from the stromatolites collected in 1996, with a DNA-DNA hybridization value of 94 ± 8%. DNA-DNA hybridization values of strain 100A6T with Halococcus morrhuae NRC 16008 and Halococcus saccharolyticus ATCC 49257T were 17 ± 6 and 11 ± 7 %, respectively. The DNA G+C content of strain 100A6T was 60.5 mol% (Tm). The main polar lipid was S-DGA-1, a sulphated glycolipid that has been detected in all strains of the genus Halococcus. Whole-cell protein profiles, enzyme composition and utilization of various carbon sources were distinct from those of all previously characterized Halococcus species. The recognition of this strain as representing a novel species within the genus Halococcus is justified, and the name Halococcus hamelinensis sp. nov. is proposed. The type strain is 100A6T (= JCM 12892T = ACM 5227T). © 2006 IUMS.

We surveyed the occurrence of toxigenic cyanobacteria, the mcyA component of the microcystin synthetase gene and microcystin in aquatic systems in temperate Australia and tropical Thailand. The survey methods, microscopy, protein phosphatase inhibition assay, enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), were evaluated for screening raw water for public health risk from microcystin producing toxigenic cyanobacteria. Three tests, ELISA, PCR and protein phosphatase inhibition (PPi), were judged very useful because of their sensitivity and speed of analysis. ELISA ranked slightly higher because of its superior specificity for microcystin. The PCR was highly sensitive, but there were three false negative results, whilst the PPi was cheap but less specific than ELISA. Some of the microcystin quantifications results were validated by high performance liquid chromatography (HPLC). The combination of a gene probe for the mcy gene complex with an ELISA or PPi assay for microcystin is proposed as a powerful screening technique for raw waters that can provide multi-level risk assessment including 'incipient risk', when microcystin producing cyanobacterial strains are rare and microcystin concentrations are below the detection limit for biochemical or chromatographic analyses. © IWA Publishing 2005.

Reduction of iron from the ferric state to the ferrous state is one strategy employed by microorganisms in near-neutral environments to increase its biological availability. In recent years, the existence of mobile reducing agents produced by microorganisms to promote iron reduction, known as electron shuttles, has been demonstrated. Production of electron shuttles has been shown for several organisms, employing a variety of mostly organic molecules as the electron carrier. Here we show that the coastal cyanobacterium Lyngbya majuscula produces iron-reducing superoxide radicals (O2.-) and that this facilitates increased iron uptake. We suggest that superoxide is a useful electron shuttle because it reacts rapidly and almost indiscriminately with Fe(III)-organic complexes and its precursor, dissolved oxygen, is ubiquitous in the photic zone. We further suggest that, for these reasons, the generation of superoxide by marine oxygenic photosynthetic microorganisms and its use in facilitating iron uptake may be a reasonably widespread process. © 2005 American Chemical Society.

In this study we investigated the presence of toxin-producing cyanobacterial contaminants in food supplements manufactured from blooms of the non-toxic freshwater cyanobacterium Aphanizomenon flos-aquae. Previous reports investigating the contamination of health food supplements with toxin-producing cyanobacteria have used chemical and or biochemical methods such as HPLC, ELISA and protein phosphatase assays. Whilst these studies have drawn attention to the presence of hepatotoxic microcystins in some commercially available food supplements, the methods used do not provide any information on the source of the contaminant. Such information would be useful for the quality control of food supplements produced for human consumption. In this study we applied a molecular technique, involving the amplification of the 16s rRNA gene, the phycocyanin operon, and two genes of the microcystin synthetase gene cluster to show that all 12 food supplement samples, sourced from various internet distributors and containing non-toxic A. flos-aquae, also contained toxigenic cyanobacteria. Sequencing of the microcystin synthetase genes detected in all of the food supplements showed that M. aeruginosa was the organism responsible for the production of microcystins in the samples. The presence of microcystins in the food supplements was confirmed by ELISA, with concentrations within the range of 0.1-4.72 µg g-1 (microcystin-LR equivalents). Given that the molecular methods applied here are highly sensitive, and show good agreement with the results obtained from ELISA, we believe that they could potentially be used as a quality control technique for food products that contain cyanobacteria. © 2005 Elsevier Ltd. All rights reserved.

