ORCID Profile
0000-0001-6461-7550
Current Organisation
Flinders University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Plant Biology not elsewhere classified | Water treatment processes | Maritime engineering | Structural Chemistry | Ecological Applications | Microbiology | Characterisation Of Macromolecules | Physical Chemistry (Incl. Structural) | Agricultural Biotechnology not elsewhere classified | Natural Resource Management | Biochemistry and Cell Biology not elsewhere classified | Microbiology not elsewhere classified | Ship and platform structures (incl. maritime hydrodynamics) | Animal Physiology - Cell | Gene Expression (incl. Microarray and other genome-wide approaches) | Comparative Physiology | Ecological Impacts of Climate Change | Physical Chemistry not elsewhere classified | Analytical Biochemistry | Animal Systematics, Taxonomy And Phylogeny | Microbial Ecology | Biological control | Analytical Biochemistry | Biological Adaptation
ANZSRC Socio-Economic Objective (SEO)
Biological sciences | Expanding Knowledge in the Biological Sciences | Ecosystem Adaptation to Climate Change | Coastal and Estuarine Flora, Fauna and Biodiversity | Land and water management | Climate Variability (excl. Social Impacts) | Wine Grapes | Land and water management | Chemical sciences | Expanding Knowledge in the Medical and Health Sciences | Physical and Chemical Conditions of Water in Coastal and Estuarine Environments | Expanding Knowledge in the Agricultural and Veterinary Sciences | Treatments (e.g. chemicals, antibiotics) |
Related Links
Publications
Export of a single drug molecule in two transport cycles by a multidrug efflux pump
Publisher: Springer Science and Business Media LLC
Date: 08-08-2014
DOI: 10.1038/NCOMMS5615
Abstract: Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of alent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of alent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.
Diversity and function of capsular polysaccharide in Acinetobacter baumannii
Publisher: Frontiers Media SA
Date: 09-01-2019
Molecular screening and characterization of Legionella pneumophila associated free-living amoebae in domestic and hospital water systems
Publisher: Elsevier BV
Date: 11-2022
DOI: 10.1016/J.WATRES.2022.119238
Abstract: Free-living amoebae are ubiquitous in the environment and cause both opportunistic and non-opportunistic infections in humans. Some genera of amoebae are natural reservoirs of opportunistic plumbing pathogens, such as Legionella pneumophila. In this study, the presence of free-living amoebae and Legionella was investigated in 140 water and biofilm s les collected from Australian domestic (n = 68) and hospital water systems (n = 72). Each s le was screened in parallel using molecular and culture-based methods. Direct quantitative polymerase chain reaction (qPCR) assays showed that 41% s les were positive for Legionella, 33% for L. pneumophila, 11% for Acanthamoeba, and 55% for Vermamoeba vermiformis gene markers. Only 7% of s les contained culturable L. pneumophila serogroup (sg)1, L. pneumophila sg2-14, and non-pneumophila Legionella. In total, 69% of s les were positive for free-living amoebae using any method. Standard culturing found that 41% of the s les were positive for amoeba (either Acanthamoeba, Allovahlk fia, Stenamoeba, or V. vermiformis). V. vermiformis showed the highest overall frequency of occurrence. Acanthamoeba and V. vermiformis isolates demonstrated high thermotolerance and osmotolerance and strong broad spectrum bacteriogenic activity against Gram-negative and Gram-positive bacteria. Importantly, all Legionella positive s les were also positive for amoeba, and this co-occurrence was statistically significant (p < 0.05). According to qPCR and fluorescence in situ hybridization, V. vermiformis and Allovahlk fia harboured intracellular L. pneumophila. To our knowledge, this is the first time Allovahlk fia and Stenamoeba have been demonstrated as hosts of L. pneumophila in potable water. These results demonstrate the importance of amoebae in engineered water systems, both as a pathogen and as a reservoir of Legionella. The high frequency of gymnamoebae detected in this study from Australian engineered water systems identifies an issue of significant public health concern. Future water management protocols should incorporate treatments strategies to control amoebae to reduce the risk to end users.
Roles of DHA2 family transporters in drug resistance and iron homeostasis in Acinetobacter spp.
Publisher: S. Karger AG
Date: 2011
DOI: 10.1159/000325367
Abstract: i Background: /i i Acinetobacter /i i baumannii /i is a major cause of nosocomial infections worldwide due to its fitness within clinical settings and recalcitrance to conventional therapies. The drug: H sup + /sup antiporter 2 (DHA2) family export systems encoded by i A. baumannii /i were investigated for their roles in promoting the success ofthis organism as a human pathogen. i Methods: /i Bioinformatic tools were used to identify the DHA2 family transporters encoded by i Acinetobacter /i spp. and establish their phylogenetic relationships. The drug resistance phenotypes conferred by the transporters were tested using both heterologously expressed proteins in i Escherichia coli /i and i Acinetobacter /i deletion mutants. The transcriptional responses of DHA2 family transporter genes to their substrates were established by qRT-PCR. i Results: /i Six highly conserved DHA2 family proteins were identified in i A. baumannii /i . Drug resistance phenotypes were established for two DHA2 family transporters. The expression of a third DHA2 family protein is highly responsive to the availability of iron. The gene encoding this protein is located within a putative siderophore biosynthesis locus, suggesting a physiological role in iron uptake, possibly via the export of a siderophore. i Conclusions: /i These results highlight functions for DHA2 family proteins in both drug resistance and the maintenance of stable cellular physiology, emphasizing their importance in i A. baumannii /i infections.
Evaluation of transparent exopolymer particles and microbial communities found post-UV light, multimedia and cartridge filtration pre-treatment in a SWRO plant
Publisher: Informa UK Limited
Date: 26-08-2015
Methicillin resistance gene diversity in staphylococci isolated from captive and free-ranging wallabies
Publisher: Informa UK Limited
Date: 2016
DOI: 10.3402/IEE.V6.31507
Abstract: Infection with methicillin-resistant staphylococci (MRS) can be life-threatening in humans and its presence in animals is a cause for public health concern. The aim of this study was to measure the prevalence of MRS in captive and free-ranging wallabies over a 16-month period in South Australia, Australia. Eighty-nine purified staphylococcal isolates recovered from 98 captive and free-ranging wallabies' anterior nasal swabs were used in this study. All isolates were tested for the presence of the mecA, mecA1, and mecC genes. Multiplex PCR-directed SCCmec-typing, ccrB-typing, and determination of the minimal inhibitory concentration of oxacillin were performed on mec-positive isolates. In total, 11 non-Staphylococcus aureus MRS were isolated from 7 out of 98 animals, corresponding to a 7.1% carriage rate. The SCCmec types I, III, and V were identified by multiplex PCR and sequencing of the ccrB gene. This is the first report of MRS carriage in both captive and free-ranging wallabies in Australia. These data demonstrate a low prevalence of MRS and no association between wallaby captivity status and MRS carriage could be assigned. These animals may act as a reservoir for the exchange of genetic elements between staphylococci. Furthermore, the mecA genes of animal isolates were identical to that found in human MRS strains and thus the possibility of zoonotic transfer must be considered.
Structural mechanisms of QacR induction and multidrug recognition
Publisher: American Association for the Advancement of Science (AAAS)
Date: 07-12-2001
Abstract: The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally erse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.
Transmembrane helix 12 of the Staphylococcus aureus multidrug transporter QacA lines the bivalent cationic drug binding pocket
Publisher: American Society for Microbiology
Date: 15-12-2007
DOI: 10.1128/JB.01492-07
Abstract: An acidic residue in transmembrane segment (TMS) 10 is important for recognition of bivalent cationic substrates by the QacA multidrug transporter. Remarkably, an acidic residue in TMS 12 compensated for the absence of such a residue in TMS 10, suggesting that TMS 12 is a component of the bivalent cation-binding region.
