Associate Professor Karl Hassan

Biolog Phenotype MicroArrays for microorganisms provide a high-throughput method for the global analysis of microbial growth phenotypes. Using a colorimetric reaction that is indicative of respiration, these microplate assays measure the response of an individual strain or microbial community to a large and diverse range of nutrients and chemicals. Phenotype MicroArrays have been used to study gene function and to improve genome annotation in single microorganisms and for physiological profiling of bacterial communities. The microplate system can be used to obtain a comprehensive overview of metabolic capability, or it can be tailored, through the use of subsets of plates, to address specific research needs. © 2014 Springer Science+Business Media, LLC.

Quantitative PCR is used to gauge the abundance of specific nucleic acid species within purified samples. Due to its high sensitivity and minimal operation costs, this method is routinely applied in modern molecular bioscience laboratories. Nonetheless, all quantitative PCR experiments must include several carefully designed, yet simple, controls to ensure the reliability of the analyses. The aim of this chapter is to provide basic quantitative PCR methods, from primer design through data analysis, that are generally applicable to studies in microbiology. These methods allow the abundance of targeted RNA or DNA molecules to be determined in nucleic acid samples purified from a variety of biological sources. © 2014 Springer Science+Business Media, LLC.

Membrane transporters are a large group of proteins that span cell membranes and contribute to critical cell processes, including delivery of essential nutrients, ejection of waste products, and assisting the cell in sensing environmental conditions. Obtaining an accurate and specific annotation of the transporter proteins encoded by a micro-organism can provide details of its likely nutritional preferences and environmental niche(s), and identify novel transporters that could be utilized in small molecule production in industrial biotechnology. The Transporter Automated Annotation Pipeline (TransAAP) (http://www.membranetransport. org/transportDB2/TransAAP_login.html) is a fully automated web service for the prediction and annotation of membrane transport proteins in an organism from its genome sequence, by using comparisons with both curated databases such as the TCDB (Transporter Classification Database) and TDB, as well as selected Pfams and TIGRFAMs of transporter families and other methodologies. TransAAP was used to annotate transporter genes in the prokaryotic genomes in the National Center for Biotechnology Information (NCBI) RefSeq; these are presented in the transporter database TransportDB (http://www. membranetransport.org) website, which has a suite of data visualization and analysis tools. Creation and maintenance of a bioinformatic database specific for transporters in all genomic datasets is essential for microbiology research groups and the general research/biotechnology community to obtain a detailed picture of membrane transporter systems in various environments, as well as comprehensive information on specific membrane transport proteins.

Acinetobacter baumannii and Klebsiella pneumoniae are opportunistic pathogens frequently co-isolated from polymicrobial infections. The infections where these pathogens co-exist can be more severe and recalcitrant to therapy than infections caused by either species alone, however there is a lack of knowledge on their potential synergistic interactions. In this study we characterise the genomes of A. baumannii and K. pneumoniae strains co-isolated from a single human lung infection. We examine various aspects of their interactions through transcriptomic, phenomic and phenotypic assays that form a basis for understanding their effects on antimicrobial resistance and virulence during co-infection. Using co-culturing and analyses of secreted metabolites, we discover the ability of K. pneumoniae to cross-feed A. baumannii by-products of sugar fermentation. Minimum inhibitory concentration testing of mono- and co-cultures reveals the ability for A. baumannii to cross-protect K. pneumoniae against the cephalosporin, cefotaxime. Our study demonstrates distinct syntrophic interactions occur between A. baumannii and K. pneumoniae, helping to elucidate the basis for their co-existence in polymicrobial infections.

Aims: This study examined the origins and evolution of the AdeABC, AdeFGH and AdeIJK efflux pumps in the Acinetobacter genus, including human and animal pathogens and species from non-clinical environments. Methods: Comparative genome analyses were performed using the reference sequences for 70 Acinetobacter species to identify putative orthologs of AdeABC, AdeFGH and AdeIJK and their regulators. Sequence similarities and the genomic locations of coding sequences were correlated with phylogeny to infer modes of evolution. Intraspecies variation was assessed in species of interest using up to 236 complete genome sequences. Mutants overproducing adeIJK in A. baylyi were examined to identify regulators of this system in a non A. baumannii species. Results: The results indicate that adeIJK has been a stable part of Acinetobacter genomes since the genesis of this genus, whereas adeABC and adeFGH were carried by less than half of the species, but showed some lineage specificity. The organisation and local genetic contexts of adeABC loci were particularly variable to the sub-species level, suggesting frequent recombination. Cognate regulatory systems were almost always found in the genomes of species encoding pumps. Mutations in adeN, which encodes a repressor of adeIJK, were selected by antibiotic exposure in A. baylyi, similar to previous findings in pathogenic lineages. Conclusions: The multidrug efflux capacity of clinical Acinetobacter strains stems from accessory and core genetic features. AdeIJK is likely to have ancient core function(s) that have promoted its maintenance, whereas recent antibiotic use may be driving the evolution of the AdeABC pump.