To measure genetic variation within and among populations of the bloom-forming cyanobacterium Microcystis aeruginosa, we surveyed a suite of lakes in the southern peninsula of Michigan that vary in productivity (total phosphorus concentrations of ~10 to 100 µg liter-1). Survival of M. aeruginosa isolates from lakes was relatively low (i.e., mean of 7% and maximum of 30%) and positively related to lake total phosphorus concentration (P = 0.014, r2 = 0.407, n = 14). In another study (D. F. Raikow, O. Sarnelle, A. E. Wilson, and S. K. Hamilton, Limnol. Oceanogr. 49:482-487, 2004), survival rates of M. aeruginosa isolates collected from an oligotrophic lake (total phosphorus of ~10 µg liter-1 and dissolved inorganic nitrogen:total phosphorus ratio of 12.75) differed among five different medium types (G test, P of <0.001), with higher survival (P = 0.003) in low-nutrient media (28 to 37% survival) than in high-nutrient media. Even with the relatively low isolate survivorship that could select against detecting the full range of genetic variation, populations of M. aeruginosa were genetically diverse within and among lakes (by analysis of molecular variance, Fsc = 0.412 [Fsc is an F-statistic derivative which evaluates the correlation of haplotypic diversity within populations relative to the haplotypic diversity among all sampled populations], P = 0.001), with most clones being distantly related to clones collected from lakes directly attached to Lake Michigan (a Laurentian Great Lake) and culture collection strains collected from Canada, Scotland, and South Africa. Ninety-one percent of the 53 genetically unique M. aeruginosa clones contained the microcystin toxin gene (mcyA). Genotypes with the toxin gene were found in all lakes, while four lakes harbored both genotypes possessing and genotypes lacking the toxin gene. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Four putative type IV pilus genes from the toxic, naturally transformable Microcystis aeruginosa PCC7806 were identified. Three of these genes were clustered in an arrangement which is identical to that from other cyanobacterial genomes. Type IV pilus-like appendages were also observed by electron microscopy. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds - Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf. © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd.

The mammalian gastrointestinal tract is covered by a layer of mucus that can harbour a range of bacterial species specifically adapted to colonize this ecological niche. Examination of 110 bacterial isolates cultivated from the gastrointestinal tract of 23 mice revealed the presence of a subgroup of 30 isolates that did not correspond genetically with genera commonly associated with this site, i.e. members of the e-Proteobacteria such as Helicobacter and Campylobacter species. Instead this group of isolates was found to lie within the phylum Deferribacteres, a completely distinct lineage in the domain Bacteria. There was a high level of consensus in results obtained from the phenotypic and genotypic characterization of a number of the isolates, which showed they were distinct from other members of the Deferribacteres. As such, they are proposed to constitute a new genus and species, Mucispirillum schaedleri gen. nov., sp. nov. These organisms are anaerobic, Gram-negative, spiral-shaped rods with bipolar flagella. The type strain is HRI I17T (= ATCC BAA-1009T = ACM 5223T). © 2005 IUMS.

The 16S-rDNA from 22 cyanobacteria isolated from biofilms on walls of modern and historic buildings in Brazil was partially sequenced (~350 bp) using specific primers. The cyanobacteria with the closest matching sequences were found using the BLAST tool. The sequences were combined with 52 other cyanobacterial sequences already deposited in public data banks and a dendrogram constructed, after deletion from each sequence of one of the variable 16S rDNA regions (VI). The newly sequenced organisms fitted well within their respective families, but their similarities to other members of the groups were generally low, less than 96%. Close matches were found only with one other terrestrial (hot dry desert) cyanobacterium, Microcoleus sociatus, and with Anabaena variabllis. Phylogenetic analysis suggested that the deletion of the hypervariable regions in the RNA structure is essential for meaningful evolutionary studies. The results support the standard phylogenetic tree based on morphology, but suggest that these terrestrial cyanobacteria are distant relatives of their equivalent aquatic genera and are, indeed, a distinct population.

Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. Microcystin is synthesized nonribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55-kb microcystin synthetase (mcy) gene cluster. Also encoded within the mcy gene cluster is a putative ATP binding cassette (ABC) transporter, McyH. This study details the bioinformatic and mutational analyses of McyH and offers functional predictions for the hypothetical protein. The transporter is putatively comprised of two homodimers, each with an N-terminal hydrophobic domain and a C-terminal ATPase. Phylogenetically, McyH was found to cluster with members of the ABC-A1 subgroup of ABC ATPases, suggesting an export function for the protein. Two mcyH null mutant (¿mcyH) strains were constructed by partial deletion of the mcyH gene. Microcystin production was completely absent in these strains. While the mcyH deletion had no apparent effect on the transcription of other mcy genes, the complete microcystin biosynthesis enzyme complex could not be detected in ¿mcyH mutant strains. Finally, expression levels of McyH in the wild type and in ¿mcyA, ¿mcyB, and ¿mcyH mutants were investigated by using immunoblotting with an anti-McyH antibody. Expression of McyH was found to be reduced in ¿mcyA and ¿mcyB mutants and completely absent in the ¿mcyH mutant. By virtue of its association with the mcy gene cluster and the bioinformatic and experimental data presented in this study, we predict that McyH functions as a microcystin exporter and is, in addition, intimately associated with the microcystin biosynthesis pathway.

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with nomology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. Tliese analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.