Biochemical characterization of the multidrug regulator QacR distinguishes residues that are crucial to multidrug binding and induction of qacA transcription
Publisher: American Chemical Society (ACS)
Date: 25-09-2009
DOI: 10.1021/BI901102H
Abstract: Staphylococcus aureus transcription factor QacR regulates expression of the qacA multidrug efflux determinant. In response to binding cationic lipophilic compounds, including ethidium and rhodamine 6G, QacR dissociates from the qacA operator alleviating repression. Such ligand binding uniformly induces a coil-to-helix transition of residues Thr(89)-Tyr(93) revealing an asymmetric binding pocket in QacR containing two distinct subpockets. Here, the functional significance of hydrophobic, aromatic, and polar residues characteristic of the rhodamine 6G pocket and the proximal Tyr(92), proposed to facilitate the transcriptionally active conformation, was examined. Notably, the presence of Tyr(92) was not essential for QacR structural changes between DNA-bound and induced conformations. Furthermore, although mutation of the majority of residues contacting rhodamine 6G exerted moderate effects on QacR-rhodamine 6G binding, mutation of Leu(54) and Gln(96), and cumulative mutations involving these with Tyr(93) and Tyr(123), imparted a dramatic decrease in QacR-rhodamine 6G binding affinity. This equated with impaired dissociation of QacR from its operator DNA in the presence of this ligand in S. aureus, delineating the important role of these residues in the QacR-rhodamine 6G interaction. Additionally, despite maintaining a high affinity for ethidium, QacR mutants involving Leu(54), Tyr(93), Gln(96), and Tyr(123), which denote the interface between the rhodamine 6G and ethidium subpockets, were unable to be induced from operator DNA in the presence of ethidium in S. aureus. This highlights the significant contribution of these residues to QacR-mediated derepression of qacA transcription following ligand binding in the distal subpocket and may be important for the general mechanism irrespective of the ligand bound.
Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii
Publisher: Springer Science and Business Media LLC
Date: 25-11-2014
Membrane topology of the metal-tetracycline/H+antiporter TetA(K) from Staphylococcus aureus
Publisher: American Society for Microbiology
Date: 06-1997
DOI: 10.1128/JB.179.11.3786-3789.1997
Abstract: A series of fusions to the reporter proteins alkaline phosphatase and beta-galactosidase have been constructed in the predicted periplasmic and cytoplasmic loops of TetA(K), a protein responsible for efflux-mediated tetracycline resistance in Staphylococcus aureus. The results support a topological model of 14 transmembrane segments for TetA(K).
Regulation of bacterial drug export systems
Publisher: American Society for Microbiology
Date: 12-2002
DOI: 10.1128/MMBR.66.4.671-701.2002
Abstract: The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.
Survival of the fittest
Publisher: Elsevier BV
Date: 10-2021
Continuous flow vortex fluidic synthesis of silica xerogel as a delivery vehicle for curcumin
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4RA15109G
Abstract: Sol–gel synthesis of silica xerogel using a continuous flow vortex fluidic device at room temperature is effective in direct incorporation of preformed curcumin particles, which has antimicrobial activity against Staphylococcus aureus .
Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1
Publisher: MDPI AG
Date: 02-08-2022
Abstract: Plant-derived pharmacological agents have been used extensively to dissect the structure–function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch cl ing of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.
Confined aquifers as viral reservoirs
Publisher: Wiley
Date: 13-06-2013
Abstract: Knowledge about viral ersity and abundance in deep groundwater reserves is limited. We found that the viral community inhabiting a deep confined aquifer in South Australia was more similar to reclaimed water communities than to the viral communities in the overlying unconfined aquifer community. This similarity was driven by high relative occurrence of the single-stranded DNA viral groups Circoviridae, Geminiviridae and Microviridae, which include many known plant and animal pathogens. These groups were present in a 1500-year-old water situated 80 m below the surface, which suggests the potential for long-term survival and spread of potentially pathogenic viruses in deep, confined groundwater. Obtaining a broader understanding of potentially pathogenic viral communities within aquifers is particularly important given the ability of viruses to spread within groundwater ecosystems.
Cationic Bactericidal Peptides
Publisher: Elsevier
Date: 1995
DOI: 10.1016/S0065-2911(08)60145-9
Abstract: The Healthy Eating Index-2015 (HEI-2015) was designed to reflect adherence to the 2015-2020 Dietary Guidelines for Americans (DGA). The study aims to examine the association between HEI-2015 and grip strength in a nationally representative s le of the U.S. adult population. This cross-sectional study used data from the National Health and Nutrition Examination Surveys of 2011-2014. Low grip strength was defined as <35.5 kg for men and <20 kg for women. HEI-2015 was computed from two days of 24-h dietary recalls and comprised 13 components. Each component was scored on the density out of 1000 calories and summed to a total score ided into quartiles. Weighted logistic regressions examined the study aim while controlling for associated covariates. The s le included 9006 eligible participants, of those, 14.4% (aged 20+ years), and 24.8% (aged ≥50 years) had low grip strength. Mean (±SD) HEI-2015 total score was 54.2 ± 13.6 and in the lowest and highest quartiles 37.3 ± 5.1 and 72.0 ± 6.5, respectively. In the multivariable model, participants in the highest vs. lowest HEI-2015 quartile had 24% lower odds of having low grip strength (Odds Ratio (OR) = 0.76 95% CI: 0.60-0.96). Specifically, participants who met the DGA for protein intakes, whole grains, greens and beans, vegetables, or whole fruits had 20-35% lower odds of having low grip strength than those who did not. Higher compliance to the DGA might reduce the risk for low grip strength as a proxy measure for sarcopenia among U.S. adults, particularly adequate intakes of proteins, whole grains, greens and beans, vegetables, and whole fruits.
Nasal colonisation of Staphylococcus spp among captive and free-ranging wallabies in South Australia
Publisher: OMICS Publishing Group
Date: 2014
The role played by drug efflux pumps in bacterial multidrug resistance
Publisher: Portland Press Ltd.
Date: 28-02-2017
DOI: 10.1042/EBC20160064
Abstract: Antimicrobial resistance is a current major challenge in chemotherapy and infection control. The ability of bacterial and eukaryotic cells to recognize and pump toxic compounds from within the cell to the environment before they reach their targets is one of the important mechanisms contributing to this phenomenon. Drug efflux pumps are membrane transport proteins that require energy to export substrates and can be selective for a specific drug or poly-specific that can export multiple structurally erse drug compounds. These proteins can be classified into seven groups based on protein sequence homology, energy source and overall structure. Extensive studies on efflux proteins have resulted in a wealth of knowledge that has made possible in-depth understanding of the structures and mechanisms of action, substrate profiles, regulation and possible inhibition of many clinically important efflux pumps. This review focuses on describing known families of drug efflux pumps using ex les that are well characterized structurally and/or biochemically.
Antimicrobial Activities of Marine Sponge-Associated Bacteria
Publisher: MDPI AG
Date: 14-01-2021
DOI: 10.3390/MICROORGANISMS9010171
Abstract: The misuse and overuse of antibiotics have led to the emergence of multidrug-resistant microorganisms, which decreases the chance of treating those infected with existing antibiotics. This resistance calls for the search of new antimicrobials from prolific producers of novel natural products including marine sponges. Many of the novel active compounds reported from sponges have originated from their microbial symbionts. Therefore, this study aims to screen for bioactive metabolites from bacteria isolated from sponges. Twelve sponge s les were collected from South Australian marine environments and grown on seven isolation media under four incubation conditions a total of 1234 bacterial isolates were obtained. Of these, 169 bacteria were tested in media optimized for production of antimicrobial metabolites and screened against eleven human pathogens. Seventy bacteria were found to be active against at least one test bacterial or fungal pathogen, while 37% of the tested bacteria showed activity against Staphylococcus aureus including methicillin-resistant strains and antifungal activity was produced by 21% the isolates. A potential novel active compound was purified possessing inhibitory activity against S. aureus. Using 16S rRNA, the strain was identified as Streptomyces sp. Our study highlights that the marine sponges of South Australia are a rich source of abundant and erse bacteria producing metabolites with antimicrobial activities against human pathogenic bacteria and fungi.