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are b-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake.

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome- wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialog between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates of B. distachyon and demonstrated the presence of multiple carbohydrates, amino acids, organic acids, and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for the Pseudomonas strains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains' pangenome, reflecting the importance of the variable gene content in the adaptation of P. fluorescens to the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.

Since the late 1990's the genome sequences for thousands of species of bacteria have been released into public databases. The release of each new genome sequence typically revealed the presence of tens to hundreds of uncharacterised genes encoding putative membrane proteins and more recently, microbial metagenomics has revealed countless more of these uncharacterised genes. Given the importance of small molecule efflux in bacteria, it is likely that a significant proportion of these genes encode for novel efflux proteins, but the elucidation of these functions is challenging. We used transcriptomics to predict that the function of a gene encoding a hypothetical membrane protein is in efflux-mediated antimicrobial resistance. We subsequently confirmed this function and the likely native substrates of the pump by using detailed biochemical and biophysical analyses. Functional studies of homologs of the protein from other bacterial species determined that the protein is a prototype for a family of multidrug efflux pumps ¿ the Proteobacterial Antimicrobial Compound Efflux (PACE) family. The general functional genomics approach used here, and its expansion to functional metagenomics, will very likely reveal the identities of more efflux pumps and other transport proteins of scientific, clinical and commercial interest in the future.

Fluoroquinolones are one of the most prescribed broad-spectrum antibiotics. However, their effectiveness is being compromised by high rates of resistance in clinically important organisms, including Acinetobacter baumannii. We sought to investigate the transcriptomic and proteomic responses of the clinical A. baumannii strain AB5075-UW upon exposure to subinhibitory concentrations of ciprofloxacin. Our transcriptomics and proteomics analyses found that the most highly expressed genes and proteins were components of the intact prophage phiOXA. The next most highly expressed gene (and its protein product) under ciprofloxacin stress was a hypothetical gene, ABUW_0098, named here the Acinetobacter ciprofloxacin tolerance (aciT) gene. Disruption of this gene resulted in higher susceptibility to ciprofloxacin, and complementation of the mutant with a cloned aciT gene restored ciprofloxacin tolerance to parental strain levels. Microscopy studies revealed that aciT is essential for filamentation during ciprofloxacin stress in A. baumannii. Sequence analysis of aciT indicates the encoded protein is likely to be localized to the cell membrane. Orthologs of aciT are found widely in the genomes of species from the Moraxellaceae family and are well conserved in Acinetobacter species, suggesting an important role. With these findings taken together, this study has identified a new gene conferring tolerance to ciprofloxacin, likely by enabling filamentation in response to the antibiotic.

Acinetobacter baumannii is one of the world's most problematic nosoco-mial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii maybeactivelyacquiredbyA. bau-mannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. Onabroaderscope, this study presents new findings on the potential health benefits of PUFA in indi-viduals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis.

Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.

The bacterial pathogen Acinetobacter baumannii has emerged as an urgent threat to health care systems. The prevalence of multidrug resistance in this critical human pathogen is closely associated with difficulties in its eradication from the hospital environment and its recalcitrance to treatment during infection. The development of resistance in A. baumannii is in part due to substantial plasticity of its genome, facilitating spontaneous genomic evolution. Many studies have investigated selective pressures imposed by antibiotics on genomic evolution, but the influence of high-abundance bioactive molecules at the host-pathogen interface on mutation and rates of evolution is poorly understood. Here, we studied the roles of host fatty acids in the gain in resistance to common antibiotics. We defined the impact of the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid on the development of resistance to erythromycin in A. baumannii strain AB5075_UW using a microevolutionary approach. We employed whole-genome sequencing and various phenotypic analyses to characterize microbelipid- antibiotic interactions. Cells exposed to erythromycin in the presence of the fatty acids displayed significantly lower rates of development of resistance to erythromycin and, importantly, tetracycline. Subsequent analyses defined diverse means by which host fatty acids influence the mutation rates. This work has highlighted the critical need to consider the roles of host fatty acids in A. baumannii physiology and antimicrobial resistance. Collectively, we have identified a novel means to curb the development of resistance in this critical human pathogen.