Pharmaceutically active substances have recently been recognised as an emerging environmental problem. Human and veterinarian therapeutic agents can contaminate aquatic ecosystems via sewage discharges (human and animal excretion), improper disposal or industrial waste. Very little is known on the effects of pharmaceutical pollutants on aquatic photosynthetic organisms. In this study the effects of erythromycin, tetracycline and ibuprofen on the growth of the cyanobacterium Synechocystis sp. PCC6803 and the duckweed Lemna minor FBR006 were studied at concentrations of 1-1000µgl-1. At dosage of 1mgl-1, erythromycin affected the growth of both Synechocystis and Lemna with a maximum inhibition of 70 and 20%, respectively. Tetracycline had inhibitory effects (20-22% reduction in growth) on Synechocystis at intermediate dosages. The same aminoglycoside antibiotic promoted growth in Lemna by 26% at 10µgl-1, while frond development was reduced at 1mgl-1 (tetracycline). The anti-inflammatory ibuprofen strongly stimulated the growth of Synechocystis at all concentrations tested (72% increase at 10µgl -1) although inhibited Lemna in a linear dose-dependent manner with a 25% reduction over control levels at a dosage of 1mgl-1. The 7 days effective concentration (EC50) calculated for Lemna were 5.6, 1 and 4gl-1, respectively, for erythromycin, tetracycline and ibuprofen. Moreover, exposure to the three pharmaceuticals resulted in the production of the stress hormone, abscisic acid (ABA), in Lemna. Erythromycin and tetracycline were more effective in promoting ABA synthesis compared to ibuprofen. The effects shown by the three therapeutic drugs on Synechocystis and Lemna growth may have potential implications in the assessments of residual environmental risks associated with the presence of pharmaceuticals in freshwater ecosystems. Promotion of ABA synthesis in Lemna by the two antibiotics and by copper suggests that the plant hormone could be a suitable (additional) indicator for future evaluation of phytotoxicity that results in plant senescence. © 2004 Elsevier B.V. All rights reserved.

The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1+2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 µg l-1. Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 µg l-1 CAM increased C1+2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1+2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1+2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3. © 2004 Elsevier B.V. All rights reserved.

A mini-Tn10:lacZ:kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in ß-galactosidase activity when grown in lactose medium. ß- Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and ß-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l-1 after 4 days incubation. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Cyanobacterial populations detected on buildings by traditional methods are mainly filamentous, whereas direct microscopy shows that they are principally coccoid morphotypes that often cannot be isolated in culture, but may grow on artificial media when the spatial biofilm relationships are maintained. The polyphasic strategy described here was to select morphologically distinct colonies from rehydrated biofilms for direct DNA amplification, allowing uncultured organisms to be sequenced and their morphology to be characterized by microscopy. DNA data banks currently contain many entries for cyanobacteria of unrecorded morphology, which does not facilitate identification, although genetic variability in a population may be assessed. The sequence homologies of the present biofilm organisms (EMBL accession numbers AJ619681 to 619690) with those in DNA databanks were low, indicating differences between xerophytic cyanobacteria on walls and aquatic species comprising the majority in the databases. Further development of databases for the populations found in this environment, subject to temperature extremes, repeated desiccation and high UV and salt levels, is required.

The biosynthesis of algicides produced by a novel Fischerella strain was investigated. Two allelochemicals were identified, the aminoacylpolyketide fischerellin A (FsA) and the alkaloid 12-epi-hapalindole F (HapF). Based on the structure of FsA, genes that could be involved in its biosynthesis, including those encoding nonribosomal peptide synthetases (NRPSs) and a polyketide synthase (PKS), were identified by the polymerase chain reaction (PCR). By showing that the expression of NRPSs and PKSs is concomitant with algicide production we suggest that the identified genes may be involved in algicide biosynthesis. Analysis of an algicide preparation of the Brazilian-Amazonian strain Fischerella sp. CENA 19 revealed the production of FsA, m/z 409 (MH +), HapF, m/z 370 (MH+), and other potential isoforms of the latter compounds, which were identified by high-performance liquid chromatography (HPLC) and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass-spectrometry. The production of HapF was confirmed after purification by HPLC, analysis by NMR, and high-resolution mass-spectrometry (HRMS). Two-NRPS and a PKS gene were identified after specific amplification using a degenerate PCR. The expression of these synthetases was confirmed by Western blot analysis employing enzyme family-specific antibodies. These analyses revealed the presence of three NRPSs and a single PKS in Fischerella sp. CENA 19. The structure of FsA indicates both aminoacyl- and polyketide moeities, suggesting that its biosynthesis may require an integrated NRPS/PKS enzyme system, possibly involving the genes and the synthetases identified. © 2004 Kluwer Academic Publishers.