Antimicrobial peptides - promising alternatives to conventional antibiotics
Publisher: S. Karger AG
Date: 2011
DOI: 10.1159/000331009
Abstract: Antimicrobial peptides (APs) have been described as evolutionary ancient weapons. Produced by a wide variety of organisms as part of a non-specific immune response, these peptides are involved in the direct destruction of various microorganisms. Several APs have been shown to have broad activity spectra against microorganisms such as Gram-positive and Gram-negative bacteria, enveloped viruses, fungi and parasites. Given that resistance to a number of antibiotics has developed in a wide range of microbes, the potential of APs as novel therapeutic agents is being evaluated. However, optimisation of APs designed for therapy will need to focus on such factors as their susceptibility to proteolytic degradation and reduction of toxicity to mammalian cells. Strict guidelines pertaining to their use should also be established to prevent or hinder future development of bacterial resistance to such peptides.
Metagenomic comparison of microbial communities inhabiting confined and unconfined aquifer ecosystems
Publisher: Wiley
Date: 18-10-2012
DOI: 10.1111/J.1462-2920.2011.02614.X
Abstract: A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (β-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.
Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification
Publisher: American Society for Microbiology
Date: 10-1994
Abstract: Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.
A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/0378-1119(95)00589-0
Abstract: The first four genes (rfbA,B,D,E) of the rfb region of Vibrio cholerae O1 are predicted to encode the enzymes required for the biosynthesis of perosamine, which constitutes the backbone structure of the O-antigen of the lipopolysaccharide. Based on homology to known proteins rotein families, the following functions are predicted: RfbA, phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase RfbB, phosphomanno-mutase RfbD, oxido reductase and RfbE, perosamine synthetase (amino-transferase). Thus, perosamine is synthesized from fructose 6-phosphate via the intermediates mannose 6-phosphate by RfbA, to mannose 1-phosphate by RfbB, to GDP-mannose by RfbA, to GDP-4-keto-6-dideoxymannose by RfbD and to GDP-perosamine by RfbE. This final product would then serve as the substrate for the addition of the tetronate, which could then be polymerized into the O-antigen for transfer to the lipid A plus core oligosaccharide and export to the cell surface. The organization of these genes are such that one would expect them to be translationally coupled as part of the rfb operon. However, the absence of readily detectable promoter sequences suggests low levels of transcription, in line with other studies. The nucleotide sequence of these genes is absolutely conserved in the two isolates 569B (classical, Inaba) and O17 (El Tor, Ogawa) which were expected to show maximal sequence variation. This suggests very tight constraints on the micro-evolution within these sequences.
Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel
Publisher: American Chemical Society (ACS)
Date: 12-2006
DOI: 10.1021/BI0614380
Abstract: The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were in idually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be hiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.
The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate)
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.GENE.2015.03.017
Abstract: Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize erse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.
The role of TMS 12 in the staphylococcal multidrug efflux protein QacA
Publisher: Oxford University Press (OUP)
Date: 26-04-2023
DOI: 10.1093/JAC/DKAD121
Abstract: To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were in idually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.
Cycliplex PCR confirmation of Fusobacterium necrophorum isolates from captive wallabies: A rapid and accurate approach
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.ANAEROBE.2012.12.003
Abstract: Isolation and identification of obligate anaerobic bacteria is labour intensive and time consuming. This has led to the increased application of molecular tools to circumvent part of this problem. We report here the development of a rapid, accurate and cost-effective method to isolate and identify Fusobacterium necrophorum species from South Australian wallaby populations using a supplemented medium (BHIRS) in conjunction with a "Cycliplex PCR" method which involves a stepwise-selective lification of target PCR products. This report demonstrates the complementation of phenotypic characterization by PCR for accurate and fast identification of F. necrophorum isolates from wildlife origin.
Annual phytoplankton dynamics in the Gulf Saint Vincent, South Australia in 2011
Publisher: Elsevier BV
Date: 2014
DOI: 10.5697/OC.56-4.757
Does anaerobic bacterial antibiosis decrease fungal diversity in oral necrobacillosis disease?
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.RVSC.2013.09.008
Abstract: Oral necrobacillosis (ON) is a model polymicrobial disease that affects macropods in captivity and livestock. Several studies in humans and animals have focused mainly on the bacterial etiology of this disease with little or no information on the role/association of fungi with ON. Using a Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay and statistical analysis of the fungal community structure in healthy and disease groups, a reduction in the species ersity and drastic reduction (>1000 fold) in the fungal population in wallabies with ON was observed. Furthermore, an in vitro assay revealed a potential anaerobic-bacteria antibiosis mechanism in the observed decrease in fungal population in ON and a synergistic bacterial-fungal interaction in wallabies with healthy oral status. This study contributes to our knowledge of the fungal community structure associated with ON and forms the basis for an investigation at an epidemiological scale in order to exploit the clinical potentials of these findings.
Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria
Publisher: Elsevier BV
Date: 11-1993
DOI: 10.1016/0378-1119(93)90168-3
Abstract: Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. To permit further studies on these peptides, bacterial expression systems were developed. Attempts to produce these peptides with an N-terminal signal sequence were unsuccessful due to the lability of the basic peptides. Therefore, a number of different fusion protein systems were tested, including fusions to glutathione-S-transferase (GST) (on plasmid pGEX-KP), Pseudomonas aeruginosa outer membrane protein OprF (on plasmid pRW5), Staphylococcus aureus protein A (on plasmid pRIT5), and the duplicated IgG-binding domains of protein A (on plasmid pEZZ18). In the first three cases, stable fusion proteins with the defensin, human neutrophil peptide 1 (HNP-1), and/or a synthetic cecropin/melittin hybrid (CEME) were obtained. In the course of these studies, we developed a novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establishing methods for preventing fusion protein proteolytic breakdown. Cationic peptides could be successfully released from the carrier protein with high efficiency by chemical means (CNBr cleavage) and with low efficiency by enzymatic cleavage (using factor Xa protease). Fusions of protein A to cationic peptides were secreted into the culture supernatant of S. aureus clones and after affinity purification, CNBr digestion and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid (aa) content, aa sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures.
Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139
Publisher: Proceedings of the National Academy of Sciences
Date: 24-10-1995
Abstract: The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.
Factors affecting the isolation and diversity of marine sponge-associated bacteria
Publisher: Springer Science and Business Media LLC
Date: 02-2022
DOI: 10.1007/S00253-022-11791-8
Abstract: Marine sponges are an ideal source for isolating as yet undiscovered microorganisms with some sponges having about 50% of their biomass composed of microbial symbionts. This study used a variety of approaches to investigate the culturable ersity of the sponge-associated bacterial community from s les collected from the South Australian marine environment. Twelve sponge s les were selected from two sites and their bacterial population cultivated using seven different agar media at two temperatures and three oxygen levels over 3 months. These isolates were identified using microscopic, macroscopic, and 16S rRNA gene analysis. A total of 1234 bacterial colonies were isolated which consisted of four phyla: Actinobacteria , Firmicutes , Proteobacteria , and Bacteroidetes , containing 21 genera . The ersity of the bacterial population was demonstrated to be influenced by the type of isolation medium, length of the incubation period and temperature, sponge type, and oxygen level. The findings of this study showed that marine sponges of South Australia can yield considerable bacterial culturable ersity if a comprehensive isolation strategy is implemented. Two sponges, with the highest and the lowest ersity of culturable isolates, were examined using next-generation sequencing to better profile the bacterial population. A marked difference in terms of phyla and genera was observed using culture-based and culture-independent approaches. This observed variation displays the importance of utilizing both methods to reflect a more complete picture of the microbial population of marine sponges. Improved bacterial ersity due to long incubations, 2 temperatures, and 3 oxygen levels. Isolates identified by morphology, restriction digests, and 16S rRNA gene sequencing. At least 70% of culturable genera were not revealed by NGS methods.