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.

Antimicrobial resistance genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Multidrug efflux pumps often transport metabolites, signals and host-derived molecules in addition to antibiotics or biocides. Understanding their ancestral physiological roles could inform the development of strategies to subvert their activity. In this study, we investigated the response of Acinetobacter baumannii to polyamines, a widespread, abundant class of amino acid-derived metabolites, which led us to identify long-chain polyamines as natural substrates of the disinfectant efflux pump AmvA. Loss of amvA dramatically reduced tolerance to long-chain polyamines, and these molecules induce expression of amvA through binding to its cognate regulator AmvR. A second clinically-important efflux pump, AdeABC, also contributed to polyamine tolerance. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.

Tigecycline, a protein translation inhibitor, is a treatment of last resort for infections caused by the opportunistic multidrug resistance human pathogen Acinetobacter baumannii. However, strains resistant to tigecycline were reported not long after its clinical introduction. Translation inhibitor antibiotics perturb ribosome function and induce the reduction of (p)ppGpp, an alarmone involved in the stringent response that negatively modulates ribosome production. Through RNA sequencing, this study revealed a significant reduction in the transcription of genes in citric acid cycle and cell respiration, suggesting tigecycline inhibits or slows down bacterial growth. Our results indicated that the drug-induced reduction of (p)ppGpp level promoted the production but diminished the degradation of ribosomes, which mitigates the translational inhibition effect by tigecycline. The reduction of (p)ppGpp also led to a decrease of transcription coupled nucleotide excision repair which likely increases the chances of development of tigecycline resistant mutants. Increased expression of genes linked to horizontal gene transfer were also observed. The most upregulated gene, rtcB, involving in RNA repair, is either a direct tigecycline stress response or is in response to the transcription de-repression of a toxin-antitoxin system. The most down-regulated genes encode two ß-lactamases, which is a possible by-product of tigecycline-induced reduction in transcription of genes associated with peptidoglycan biogenesis. This transcriptomics study provides a global genetic view of why A. baumannii is able to rapidly develop tigecycline resistance.

Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, FAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.

Clostridium strains from six phylogenetic groups, C. botulinum groups I to IV, C. baratii, and C. butyricum, display the capacity to produce botulinum neurotoxin. Here, we present the genome sequence of a C. butyricum isolate, the neurotoxigenic strain 5521, which encodes the type E botulinum neurotoxin.

The neurotoxins produced by Clostridium botulinum strains are among the world's most potent toxins and are the causative agents of paralytic botulism. Here, we present the draft genome sequence of the group III C. botulinum strain Eklund-C, including a pseudolysogen-like bacteriophage that harbors the type C neurotoxin operon.

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis. © 2009 Tsolis et al.

Background. The staphylococcal QacA multidrug efflux protein confers resistance to an exceptional number of structurally unrelated antimicrobial compounds. Aromatic amino acid residues have been shown to be highly important for the transport function of several multidrug transporters and are intimately involved in multidrug binding. This study investigated the structural and functional importance of the seven tyrosine residues in QacA by examining the phenotypic effect of incorporating conservative (aromatic) and non-conservative (non-aromatic) substitutions for these residues. Results. Determination of the resistance profiles and analysis of drug transport assays revealed that non-conservative substitutions for most tyrosine residues influenced the QacA drug recognition spectrum. However, an aromatic residue at three tyrosine positions, 63, 410 and 429, was of importance for QacA-mediated transport and resistance to the majority of substrates tested. Conclusion. A tyrosine or phenylalanine residue at amino acid positions corresponding to 63 of QacA in related drug efflux proteins is found to be highly conserved. Therefore, an aromatic side chain at this position is likely to partake in a function common to these drug transporters, such as proton translocation or essential intramolecular contacts, whereas aromatic residues at the non-conserved 410 and 429 positions are expected to mediate a QacA-specific function, possibly forming or stabilising part of the QacA drug binding region. © 2008 Wu et al; licensee BioMed Central Ltd.

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism. © 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.