The effects of procaine and some related agents on the growth rate of the cyanobacterium Synechococcus leopoliensis were investigated under photoautotropic growth conditions and the data compared with those for selected phytohormones. Low concentrations (0.1-1 µM) of synthetic local anaesthetics (procaine, procainamide, tetracaine, lidocaine, dibucaine) enhanced photoautotrophic growth. Lidocaine was the most effective growth stimulant, followed in decreasing order by procainamide, tetracaine, dibucaine, and procaine. Increases in growth rate were comparable to those elicited by similar concentrations of the auxins indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid and of the cytokinin kinetin. Quantitative structure-activity relationships between the anaesthetics and phytohormones were sought. Score plots of principal components formulated from twelve molecular descriptors suggest that the proliferative effects evoked by the anaesthetics and phytohormones may result from similar affinities for binding receptor sites on the cyanobacterial cell surface. Cyanobacterial growth assays could therefore be utilised as screens for compounds with potential applications in agriculture and medicine. evolution.

Cyanobacteria are ubiquitous in the freshwater environment. Their success as a group in a wide range of aquatic habitats has been attributed to their unique physiological characteristics and their high adaptive ability over a wide range of environmental conditions. They are capable of reaching very high biomass levels, often dominating the other aquatic biota, and under some circumstances can accumulate near the water surface, producing scums. Such cyanobacterial "blooms" are of particular concern in reservoirs used to supply potable water. Dense aggregations of cyanobacterial cells may block water filters, and many species produce compounds that affect the taste and odor of water supplies. Of greatest concern, however, is the potential of many bloom-forming cyanobacteria to produce a wide range of toxic substances. These natural compounds, known as cyanotoxins, are chemically diverse and are usually either neuro- or hepatotoxic in pathology.

A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms.

Aims: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). Methods and Results: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. Conclusions: The results indicate that cereulide is produced by a NRPS complex. Significance and Impact of the Study: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.

Stromatolites have been present on Earth, at various levels of distribution and diversity, for more than 3 billion years. Today, the best examples of stromatolites forming in hypersaline marine environments are in Hamelin Pool at Shark Bay, Western Australia. Despite their evolutionary significance, little is known about their associated microbial communities. Using a polyphasic approach of culture-dependent and culture-independent methods, we report the discovery of a wide range of microorganisms associated with these biosedimentary structures. There are no comparable reports combining these methodologies in the survey of cyanobacteria, bacteria, and archaea in marine stromatolites. The community was characterized by organisms of the cyanobacterial genera Synechococcus, Xenococcus, Microcoleus, Leptolyngbya, Plectonema, Symploca, Cyanothece, Pleurocapsa and Nostoc. We also report the discovery of potentially free-living Prochloron. The other eubacterial isolates and clones clustered into seven phylogenetic groups: OP9, OP10, Marine A group, Proteobacteria, Low G+C Gram-positive, Planctomycetes and Acidobacteria. We also demonstrate the presence of sequences corresponding to members of halophilic archaea of the divisions Euryarchaeota and Crenarchaeota and methanogenic archaea of the order Methanosarcinales. This is the first report of such archaeal diversity from this environment. This study provides a better understanding of the microbial community associated with these living rocks.

The global pattern of growth-phase-dependent gene expression of Helicobacter pylori during in vitro culture was analyzed by using a high-density DNA microarray. To detect consistent coordinated gene expression in this bacterium, temporal changes in transcription were assessed in two independent time courses. Cluster analysis of the expression profiles highlighted a major switch in gene expression during the late log-to-stationary phase transition that we have termed the Log-Stat switch. Statistical analysis of the genes that were significantly induced or repressed during the Log-Stat switch revealed that many of these genes were related to virulence. Among these, expression of the genes for the neutrophil activating protein (napA) and the major flagellin subunit (flaA) were significantly induced. Additionally, the expression of a number of genes involved in iron homeostasis changed dramatically at this switch; the gene for the iron-storage protein, pfr, was induced, while the genes for two putative iron uptake proteins, fecA and frpB, were significantly repressed. These data suggest that the late log phase may correspond to the most virulent phase of growth in H. pylori and may be intimately related to its pathogenesis. The use of microarrays to analyze the kinetics of the transcriptional response of a bacterial pathogen to a changing environment has enabled the discovery of previously unappreciated relationships between genes by elucidation of coordinated gene expression profiles.

Type I polyketide synthases are known to produce a wide range of medically and industrially important polyketides. The ketosynthase (KS) domain is required for the condensation of an extender unit onto the growing polyketide chain during polyketide biosynthesis. KSs represent a superfamily of complex biosynthetic pathway-associated enzymes found in prokaryotes, fungi, and plants. Although themselves functionally conserved, KSs are involved in the production of a structurally diverse range of metabolites. Degenerate oligonucleotide primers, designed for the amplification of KS domains, amplified KS domains from a range of organisms including cyanobacterial and dinoflagellates. KS domains detected in dinoflagellate cultures appear to have been amplified from the less than 3-µm filtrate of the nonaxenic culture. Phylogenetic analysis of sequences obtained during this study enabled the specific identification of KS domains of hybrid or mixed polyketide synthase/peptide synthetase complexes, required for the condensation of an extender unit onto an amino acid starter unit. The primer sets described in this study were also used for the detection of novel KS domains directly from environmental samples. The ability to predict function based on primary molecular structure will be critical for future discovery and rational engineering of polyketides.

