Associate Professor Ian Grainge

Two classes of conservative site-specific recombinases, those belonging to the tyrosine and serine families, have been identified, and several of its members characterized in genetic and biochemical detail. These families are named after the active site amino acid, tyrosine or serine, that is utilized as the nucleophile during the strand breaking step of recombination. The Flp recombinase encoded by the 2 micron plasmid of Saccharomyces cerevisiae and related recombinases encoded by similar plasmids found in other yeast species belong to the tyrosine family. The Flp protein has provided several insights into the mechanism of target DNA recognition, strand cleavage and strand exchange during the recombination reaction. Here we describe how the Flp system has been used as a tool for tackling basic and applied problems in biology. © 2005 Elsevier B.V. All rights reserved.

Microplastics (MPs) exposed to the natural environment provide an ideal surface for biofilm formation, which potentially acts as a reactive phase facilitating the sorption of hazardous contaminants. Until now, changes in the contaminant sorption capacity of MPs due to biofilm formation have not been quantified. This is the first study that compared the capacity of naturally aged, biofilm-covered microplastic fibers (BMFs) to adsorb perfluorooctane sulfonate (PFOS) and lead (Pb) at environmentally relevant concentrations. Changes in the surface properties and morphology of aged microplastic fibers (MF) were studied by surface area analysis, infrared spectroscopy, and scanning electron microscopy. Results revealed that aged MFs exhibited higher surface areas because of biomass accumulation compared to virgin samples and followed the order polypropylene>polyethylene>nylon>polyester. The concentrations of adsorbed Pb and PFOS were 4-25% and 20-85% higher in aged MFs and varied among the polymer types. The increased contaminant adsorption was linked with the altered surface area and the hydrophobic/hydrophilic characteristics of the samples. Overall, the present study demonstrates that biofilms play a decisive role in contaminant-plastic interactions and significantly enhance the vector potential of MFs for toxic environmental contaminants. We anticipate that knowledge generated from this study will help refine the planetary risk assessment of MPs.

The long-term aging of plastic leads to weathering and biofouling that can influence the behavior and fate of plastic in the marine environment. This is the first study to fingerprint the contaminant profiles and bacterial communities present in plastic-associated inorganic and organic matter (PIOM) isolated from 10 year-aged plastic. Plastic sleeves were sampled from an oyster aquaculture farm and the PIOM was isolated from the intertidal, subtidal, and sediment-buried segments to investigate the levels of metal(loid)s, polyaromatic hydrocarbons (PAHs), per-fluoroalkyl substances (PFAS) and explore the microbial community composition. Results indicated that the PIOM present on long-term aged high-density polyethylene plastic harbored high concentrations of metal(loid)s, PAHs, and PFAS. Metagenomic analysis revealed that the bacterial composition in the PIOM differed by habitat type, which consisted of potentially pathogenic taxa including Vibrio, Shewanella, and Psychrobacter. This study provides new insights into PIOM as a potential sink for hazardous environmental contaminants and its role in enhancing the vector potential of plastic. Therefore, we recommend the inclusion of PIOM analysis in current biomonitoring regimes and that plastics be used with caution in aquaculture settings to safeguard valuable food resources, particularly in areas of point-source contamination.

Conditioning films (CFs) are surface coatings formed by the adsorption of biomolecules from the surrounding environment that can modify the material-specific surface properties and precedes the attachment of microorganisms. Hence, CFs are a biologically relevant identity that could govern the behavior and fate of microplastics in the aquatic environment. In the present study, polyethylene terephthalate (PET) and polylactic acid (PLA) plastic cards were immersed in natural seawater to allow the formation of CFs. The changes in the surface roughness after 24 h were investigated by atomic force microscopy (AFM), and the surface changes were visualized by scanning electron microscopy (SEM). The global elemental composition of the conditioned surface was investigated by energy dispersive spectroscopy (EDS). Results indicated that marine conditioning of PET and PLA samples for 24 h resulted in an increase of ~11 and 31% in the average surface roughness, respectively. SEM images revealed the attachment of coccoid-shaped bacterial cells on the conditioned surfaces, and the accumulation of salts of sodium and phosphate-containing precipitates was revealed through the EDS analysis. The results indicate that the increase in surface roughness due to conditioning is linked to a material¿s hydrophilicity leading to a rapid attachment of bacteria on the surfaces. Further investigations into the CFs can unfold crucial knowledge surrounding the plastic-microbe interaction that has implications for medical, industrial, and environmental research.