Optimized production and analysis of the staphylococcal multidrug efflux protein QacA
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.PEP.2008.11.009
Abstract: The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression urification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.
Legionella persistence in manufactured water systems: pasteurization potentially selecting for thermal tolerance
Publisher: Frontiers Media SA
Date: 19-07-2017
A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae
Publisher: American Society for Microbiology
Date: 31-08-2021
Abstract: The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance.
Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA
Publisher: Elsevier BV
Date: 2006
Active export proteins mediating drug resistance in staphylococci
Publisher: S. Karger AG
Date: 2007
DOI: 10.1159/000099640
Abstract: Drug resistance mediated by integral membrane transporters is an important mode of cellular resistance to cytotoxic agents across all classes of living organisms. Gram-positive bacteria, such as staphylococcal species, are not encapsulated by a selective outer membrane permeability barrier. Therefore, these organisms often employ integral membrane drug transport systems to maintain cellular concentrations of antimicrobials at subtoxic levels. Staphylococcal species, including the opportunistic human pathogen i Staphylococcus aureus /i , encode a multitude of drug exporters, encompassing transporters from each of the five currently recognized families of bacterial drug resistance transporters. A number of these transporters are chromosomally encoded and allow the host cell to realize clinically significant levels of drug resistance after minor mutations to regulatory regions. Others are plasmid-encoded and can be easily passed between staphylococcal strains and species, or acquired from other Gram-positive genera. In combination, staphylococcal drug transporters potentiate resistance to a vast array of antimicrobial compounds, including macrolide, quinolone, tetracycline and streptogramin antibiotics, as well as a broad range of biocides, such as quaternary ammonium compounds, biguanidines and diamidines. An understanding of the genetic and molecular properties of drug transporters will lead to effective treatments of staphylococcal infections. Here we provide a detailed review of the active drug transporters of the staphylococci.
The StkSR Two-Component System Influences Colistin Resistance in Acinetobacter baumannii
Publisher: MDPI AG
Date: 08-05-2022
DOI: 10.3390/MICROORGANISMS10050985
Abstract: Acinetobacter baumannii is an opportunistic human pathogen responsible for numerous severe nosocomial infections. Genome analysis on the A. baumannii clinical isolate 04117201 revealed the presence of 13 two-component signal transduction systems (TCS). Of these, we examined the putative TCS named here as StkSR. The stkR response regulator was deleted via homologous recombination and its progeny, ΔstkR, was phenotypically characterized. Antibiogram analyses of ΔstkR cells revealed a two-fold increase in resistance to the clinically relevant polymyxins, colistin and polymyxin B, compared to wildtype. PAGE-separation of silver stained purified lipooligosaccharide isolated from ΔstkR and wildtype cells ruled out the complete loss of lipooligosaccharide as the mechanism of colistin resistance identified for ΔstkR. Hydrophobicity analysis identified a phenotypical change of the bacterial cells when exposed to colistin. Transcriptional profiling revealed a significant up-regulation of the pmrCAB operon in ΔstkR compared to the parent, associating these two TCS and colistin resistance. These results reveal that there are multiple levels of regulation affecting colistin resistance the suggested ‘cross-talk’ between the StkSR and PmrAB two-component systems highlights the complexity of these systems.
A molecular ecological approach to the detection and designation of the etiological agents of a model polymicrobial disease
Publisher: SAGE Publications
Date: 18-06-2013
Abstract: The application of the original Koch postulates and the molecular Koch postulates in the definition of the etiological agents of polymicrobial diseases has received little or no attention. In the present study, denaturing gradient gel electrophoresis (DGGE) of oral s les ( n = 3) from each of 3 categories of animals (healthy, diseased [gingivitis], and then oxytetracycline-treated) was used and revealed different bacterial community structures in a model polymicrobial disease (gingivitis) and after clinical cure. Potential microbes associated with the disease and belonging to the following families were identified: Fusobacteriaceae, Porphyromonadaceae, Flavobacteriaceae, Alcanivoracaceae, Bacteroidaceae, Xanthomonadaceae, and Neisseriaceae. Liquid chromatography–mass spectrophotometric analysis of culturable anaerobic bacteria culture supernatant revealed 3 major compounds (2-hydroxycaproic acid, phenyllactic acid, and indole acetic acid) that differentiated the healthy and disease groups. Results indicate that different microbial community structures were associated with the healthy and disease oral states. The results demonstrate the potential of DGGE as a tool in the detection and designation of etiological agents of polymicrobial diseases.
Hospital water as the source of healthcare-associated infection and antimicrobial-resistant organisms
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-07-2022
Interactions of the QacR Multidrug-Binding Protein with Structurally Diverse Ligands: Implications for the Evolution of the Binding Pocket
Publisher: American Chemical Society (ACS)
Date: 12-2003
DOI: 10.1021/BI035447+
Abstract: The QacR multidrug-binding repressor protein regulates the expression of the Staphylococcus aureus qacA gene, a multidrug resistance (MDR) locus that is prevalent in clinical isolates of this important human pathogen. In this paper we demonstrate that the range of structurally erse compounds capable of inducing qacA transcription is significantly more varied than previously appreciated, particularly in relation to bivalent cations. For all of the newly identified inducing compounds, induction of qacAexpression was correlated with a matching ability to dissociate QacR from operator DNA. Development of a ligand-binding assay based on intrinsic tryptophan fluorescence permitted dissociation constants to be determined for the majority of known QacR ligands, with values ranging from 0.2 to 82 microM. High-affinity binding of a compound to QacR in vitro was not found to correlate very strongly with either its in vivo inducing abilities or its structure. The latter observation indicated that the QacR ligand-binding pocket appears to have evolved to accommodate a wide range of toxic hydrophobic cations, rather than a specific class of compound. Importantly, the antimicrobial ligands of QacR included several plant alkaloids that share structural similarities with synthetic MDR substrates. This is consistent with the suggestion that the qacA-qacR MDR locus was recently derived from genes that protect against natural antimicrobial compounds.
A Single Acidic Residue Can Guide Binding Site Selection but Does Not Govern QacR Cationic-Drug Affinity
Publisher: Public Library of Science (PLoS)
Date: 17-01-2011
Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978
Publisher: Public Library of Science (PLoS)
Date: 11-05-2018
Stable low-copy-number Staphylococcus aureus shuttle vectors
Publisher: Microbiology Society
Date: 03-2003
Abstract: A series of Staphylococcus aureus-Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.
A new antibiotic with potent activity targets MscL
Publisher: Springer Science and Business Media LLC
Date: 04-02-2015
DOI: 10.1038/JA.2015.4
Analysis of tryptophan residues in the staphylococcal multidrug transporter QacA reveals long-distance functional associations of residues on opposite sides of the membrane
Publisher: American Society for Microbiology
Date: 04-2008
DOI: 10.1128/JB.01864-07
Abstract: Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were in idually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein.
Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism
Publisher: American Chemical Society (ACS)
Date: 13-05-2021
DOI: 10.1021/ACSINFECDIS.1C00149
Abstract: Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium
H-NS Plays a Role in Expression of Acinetobacter baumannii Virulence Features
Publisher: American Society for Microbiology
Date: 07-2013
DOI: 10.1128/IAI.00065-13
Abstract: Acinetobacter baumannii has become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success of A. baumannii as a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative of A. baumannii strain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality toward Caenorhabditis elegans nematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted an hns -like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS.
QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA
Publisher: Elsevier BV
Date: 07-1998
A putative pathway for biosynthesis of the O-antigen component, 3-deoxy-L-glycero-tetronic acid, based on the sequence of the Vibrio cholerae 01 rfb region
Publisher: Elsevier BV
Date: 12-1995
DOI: 10.1016/0378-1119(95)00588-9
Abstract: The nucleotide sequence of a region of the rfb genes, encoding biosynthesis of the Vibrio cholerae (Vc) O1 O-antigen, was determined. Analysis of the open reading frames (ORFs) within this region has revealed similarities with a number of different classes of biosynthetic proteins and enzymes. The ORFs have been designated RfbK, RfbL, RfbM, RfbN and RfbO. RfbK is a small, acidic protein which has similarity to the family of proteins known as acyl-carrier proteins (ACP). The RfbL protein has similarity to a super-family of enzymes which adenylate their substrates as a part of their reaction mechanism. Included in these are several acetyl-CoA ligases. Alignment of RfbL with these proteins reveals a highly conserved domain containing the motif GlyXaaXaaGlyXaaPro. This resembles the ATP-binding site motif and may represent a variant of the usual motif, except that Pro replaces Gly. The VcRfbM protein has similarity with a family of long-chain, iron-containing alcohol dehydrogenases, of which the Escherichia coli K-12 fucO and adhE gene products are also members. The RfbN protein has sequence homology with LuxE and LuxC of Vibrio harveyi (Vh) and other bioluminescent bacterial species. The latter are two components of the enzyme complex which synthesizes the long-chain aldehyde used in the V. harveyi bioluminescence system. Finally, the VcRfbO protein has sequence similarity with acetyl-CoA transferases. We were able to identify a number of the gene products using a T7 expression system, confirming several of the allocated ORFs. A biosynthetic pathway for the Vc O-antigen component 3-deoxy-L-glycero-tetronic acid, based on the enzymatic functions predicted for the RfbK, RfbL, RfbM, RfbN and RfbO proteins, is presented.
Arabidopsis phospholipase Dδ as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/FP08222
Abstract: Phospholipase D (PLD), in combination with the cytoskeleton, plays a key role in plant signal transduction. One isotype of the multigene Arabidopsis PLD family, AtPLDδ, has been implicated in binding microtubules, although the molecular details of the mechanism and identities of potential interaction partners are unclear. We constructed a GFP-AtPLDδ reporter gene, stably transformed it into an Arabidopsis suspension cell line, and used epitope-tagged affinity pull-down assays to isolate a complex of co-purifying proteins. Mass spectrometry analysis of the complex revealed a set of proteins including β-tubulin, actin 7, HSP70, clathrin heavy chain, ATP synthase subunits, and a band 7–4/flotillin homologue. Sequence alignments with defined tubulin- and actin-binding regions from human HsPLD2 revealed highly homologous regions in all 12 AtPLD isotypes, suggesting direct interactions of AtPLDδ with tubulin and actin, while interactions with the remaining partners are likely to be mediated by the cytoskeleton. We propose that AtPLDδ acts through a complex of cytoskeletal and partner proteins to modulate fundamental cellular processes such as cytoskeletal rearrangements, vesicular trafficking, assembly of Golgi apparatus, mitosis and cytokinesis.
A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases
Publisher: Elsevier BV
Date: 05-2013
DOI: 10.1016/J.VETMIC.2013.01.012
Abstract: Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.
Clonal diversity of methicillin-sensitive Staphylococcus aureus from South Australian wallabies
Publisher: Elsevier BV
Date: 12-2016
MITE Aba12 , a Novel Mobile Miniature Inverted-Repeat Transposable Element Identified in Acinetobacter baumannii ATCC 17978 and Its Prevalence across the Moraxellaceae Family
Publisher: American Society for Microbiology
Date: 27-02-2019
DOI: 10.1128/MSPHEREDIRECT.00028-19
Abstract: One of the most important weapons in the armory of Acinetobacter is its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from the Moraxellaceae family and its recent translocation into the hns locus in the A. baumannii ATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented ex les of MITEs in the literature and underscores the plastic nature of the hns locus in A. baumannii . MITE Aba12 , and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.
Regions of the cloned Vibrio cholerae rfb genes needed to determine the Ogawa form of the O-antigen
Publisher: Springer Science and Business Media LLC
Date: 12-1990
DOI: 10.1007/BF00262435
Abstract: The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase.
Investigation of the human pathogen Acinetobacter baumannii under iron limiting conditions
Publisher: Springer Science and Business Media LLC
Date: 23-02-2011
Abstract: Iron acquisition systems are important virulence factors in pathogenic bacteria. To identify these systems in Acinetobacter baumannii , the transcriptomic response of the completely sequenced strain ATCC 17978 under iron limiting conditions was investigated using a genomic microarray that contained probes for all annotated open reading frames. Under low iron conditions, transcription levels were more than 2-fold up-regulated for 463 genes, including 95 genes that were up-regulated more than 4-fold. Of particular significance, three siderophore biosynthesis gene clusters, including one novel cluster, were highly up-regulated. Binding sites for the ferric uptake regulator were identified in the promoter regions of many up-regulated genes, suggesting a prominent role for this regulator in the Acinetobacter iron acquisition response. Down-regulation under iron limitation was less dramatic as the transcription of only 202 genes varied more than 2-fold. Various genes involved in motility featured prominently amongst the genes down-regulated when iron was less readily available. Motility assays confirmed that these transcriptional changes are manifested at the phenotypic level. The siderophore biosynthesis gene clusters were further investigated by means of comparative genomic analysis of 10 sequenced Acinetobacter isolates. These analyses revealed important roles for mobile genetic elements in shaping the siderophore meditated iron acquisition mechanisms between different Acinetobacter strains. A. baumannii grown under iron limited conditions resulted in major transcriptional changes of not only many iron acquisition related genes, but also genes involved in other processes such as motility. Overall, this study showed that A. baumannii is well adaptable to growth in an environment which has limiting iron availability.
Functional effects of intramembranous proline substitutions in the staphylococcal multidrug transporter QacA
Publisher: Oxford University Press (OUP)
Date: 10-2006
DOI: 10.1111/J.1574-6968.2006.00411.X
Abstract: 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.
Cloning of the structural gene (hly) for the haemolysin of Vibrio cholerae El Tor strain 017
Publisher: Elsevier BV
Date: 11-1984
DOI: 10.1016/0378-1119(84)90213-0
Abstract: We have cloned the DNA encoding the haemolysin of Vibrio cholerae El Tor strain 017 into the plasmid vector pBR322. The resultant plasmid, pPM431, has a 6.2-kb PstI DNA insert which leads to the production of the haemolysin in Escherichia coli K-12. Deletion analysis and transposon mutagenesis have allowed us to localize several regions affecting haemolysin production. A number of these mutants have been analysed in E. coli K-12 minicells. Three proteins have been identified: A, 80 kDal B, 71 kDal and C, 22 kDal. A is the haemolysin which appears to be cell-associated in E. coli K-12, and B and C are required for its efficient production. We suggest that the genes for proteins A, B and C be designated hlyA, hlyB and hlyC, respectively.