The metabolic effect of three different concentrations of lidocaine hydrochloride (0.01, 0.1, and 1 µM) on growth and saxitoxin (STX) production of the freshwater cyanobacterium Cylindrospermopsis raciborskii (Wolosznska) T3 was analyzed. Lidocaine hydrochloride increased both the growth rate and the final growth yield in the toxic cyanobacterium, with a maximum of 25% and 18% for a 1-µM dose, respectively. Moreover, C. raciborskii T3 samples harvested at the end of the growth phase and analyzed for STX content by HPLC showed an increase in STX intracellular concentration of 14.3% and 49.3% after exposure to 0.01 and 0.1 µM lidocaine hydrochloride, respectively, whereas 1 µM lidocaine hydrochloride resulted in a 114% incremental change in STX content. The time course of the 1-µM lidocaine hydrochloride effect showed the highest rate of increase in mean STX intracellular concentration (298%) within the first 2 h after induction. The increase in STX content induced by lidocaine hydrochloride in C. raciborskii T3 was dependent on the concentration of Na+ ions in the culture medium and alkaline pH. The results suggest a possible action of lidocaine hydrochloride on membrane ion fluxes and the hypothesis of a potential linkage between cyanobacterial homeostasis and STX regulation.

This paper describes the purification and characterization of microviridin J, a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Following sample collection and screening at a number of Mn-associated mine sites in Northern Australia, a microbial strain was selected for its enhanced rate of Mn uptake. The strain was identified by phylogenetic analysis as a Rhizobium sp. Kinetic studies of Mn(II) uptake and oxidation by this strain in glucose-based media established that the uptake of Mn(II) was much greater than the conversion of Mn(II) to Mn oxide. Chemical analysis and scanning electron microscopy confirmed the production of significant amounts of polysaccharides by this strain. These polysaccharides may play a role both in enhancing Mn(II) accumulation and in minimizing Mn oxide production.

The freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in freshwaters worldwide. This species is a concern from a water quality perspective due to its known ability to produce a potent hepatotoxic alkaloid cylindrospermopsin, which has been implicated in outbreaks of human sickness and cattle mortality. C. raciborskii strains isolated from Brazil have also been found to produce the highly toxic paralytic shellfish poisons (PSPs). This article reports the toxicity of four strains of C. raciborskii taken from three reservoirs and one river in Portugal, as well as the occurrence of this species in other water bodies used for potable and recreational purposes. All four strains grown in pure culture in the laboratory were found to be toxic in the mouse bioassay at 8-24h after intraperitoneal administration of single doses ranging from 1337 to 1572mgkg-1 Histological examination indicated that liver damage was the primary lesion; in addition, there was inflammation in the intestine. HPLC/MS tests for the presence of cylindrospermopsin, microcystins, and PSP toxins were negative. The available evidence suggests that another toxin may be present. This constitutes the first report of toxic C. raciborskii in Europe and draws attention to the need for increased monitoring of this cyanobacterium in water bodies used for potable and recreational purposes. © 2003 Elsevier Science (USA). All rights reserved.

A major factor limiting application of bacterial sulfate reduction to removal of sulfate and heavy metals in wetland systems is the requirement to supply carbon and energy to drive the process. Primary production by aquatic plants and algae is a cheap option for driving sustainable bacterial sulfate reduction and most operational systems have relied on plants. The use of harvested, non-growing algal biomass to support bacterial sulfate reduction was investigated. Two genera of green algae, strains N9 and A3, were isolated from treatment cells from the Artificial Wetland Filter at the Ranger uranium mine (Northern Territory, Australia) which successfully removes UO22+, Mn2+ and nitrate, but little sulfate, from mine waters. These algae were identified as Carteria sp. and Scenedesmus sp. and were used as the sole carbon and energy source to enrich a sulfate-reducing mixed bacterial culture from the constructed wetland. Bacterial sulfate reduction supported solely by degradation of algal biomass was demonstrated at laboratory scale using both algae. In excess of 300 mg/L, sulfate was reduced in 17 days following an initial period of approximately 8 days during which sulfate levels did not decrease. The amount and rate of reduction was shown to be dependent on the concentration of algal biomass added. Carteria algae at low concentration showed reduction earlier; however, yields at higher concentration were affected by unknown inhibition. Scenedesmus strain N9 produced a maximum specific yield of 94.3 g of sulfate reduced per gram biomass added compared with 43.5 for Carteria strain A3. Sequence analysis of the 16S rRNA gene of members of the bacterial consortium indicated that the sulfate-reducing bacteria (SRB) showed highest homology (98.5%) with Desulfovibrio alcoholovorans. A second bacterium, which showed homologies of 91-92% with organisms of the Clostridial assemblage, was also present in the culture and represents a new species, or possibly a new genus. Crown Copyright © 2003 Published by Elsevier B.V. All rights reserved.