Metabolic uptake of lead (Pb) is controlled by its bioaccessibility. Most studies have examined bioaccessibility of Pb in the absence of gut microbes, which play an important role in the metabolic uptake of nutrients and metal(loid)s in intestine. In this study, we examined the effect of three gut microbes, from various locations in the gut, on the bioaccessibility of soil ingested Pb. The gut microbes include Lactobacillus acidophilus, Lactobacillus rhamnosus and Escherichia coli. Lead toxicity to these three microbes was also examined at various pH values. Bioaccessibility of Pb was measured using gastric and intestinal extractions. Both Pb spiked and Pb-contaminated shooting range field soils were used to measure Pb bioaccessibility in the presence and absence of gut microbes. The results indicated that Pb toxicity to gut microbes, as measured by LD50 value, decreased with increasing pH, and was higher for Lactobacillus species. Gut microbes decreased the bioaccessible Pb; the effect was more pronounced at low pH, mimicking gastric conditions than in conditions closer to the intestine. Lead adsorption by these microbes increased at the higher pH tested, and E. coli adsorbed higher amounts of Pb than did the Lactobacillus species. The effect of gut microbes on reducing Pb bioaccessibility may be attributed to microbially-induced immobilization of Pb through adsorption, precipitation, and complexation reactions. The study demonstrates that bioaccessibility and subsequently bioavailability of metal(loid)s can be modulated by gut microbes, and it is important to undertake bioaccessibility measurements in the presence of gut microbes.

In this study, the intestinal permeability of metal(loid)s (MLs) such as arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) was examined, as influenced by gut microbes and chelating agents using an in vitro gastrointestinal/Caco-2 cell intestinal epithelium model. The results showed that in the presence of gut microbes or chelating agents, there was a significant decrease in the permeability of MLs (As-7.5%, Cd-6.3%, Pb-7.9% and Hg-8.2%) as measured by apparent permeability coefficient value (Papp), with differences in ML retention and complexation amongst the chelants and the gut microbes. The decrease in ML permeability varied amongst the MLs. Chelating agents reduce intestinal absorption of MLs by forming complexes thereby making them less permeable. In the case of gut bacteria, the decrease in the intestinal permeability of MLs may be associated to a direct protection of the intestinal barrier against the MLs or indirect intestinal ML sequestration by the gut bacteria through adsorption on bacterial surface. Thus, both gut microbes and chelating agents can be used to decrease the intestinal permeability of MLs, thereby mitigating their toxicity.

Besides the ecotoxicological consequences of microplastics and associated chemicals, the association of microbes on plastics has greater environmental implications as microplastics may select for unique microbiome participating in environmentally significant functions. Despite this, the functional potential of the microbiome associated with different types of plastics is understudied. Here, we investigate the interaction between plastic and marine biofilm-forming microorganisms through a whole-genome sequencing approach on four types of microplastics incubated in the marine environment. Taxonomic analysis suggested that the microplastic surfaces exhibit unique microbial profiles and niche partitioning among the substrates. In particular, the abundance of Vibrio alginolyticus and Vibrio campbellii suggested that microplastic pollution may pose a potential risk to the marine food chain and negatively impact aquaculture industries. Microbial genera involved in xenobiotic compound degradation, carbon cycling, and genes associated with the type IV secretion system, conjugal transfer protein TraG, plant-pathogen interaction, CusA/CzcA family heavy metal efflux transfer proteins, and TolC family proteins were significantly enriched on all the substrates, indicating the variety of processes operated by the plastic-microbiome. The present study gives a detailed characterization of the rapidly altering microbial composition and gene pools on plastics and adds new knowledge surrounding the environmental ramifications of marine plastic pollution.

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell¿s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Biomotors are extensively involved in biological processes including cell mitosis, bacterial binary fission, DNA replication, DNA repair, homologous recombination, Holliday junction resolution, RNA transcription, and viral genome packaging. Traditionally, they were classified into two categories including linear and rotation motors. In 2013, a third class of motor by revolution mechanism without rotation was discovered. In this issue of " Structure and mechanisms of nanomotors in the cells" , four comprehensive reviews are published to address the latest advancements of the structure and motion mechanism of a variety of biomotors in archaea, animal viruses, bacteria, and bacteriophages.

Site-specific recombination is a reaction in which a pair of genetically defined sites undergoes reciprocal exchange ("crossing-over") via a recombinase-mediated DNA breakage and joining process. Such reactions have a wide range of biological outcomes, from integration and excision of virus genomes into and out of host chromosomes, to acquisition of novel genes and drug resistance, and even facilitating bacterial chromosome segregation. Two distinct families of recombinases exist, designated by their active site residues. In both these families recombination is carried out by a core of four recombinase monomers acting at two synapsed DNA sites. In many cases additional recombinase monomers and/or accessory proteins act at adjacent DNA sites to facilitate synapsis and often play a critical role in determining reaction topology. Here, the mechanism of site-specific recombination reactions is examined for both site-specific recombinase families, as well as for related proteins that mediate variant reactions, such as integrons and the integrases of conjugative transposons. © 2006 Springer-Verlag Berlin Heidelberg.