Environmental variability and phytoplankton dynamics in a South Australian inverse estuary
Publisher: Elsevier BV
Date: 12-2014
Identification of genes essential for pellicle formation in Acinetobacter baumannii
Publisher: Springer Science and Business Media LLC
Date: 27-07-0027
The Presence of Opportunistic Premise Plumbing Pathogens in Residential Buildings: A Literature Review
Publisher: MDPI AG
Date: 04-2022
DOI: 10.3390/W14071129
Abstract: Opportunistic premise plumbing pathogens (OPPP) are microorganisms that are native to the plumbing environment and that present an emerging infectious disease problem. They share characteristics, such as disinfectant resistance, thermal tolerance, and biofilm formation. The colonisation of domestic water systems presents an elevated health risk for immune-compromised in iduals who receive healthcare at home. The literature that has identified the previously described OPPPs (Aeromonas spp., Acinetobacter spp., Helicobacter spp., Legionella spp., Methylobacterium spp., Mycobacteria spp., Pseudomonas spp., and Stenotrophomonas spp.) in residential drinking water systems were systematically reviewed. By applying the Preferred reporting items for systematic reviews and meta-analyses guidelines, 214 studies were identified from the Scopus and Web of Science databases, which included 30 clinical case investigations. Tap components and showerheads were the most frequently identified sources of OPPPs. Sixty-four of these studies detected additional clinically relevant pathogens that are not classified as OPPPs in these reservoirs. There was considerable variation in the detection methods, which included traditional culturing and molecular approaches. These identified studies demonstrate that the current drinking water treatment methods are ineffective against many waterborne pathogens. It is critical that, as at-home healthcare services continue to be promoted, we understand the emergent risks that are posed by OPPPs in residential drinking water. Future research is needed in order to provide consistent data on the prevalence of OPPPs in residential water, and on the incidence of waterborne homecare-associated infections. This will enable the identification of the contributing risk factors, and the development of effective controls.
Characterization of a Halotolerant Fungus from a Marine Sponge
Publisher: Hindawi Limited
Date: 23-11-2019
DOI: 10.1155/2019/3456164
Abstract: Introduction . Marine sponges have established symbiotic interactions with a large number of microorganisms including fungi. Most of the studies so far have focussed on the characterization of sponge-associated bacteria and archaea with only a few reports on sponge-associated fungi. During the isolation and characterization of bacteria from marine sponges of South Australia, we observed multiple types of fungi. One isolate in particular was selected for further investigation due to its unusually large size and being chromogenic. Here, we report on the investigations on the physical, morphological, chemical, and genotypic properties of this yeast-like fungus. Methods and Materials . Sponge s les were collected from South Australian marine environments, and microbes were isolated using different isolation media under various incubation conditions. Microbial isolates were identified on the basis of morphology, staining characteristics, and their 16S rRNA or ITS/28S rRNA gene sequences. Results . Twelve types of yeast and fungal isolates were detected together with other bacteria and one of these fungi measured up to 35 μ m in diameter with a unique chromogen compared to other fungi. Depending on the medium type, this unique fungal isolate appeared as yeast-like fungi with different morphological forms. The isolate can ferment and assimilate nearly all of the tested carbohydrates. Furthermore, it tolerated a high concentration of salt (up to 25%) and a range of pH and temperature. ITS and 28S rRNA gene sequencing revealed a sequence similarity of 93% and 98%, respectively, with the closest genera of Eupenidiella , Hortaea, and Stenella . Conclusions . On the basis of its peculiar morphology, size, and genetic data, this yeast-like fungus possibly constitutes a new genus and the name Magnuscella marinae , gen nov., sp. nov., is proposed. This study is the first of its kind for the complete characterization of a yeast-like fungus from marine sponges. This novel isolate developed a symbiotic interaction with living hosts, which was not observed with other reported closest genera (they exist in a saprophytic relationship). The observed unique size and morphology may favour this new isolate to establish symbiotic interactions with living hosts.
QacR-cation recognition is mediated by a redundancy of residues capable of charge neutralization
Publisher: American Chemical Society (ACS)
Date: 11-07-2008
DOI: 10.1021/BI8008246
AbGRI1-5, a novel AbGRI1 variant in an Acinetobacter baumannii GC2 isolate from Adelaide, Australia
Publisher: Oxford University Press (OUP)
Date: 16-11-2019
DOI: 10.1093/JAC/DKY459
Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins
Publisher: Proceedings of the National Academy of Sciences
Date: 25-11-2013
Abstract: Drug resistance is an increasing problem in clinical settings with some bacterial pathogens now resistant to virtually all available drugs. Chlorhexidine is a commonly used antiseptic and disinfectant in hospital environments, and there is increasing resistance to chlorhexidine seen in some pathogenic bacteria, such as Acinetobacter baumannii . This paper examines the global gene expression of A. baumannii in response to chlorhexidine exposure and identifies a gene that we demonstrate to mediate chlorhexidine resistance. Biochemical investigation reveals that this gene encodes a previously uncharacterized type of drug efflux pump that actively transports chlorhexidine out of the cell.
29Si{1H} CP-MAS NMR comparison and ATR-FTIR spectroscopic analysis of the diatoms Chaetoceros muelleri and Thalassiosira pseudonana grown at different salinities
Publisher: Springer Science and Business Media LLC
Date: 31-01-2013
DOI: 10.1007/S00216-013-6746-Z
Abstract: Diatoms are key indicators of marine environmental health. To further understand how diatoms respond to varying degrees of salinity, either due to climate change or brine waste discharge into marine environments, two different diatom species were studied. Thalassiosira pseudonana and Chaetoceros muelleri were cultured at three different salinities namely, 26 practical salinity units (PSU or parts per thousand), 36 PSU (standard salinity for culturing of seawater species) and 46 PSU. Changes in silica and organic content within the cultured diatoms were analysed using solid-state (29)Si{(1)H} cross-polarization-magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopies coupled with analysis of variance. (29)Si CP-MAS NMR showed that qualitatively the Q4:Q3 area ratios of C. muelleri, grown away from standard salinities, increased in response to the formation of more condensed (2 ≡SiOH → ≡Si-O-Si≡ + H2O) and/or an increase in closely associated organic matter to the Q4 component of the diatoms. This was not observed for T. pseudonana. However, both species showed the appearance of a new peak centered at 1575-1580 cm(-1) in the ATR-FTIR spectra, designated as the C═N band of nitrogenous purine-type compounds. Further, the C. muelleri species was shown to produce more extracellular polymeric substances at non-standard salinities. On this basis, results suggest that there is a strong relationship between diatom composition and salinity and that C. muelleri is more sensitive to its environment than T. pseudonana.
Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR
Publisher: Frontiers Media SA
Date: 30-01-2023
DOI: 10.3389/FMICB.2023.1094877
Abstract: Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella . The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water s les using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water s les. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease.
Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems
Publisher: MDPI AG
Date: 15-04-2020
Abstract: Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires’ disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.