mlrA is the only microcystin-degrading gene detected in Sphingomonas sp. MJ-PV. The gene has an extremely rare nucleotide sequence and homologous genes have not yet been discovered in the DNA database. We discovered the existence of a gene homologous to mlrA in new microcystin-degrading bacteria, MD-1 and Y2. These strains possessed mlrA homologues, and the identities of the genes of MD-1 and Y2 with the corresponding MJ-PV exceeded 98% and 84%, respectively. On the other hand, the mlrA gene was not detected in laboratory strains of the closely related Sphingomonas spp. strains employing hemi-nested polymerase chain reaction detection using two primer sets. Although the microcystin-degrading bacteria were closely related strains, they did not cluster together as the same species. We can conclude that the mlrA gene is conserved in three different bacterial species, and it is unique to microcystin degraders but not to the genus Sphingomonas. © 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains: The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.

Rapid and sensitive methods for the detection and genetic characterization of cyanobacteria have been developed based on DNA amplification techniques. This article describes the molecular methods that have been used to characterize cyanobacteria and their use as tools to identify toxin-producing strains. Different species and strains were compared using restriction fragment length polymorphism (RFLP) of amplified fragments of the phycocyanin gene and the 16S-23S rRNA internal transcribed spacer.

The mcyABCDEFGHIJ gene cluster of Microcystis aeruginosa encodes the mixed polyketide synthase/nonribosomal peptide synthetase (microcystin synthetase) which is responsible for biosynthesis of the potent liver toxin microcystin. The sequence and orientation of the mcy genes have previously been reported, but no transcriptional analysis had been performed prior to this study. The mcyABCDEFGHIJ genes are transcribed as two polycistronic operons, mcyABC and mcyDEFGHIJ, from a central bidirectional promoter between mcyA and mcyD. Two transcription start sites were detected for both mcyA and mcyD when cells were exposed to light intensities of 68 and 16 µmol of photons m-2 S-1. The start sites, located 206 and 254 bp upstream of the translational start for mcyD under high and low light conditions, respectively, indicate long untranslated leader regions. Putative transcription start sites were also identified for mcyE, mcyF, mcyG, mcyH, mcyI, and mcyJ but not for mcyB and mcyC. A combination of reverse transcription-PCR and rapid amplification of cDNA ends was employed throughout this work, which may have been one of the first transcriptional analyses of a large nonribosomal polyketide gene cluster.

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the ß and a subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.

The potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO3-, NH4+, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH4+ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH4+. Cultures supplied with NO3- were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH4+ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO3- < NH4+). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for Cylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular non-ribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.

Nodularin is a hepatotoxin produced by the bloom-forming cyanobacterial species Nodularia spumigena. Putative peptide synthetase and polyketide synthase genes were detected in toxic strains of Nodularia by degenerate PCR. Using specific primer sets, peptide synthetase and polyketide synthase gene homologues were detected in nodularin-producing strains indicating a possible role of peptide synthetase and polyketide synthase enzyme complexes in the biosynthesis of nodularin. Strains of Nodularia isolated from around the world were also analyzed for the production of nodularin by the protein phosphatase 2A inhibition assay. The protein phosphatase inhibition assay and the molecular detection of peptide synthetase and polyketide synthase genes in Nodularia may be useful techniques for the assessment of nodularin-producing cyanobacteria in the environment. © 2001 Federation of European Microbiological Societies.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

Microcystis aeruginosa strain MRC is unique in its' possession of the mcyA-J gene cluster, which encodes microcystin synthetase, but its' inability to produce microcystins. M. aeruginosa strain MRD is genetically identical to MRC at numerous genomic loci examined, but produces a variety of microcystins, mainly with the amino acid tyrosine in the molecule. Zooplankton studies with Daphnia galeata and D. pulicaria, using the mutant (MRC) and its' wild type (MRD), showed for the first time that microcystins other than microcystin-LR can be responsible for the poisoning of Daphnia by Microcystis. Regardless of microcystin content, both Daphnia exhibited significantly reduced ingestion rates when fed with either strain of M. aeruginosa compared with the green alga Scenedesmus acutus. A disruption of the molting process in both Daphnia spp. was noted when these species were fed with MRC cells. Such symptoms on Daphnia have not been previously reported for cyanobacteria and may point to a bioactive compound, other than microcystin, which inhibits the hardening of protein-chitin complexes in Daphnia.

Nodularia, a member of the order Nostocales, is a bloom-forming filamentous cyanobacterium that possesses the ability to form toxic blooms. The toxin produced by Nodularia, nodularin, is a hepatotoxin, similar in structure to the heptapeptide toxin microcystin. Twenty-one strains of Nodularia, representing the species Nodularia spumigena, Nodularia harveyana and Nodularia sphaerocarpa, were analysed for toxin production by protein phosphatase inhibition assay and sequenced over the 16S rDNA region. Phylogenetic analysis of Nodularia 16S rDNA sequences found that Nodularia clustered into two main groups. An N. spumigena cluster was distinct from the benthic species N. harveyana and N. sphaerocarpa. There was no distinction between strains isolated from globally diverse locations. Nodularin-producing species were restricted to the single, evolutionally distinct cluster of N. spumigena. This observation has enabled the design of a specific 16S rRNA PCR for the rapid detection of nodularin-producing strains. Alignment of 16S rDNA sequences from toxic and non-toxic Nodularia with other members of the cyanobacteria allowed the design of both Nodularia generic and toxic N. spumigena-specific primers.

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYP020B were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. © 2001 John Wiley & Sons, Inc.

We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the ß- and a-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity. © 2001 by John Wiley & Sons, Inc.

The production of defined isogenic Helicobacter pylori pyrB mutants was undertaken to investigate the role of aspartate carbamoyltransferase (encoded by pyrB) in the survival of the bacterium. The complete structural gene for aspartate carbamoyltransferase from H. pylori strain RU1 was cloned into Escherichia coli by complementation of a pyrB auxotrophic mutant to facilitate the construction of a pyrB-disrupted copy in E. coli. The H. pylori pyrB gene had high similarity to other bacterial pyrB genes, and the phylogenetic clustering with different species was consistent with functional characteristics of the ACTase. The transcription initiation site for H. pylori pyrB-mRNA was mapped 25 bp upstream of the ATG start codon, and potential promoter regions were identified. In order to construct an isogenic pyrB H. pylori mutant by natural transformation and allelic exchange, the plasmid in sert containing pyrB was disrupted by insertional mutagenesis of a chloramphenicol transferase gene cassette. In multiple transformations of H. pylori cells, no chloramphenicol-resistant pyrB mutants were isolated. Successful mutagenesis of other H. pylori genes and PCR amplification of the recombined gene demonstrated that the ACTase-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the pyrB gene with the chloram-phenicol-pyrB-disrupted copy. These findings suggested that the ACTase enzyme is essential for the survival of H. pylori. (C) 2000 Academic Press.

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na+ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na+ current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis, Lyngbya, Pseudanabaena, Aphanizomenon, Nodularia, Anabaena, and Nostoc, based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gramnegative bacteria and the Archea.

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.

Microcystin, a hepatotoxin known to be the cause of animal and human deaths, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. The toxin is produced nonribosomally via a multifunctional enzyme complex, consisting of both peptide synthetase and polyketide synthase modules coded for by the mcy gene cluster. The recent identification of the mcy genes in the production of microcystin synthetase for the first time provides an avenue to study the regulation of microcystin production at a genetic level. In this study, M. aeruginosa PCC7806 was grown either under continuous light of various intensities or under low light with subsequent short-term exposure to different light intensities and qualities and various stress factors. RNase protection assays were employed to observe the level of mcyB and mcyD transcription under each condition. Both mcyB and mcyD transcript levels were increased under high light intensities and red light. Blue light and certain artificial stress factors (methylviologen and NaCI) led to reduced transcript amounts. There appeared to be two light thresholds, between dark and low light (16 µmol of photons m-2 s-1, and medium (31 µmol of photons m-2 s-1) and high light (68 µmol of photons m-2 s-1), at which a significant increase in transcription occurred. Our findings show that the effect of light on microcystin synthetase production is due to light quality and is initiated at certain threshold intensities, which are not necessarily reflected by observed intracellular toxin bioactivity.

Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems. Copyright (C) 2000 Federation of European Microbiological Societies.

A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 µg of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non- polyadenylated eukaryotic RNA.

Background: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the ß-amino polyketide moiety Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. Results: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (Mcyl), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. Conclusions: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a ß-amino-pentaketide precursor and the formation of ß- and ¿-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Numerous important chemotherapeutic agents are non-ribosomal peptides (NRPs). Synthesised as secondary metabolites, they include a range of antimicrobials and recent work examining the biosynthetic pathways of non-ribosomal peptides suggests that the paths may be manipulated at a molecular genetic level. This provides a fantastic opportunity for the rapid and cost-effective discovery of novel antibiotic agents.