Transcriptional regulation of multidrug efflux pumps in bacteria
Publisher: Elsevier BV
Date: 06-2001
Erratum: Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR (EMBO Journal (2002) 21 (1210-1218))
Publisher: Wiley
Date: 05-2002
Water as a Source of Antimicrobial Resistance and Healthcare-Associated Infections
Publisher: MDPI AG
Date: 18-08-2020
Abstract: Healthcare-associated infections (HAIs) are one of the most common patient complications, affecting 7% of patients in developed countries each year. The rise of antimicrobial resistant (AMR) bacteria has been identified as one of the biggest global health challenges, resulting in an estimated 23,000 deaths in the US annually. Environmental reservoirs for AMR bacteria such as bed rails, light switches and doorknobs have been identified in the past and addressed with infection prevention guidelines. However, water and water-related devices are often overlooked as potential sources of HAI outbreaks. This systematic review examines the role of water and water-related devices in the transmission of AMR bacteria responsible for HAIs, discussing common waterborne devices, pathogens, and surveillance strategies. AMR strains of previously described waterborne pathogens including Pseudomonas aeruginosa, Mycobacterium spp., and Legionella spp. were commonly isolated. However, methicillin-resistant Staphylococcus aureus and carbapenem-resistant Enterobacteriaceae that are not typically associated with water were also isolated. Biofilms were identified as a hot spot for the dissemination of genes responsible for survival functions. A limitation identified was a lack of consistency between environmental screening scope, isolation methodology, and antimicrobial resistance characterization. Broad universal environmental surveillance guidelines must be developed and adopted to monitor AMR pathogens, allowing prediction of future threats before waterborne infection outbreaks occur.
Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.MIMET.2007.05.011
Abstract: Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus.
The complete genome and phenome of a community-acquired Acinetobacter baumannii
Publisher: Public Library of Science (PLoS)
Date: 19-03-2013
The composition of planktonic prokaryotic communities in a hospital building water system depends on both incoming water and flow dynamics
Publisher: Elsevier BV
Date: 09-2023
Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains
Publisher: Oxford University Press (OUP)
Date: 10-1985
Efflux Pump Mediated Antimicrobial Resistance by Staphylococci in Health-Related Environments: Challenges and the Quest for Inhibition
Publisher: MDPI AG
Date: 07-12-2021
DOI: 10.3390/ANTIBIOTICS10121502
Abstract: The increasing emergence of antimicrobial resistance in staphylococcal bacteria is a major health threat worldwide due to significant morbidity and mortality resulting from their associated hospital- or community-acquired infections. Dramatic decrease in the discovery of new antibiotics from the pharmaceutical industry coupled with increased use of sanitisers and disinfectants due to the ongoing COVID-19 pandemic can further aggravate the problem of antimicrobial resistance. Staphylococci utilise multiple mechanisms to circumvent the effects of antimicrobials. One of these resistance mechanisms is the export of antimicrobial agents through the activity of membrane-embedded multidrug efflux pump proteins. The use of efflux pump inhibitors in combination with currently approved antimicrobials is a promising strategy to potentiate their clinical efficacy against resistant strains of staphylococci, and simultaneously reduce the selection of resistant mutants. This review presents an overview of the current knowledge of staphylococcal efflux pumps, discusses their clinical impact, and summarises compounds found in the last decade from plant and synthetic origin that have the potential to be used as adjuvants to antibiotic therapy against multidrug resistant staphylococci. Critically, future high-resolution structures of staphylococcal efflux pumps could aid in design and development of safer, more target-specific and highly potent efflux pump inhibitors to progress into clinical use.
In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity
Publisher: American Society for Microbiology
Date: 06-2000
DOI: 10.1128/IAI.68.6.3548-3553.2000
Abstract: Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus . We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729–732, 1994 A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169–3172, 1998 V. K. Dhawan et al., Infect. Immun. 65:3293–3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1 r ) strains and their genetically related, tPMP-1-susceptible (tPMP-1 s ) counterpart strains. The tPMP-1 r strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1 r strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1 s counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1 r strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.
The oral microbial community of gingivitis and lumpy jaw in captive macropods
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.RVSC.2013.08.010
Abstract: Gingivitis and lumpy jaw are diseases of polymicrobial aetiology. Although Fusobacterium necrophorum has been associated with these diseases in macropods, little is known about other organisms associated with these diseases in this animal species. PCR-DGGE analysis revealed the potential pathogens associated with gingivitis and lumpy jaw in macropods. PCR-DGGE profile comparison between the healthy and disease groups indicated a shift in the oral bacterial community structures with similarity coefficients of 48% and 35% for gingivitis and lumpy jaw respectively. Moreover, gingivitis was associated with increase in bacterial ersity (Shannon index = 2.87 PL curve = 45%) while lumpy jaw resulted in a decline in bacterial ersity (Shannon index = 2.47 PL curve = 74%). This study suggest that the establishment of gingivitis and lumpy jaw diseases follows the ecological plaque hypothesis. This forms the basis for an expanded investigation in an epidemiological scale and suggests the need for the appropriate choice of antimicrobial agent(s) and for the effective management and control of polymicrobial diseases.
Rapid multiplexed phenotypic screening identifies drug resistance functions for three novel efflux pumps in Acinetobacter baumannii
Publisher: Oxford University Press (OUP)
Date: 31-01-2016
DOI: 10.1093/JAC/DKV460
Abstract: Drug efflux pumps are one of the key machineries in bacterial drug resistance. Although quite a few of these transport systems have been functionally characterized in various organisms, due to large-scale genome sequencing efforts and improved prediction pipelines there are increasing numbers of putative drug efflux genes annotated. For phenotype identification of the proteins encoded by these genes, we developed a novel high-throughput phenotype screening strategy and demonstrated its utility in identifying phenotypes for putative efflux systems encoded by the human pathogen Acinetobacter baumannii. Seventeen putative drug efflux systems from A. baumannii were heterologously expressed in Escherichia coli. For rapid and economical phenotype screening we employed a combination of multiplexed Biolog Phenotype Microarrays and quantitative PCR. Using this method we screened these putative drug efflux pumps against 240 antimicrobial conditions, equating to 4080 simultaneous phenotypic tests. Of the 17 putative drug efflux systems, phenotypes were confirmed for two pumps and novel drug resistance phenotypes were identified for three new A. baumannii drug transporters, which exemplified the power of this method as a high-throughput screening technique. One of the phenotypically characterized putative drug efflux systems was classified within the ATP-binding cassette superfamily of transport proteins and represents the first drug resistance protein characterized from this superfamily in A. baumannii. The remaining two proteins were members of the major facilitator superfamily of efflux pumps. This method has broad potential for high-throughput phenotype characterization of putative drug efflux systems in a range of organisms.
Molecular characterization of the staphylococcal multidrug resistance export protein QacC
Publisher: American Society for Microbiology
Date: 05-1995
DOI: 10.1128/JB.177.10.2827-2833.1995
Abstract: The QacC polypeptide is a member of a family of small membrane proteins which confer resistance to toxic compounds. The staphylococcal qacC gene confers resistance to toxic organic cations via proton-dependent export. The membrane topology of the QacC polypeptide was investigated by constructing and analyzing a series of qacC-phoA and qacC-lacZ fusions. From these analyses, most of the predicted features of the QacC protein were verified, although data regarding the possible orientation of the COOH region were not conclusive. The role of the sole cysteine residue, Cys-42, in QacC was studied by using the sulfhydryl reagent N-ethylmaleimide and site-directed mutagenesis. N-Ethylmaleimide was shown to inhibit qacC-mediated ethidium export. Multiple amino acid substitutions were made for Cys-42, and mutations at this location had various effects on resistance specificity. This suggests that the Cys-42 residue may be located near a region of QacC that is involved in substrate recognition. Mutagenesis of conserved residues in QacC indicated that Tyr-59 and Trp-62 also play an essential structural or functional role in QacC.
Adherence and motility characteristics of clinical Acinetobacter baumannii isolates
Publisher: Oxford University Press (OUP)
Date: 09-08-2011
DOI: 10.1111/J.1574-6968.2011.02362.X
Abstract: Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.