Cylindrospermopsis raciborskii Woloszynska is a prominent constituent of a number of water bodies in northern Australia. In Solomon Dam, this species occurs as two distinct morphological forms, one with straight trichomes and one with coiled trichomes. Isolates of the two forms have been grown in pure culture and have been shown to maintain their respective characteristic form over successive generations. Both forms were similar with respect to cylindrospermopsin content expressed as a percentage of freeze-dried culture material, ranging from 0.14% to 0.20%, depending on the N source provided in the medium. A morphological comparison between natural populations of the two forms showed significant differences in vegetative, heterocyst, and akinete cell dimensions. These characteristics are the primary taxonomic criteria at the species level in this genus. In culture, the coiled form grew slightly faster than the straight form over the range of conditions investigated in this study. The coiled form was better suited to growth under low-light conditions. These clear and consistent morphological and physiological differences contrast with the 99.8% similarity between the two forms in their 16S rRNA gene nucleotide sequences. It is concluded that although taxonomic separation of the two forms at the species level might not be warranted, the two strains investigated are clearly distinct morphotypes, and it is recommended that they be so recorded in monitoring programs.

We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation-hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This 'enzyme-free cloning' procedure is highly efficient, and is not constrained by the need for the presence of suitable restriction enzyme sites within the plasmid vector. The avoidance of post-amplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps.

Cellulose-producing bacteria used for Nata de Coco production in the Philippines were isolated and characterized. Two distinct wild-type strains (ITDI 2.1 and PA 2.2) were selected from thirty-eight isolates obtained. The two strains, and the rest of the cellulose producers, were established to be members of the genus Acetobacter based on rapid phenotypic characterization. They were characterized and differentiated based on pellicle type and colony morphology, carbon-utilization pattern (Biolog® assay), amount of cellulose production and 16S rDNA sequence. The thick pellicle cellulose-producing strain (ITDI 2.1) has a stable and consistent production of a very thick pellicle in any sugar-based, acidic medium, with a dry weight up to twenty-six times that of the thin pellicle cellulose-producing strain (PA 2.2) after 7-8 days static batch culture. Species identification using 16S rDNA sequencing revealed that the two strains belong to two different species of Acetobacter, Acetobacter xylinus and A. hansenii and may be new subspecies under these species designation.

Background. Plasmids are extra-chromosomal DNA that may encode products that aid in virulence, pathogenesis, and the spread of antibiotic resistance among a wide spectrum of bacteria. Plasmids have been detected in Helicobacter pylori, H. felis, H. fennelliae, and H. cinaedi. However, no function has been attributed to the Helicobacter plasmids studied to date. Moreover, the characterization of plasmids in other Helicobacter species is an as yet unexplored area of research. Several laboratories have reported difficulties in the extraction and isolation of plasmid DNA from H. pylori and H. felis isolates due to the presence of large amounts of DNase, necessitating cumbersome and time-consuming purification steps. The development of a method for extracting plasmid DNA from Helicobacter species would be useful for future systematic studies of plasmids in this important group of microorganisms. Materials and Methods. Eight H. pylori isolates, including the Sydney Strain SS1, three H. felis isolates, and one isolate each of H. hepaticus, H. bilis, H. mustelae, and H. rodentium, were screened for plasmid DNA using a novel method that includes a potassium xanthogenate-sodium dodecyl sulfate-phenol (XSP) buffer. A specific PCR targeting a highly conserved plasmid replication protein gene, repA, was used to confirm the presence of plasmids in the H. pylori isolates examined. The PCR primers used were designed based on the sequence of the H. pylori plasmid pHPM180. To demonstrate the effec-tiveness of this method, plasmid DNA extracted from SS1 using XSP buffer was digested using three restriction enzymes (DdeI, SpeI and MaeIII). The relative amount of DNA obtained using the protocol was also compared to the yield derived from four commercial kits commonly used in many laboratories. Results. High and low molecular weight plasmids were extracted from H. pylori (n = 8) and H. felis (n = 3) isolates. The size range of these plasmids was from 3 kb to > 16 kb. Attempts to isolate plasmids from H. hepaticus ATCC 51488, H. bilis ATCC 51630, H. rodentium MIT-95-2060, and H. mustelae NCTC 11574 were not successful, which was most likely due to the absence of endogenous plasmids from the strains examined. The relative amount of DNA obtained using the XSP buffer protocol was comparable to that obtained from commercial kits as assessed by direct examination of plasmid profiles on agarose gels. Plasmid DNA extracted from H. pylori SS1 using XSP buffer was successfully digested with restriction enzymes. Conclusion. This study reports the development of an efficient, inexpensive, and rapid method for extracting high and low molecular weight plasmids from Helicobacter species. Application of this novel method for the isolation and future characterization of plasmids from different Helicobacter species could promote a better understanding of the role of plasmids in the basic microbial physiology and ecology of this group of microorganisms. © Blackwell Science, Inc.