Characterisation of β-lactam resistance mediated by blaZ in staphylococci recovered from captive and free-ranging wallabies
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.JGAR.2015.05.002
Abstract: Staphylococci are commensal organisms of animals, but some species are opportunistic pathogens that are resistant to almost all antimicrobial agents in clinical use. Bacterial resistance to β-lactam antimicrobial agents is widespread and has been investigated in species isolated from humans in addition to food production and companion animals. However, minimal progress has been made towards identifying reservoirs of β-lactam-resistant staphylococci in wildlife. This study was aimed at investigating and characterising β-lactamase resistance from staphylococci of wallaby origin. Staphylococci from free-ranging and captive wallabies were assessed for their phenotypic susceptibility to β-lactam antimicrobial agents prior to sequence analysis of their blaZ and blaR1 genes. Deduced amino acid sequences were classified according to the Ambler molecular characterisation method, assigned a protein signature type and compared with sequences generated from previous studies involving isolates from humans, cattle and companion animals. All BlaZ sequences identified in this study were assignable to a pre-existing β-lactamase class and protein signature type, including the more recently discovered protein signature type 12. Three major phylogenetic groups were resolved upon phylogenetic analysis against published BlaZ sequences. This study has found antibiotic-resistant staphylococci both in free-ranging and captive wallaby populations and these bacteria harbour blaZ variants that are different to those recovered from humans, cattle and companion animals. Further studies of staphylococci from non-traditional sources are required in order to enhance our knowledge of the epidemiology of antibiotic resistance genes.
Bioenergetics of the staphylococcal multidrug export protein QacA
Publisher: Elsevier BV
Date: 02-1999
Microbial drug efflux proteins of the major facilitator superfamily
Publisher: Bentham Science Publishers Ltd.
Date: 07-2006
DOI: 10.2174/138945006777709575
Abstract: Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the lified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, C ylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed ex les. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.
Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR
Publisher: Wiley
Date: 03-2002
The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers
Publisher: American Society for Microbiology
Date: 15-12-2001
DOI: 10.1128/JB.183.24.7102-7109.2001
Abstract: Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the ergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.
The TetA(K) tetracycline/H+ antiporter from Staphylococcus aureus: Mutagenesis and functional analysis of motif C
Publisher: American Society for Microbiology
Date: 15-03-2000
DOI: 10.1128/JB.182.6.1492-1498.2000
Abstract: Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 in idual amino acid replacements were introduced however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H + activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.
Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system
Publisher: Frontiers Media SA
Date: 07-06-2023
DOI: 10.3389/FCIMB.2023.1190631
Abstract: Hospital water systems are a significant source of Legionella , resulting in the potentially fatal Legionnaires’ disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water s les were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers s led was measured using the Enware Smart Flow ® monitoring system. qPCR analysis demonstrated that 21.8% s les were positive for Legionella spp., 21% for L. pneumophila , 40.9% for V. vermiformis and 4.2% for Acanthamoeba . All s les that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp. however, only 2.5% of s les were positive for culturable Legionella spp. 18.1% of the s les were positive for free-living amoebae (FLA) using culture. All s les positive for Legionella spp. were also positive for FLA. S les with a high heterotrophic plate count (HPC ≥ 5 × 10 3 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./ L. pneumophila ( p & 0.01) and V. vermiformis ( p & 0.05). Temporary water stagnation arising through intermittent usage (& 2 hours of usage per month) significantly ( p & 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./ L. pneumophila , and V. vermiformis however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis , heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.
The multidrug efflux protein NorM is a prototype of a new family of transporters [2]
Publisher: Wiley
Date: 1999
Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: Membrane topology and identification of residues involved in substrate specificity
Publisher: Proceedings of the National Academy of Sciences
Date: 16-04-1996
Abstract: The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to alent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to alent cations. Both QacA and the QacB mutants capable of conferring resistance to alent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to alent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.
Development of a high-throughput cloning strategy for characterization of acinetobacter baumannii drug transporter proteins
Publisher: S. Karger AG
Date: 2011
DOI: 10.1159/000329836
Abstract: Heterologous expression of membrane proteins in i Escherichia coli /i often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell ision superfamily encoded by the human pathogen i Acinetobacter baumannii /i . Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in i A. baumannii /i . Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific i A. baumannii /i insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins.
The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes
Publisher: Oxford University Press (OUP)
Date: 16-09-2009
DOI: 10.1093/NAR/GKP756
Functional analyses reveal an important role for tyrosine residues in the staphylococcal multidrug efflux protein QacA
Publisher: Springer Science and Business Media LLC
Date: 16-09-2008
Abstract: 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. 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. 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.
Low-level resistance of staphylococcus aureus to thrombin-induced platelet microbicidal protein 1 in vitro associated with qacA gene carriage is independent of multidrug efflux pump activity
Publisher: American Society for Microbiology
Date: 07-2006
DOI: 10.1128/AAC.00028-06
Abstract: Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus , the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1 r ) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1 r and (ii) tPMP-1 r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1 r qacA- bearing strains, the parental strain and its isogenic qacB - and qacC -containing strains were tPMP-1 susceptible (tPMP-1 s ). Efflux pump inhibition by cyanide m -chlorophenylhydrazone abrogated Et r , but not tPMP-1 r , in the qacA- bearing strain. In synergy assays, exposure of the qacA -bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA -bearing and parental strains: contents of the major phospholipids asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol fatty acid composition and relative surface charge. Of note, the qacA -bearing strain exhibited greater membrane fluidity than that of the parental, qacB -, or qacC -bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1 r phenotype. These data suggest that qacA -encoded tPMP-1 r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.
Detection of Fusobacterium necrophorum leukotoxin (lkta) gene sequence in the oral cavity of captive Macropods
Publisher: OMICS Publishing Group
Date: 2013
Water Stagnation and Flow Obstruction Reduces the Quality of Potable Water and Increases the Risk of Legionelloses
Publisher: Frontiers Media SA
Date: 14-12-2020
DOI: 10.3389/FENVS.2020.611611
Abstract: Legionella is an opportunistic waterborne pathogen associated with Legionnaires' disease and Pontiac fever. Despite improved public awareness, the incidence of Legionella associated infections has been increasing. Aerosols generated from engineered potable water systems are a demonstrated cause of both nosocomial and community-acquired legionellosis. The ecology of Legionella in these systems is complex with multiple factors impacting their colonization and persistence. Flow dynamics has been identified as an important factor and stagnation in cooling towers is an accepted risk for increased Legionella growth however, less is known about the impact of flow dynamic on Legionella in potable water systems. This is especially complex due to the inherent intermittent and variable usage observed within outlets of a potable water system. This systematic literature review examines the role of fluid dynamics and stagnation on the colonization and growth of Legionella in potable water systems. Twenty two of 24 identified studies show a positive association between stagnation zones and increased colonization of Legionella . These zones included dead legs, dead ends, storage tanks, and obstructed water flow (such as intermittent usage or flow restriction). Prolonged stagnation in building plumbing systems also deteriorates the quality of thermally or chemically treated potable water. This stimulates the colonization of Legionella established biofilms. Such biofilms are intrinsically resistant to disinfection procedures and accelerate the rate of decay of chemical disinfectants. Sub-lethal doses of disinfectants and the presence of protozoan hosts in stationary water promote generation of viable but non-culturable Legionella cells. This results in false negatives in surveillance methods that use culture methodology. In conclusion, elimination of temporal and permanent stagnation points can improve the quality of potable water, efficacy of disinfectants, and reduce the risk of legionellosis. Current guidelines and water safety plans recognize the risks associated with permanent stagnation point (dead ends and dead legs) however, there is a need for greater emphasis on controlling temporal stagnation arising from intermittent usage.
Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein
Publisher: American Society for Microbiology
Date: 24-12-2019
Abstract: With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae , the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.
Diatom adaptability to environmental change: A case study of two Cocconeis species from high-salinity areas
Publisher: Informa UK Limited
Date: 18-10-2013
Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherixhia coli K-12
Publisher: Oxford University Press (OUP)
Date: 08-1991
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