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ABSTRACT: Bioremediation efforts worldwide are faced with the problem of metals interfering with the degradation of organic pollutants. There has been little systematic investigation into how the important environmental factors of media composition, buffering agent, and carbon source affect the exertion of metal toxicity on bacteria. This study aimed to systematically separate and investigate the influence of these factors by examining planktonic and biofilm establishment and growth. Two Pseudomonads were chosen, the PCB degrader P. pseudoalcaligenes KF707 and P. fluorescens. The two strains were grown in the presence of Al(3+) and Cu(2+) under different media conditions of carbon source (Lysogeny broth, biphenyl, succinate, aspartic acid, butyric acid, oxaloacetic acid, putrescine and benzoic acid) and under different buffering conditions (high and low phosphate or MOPS). These experiments allowed for the elucidation of an effect of different metabolic conditions and metal speciation on planktonic bacteria growth and biofilm establishment and development under metal stress. Here we show that the nature of bacterial growth (planktonic and biofilm development) is dramatically affected by the interplay between toxic metals, carbon source and media composition. The capacity of a media to bind toxic metals as well as quality of carbon source greatly influences the amount of metal that bacteria can tolerate, depending on both the bacterium and metal. Future studies evaluating metal ion toxicity should consider these effects, as well as their interactions with specific environments into account in order to improve clean-up success.
Metallomics 04/2013; · 3.90 Impact Factor
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ABSTRACT: Bioremediation efforts worldwide are faced with the problem of metals interfering with the degradation of organic pollutants. There has been little systematic investigation into how the important environmental factors of media composition, buffering agent, and carbon source affect the exertion of metal toxicity on bacteria. This study aimed to systematically separate and investigate the influence of these factors by examining planktonic and biofilm establishment and growth. Two Pseudomonads were chosen, the PCB degrader P. pseudoalcaligenes KF707 and P. fluorescens. The two strains were grown in the presence of Al3+ and Cu2+ under different media conditions of carbon source (Lysogeny broth, biphenyl, succinate, aspartic acid, butyric acid, oxaloacetic acid, putrescine and benzoic acid) and under different buffering conditions (high and low phosphate or MOPS). These experiments allowed for the elucidation of an effect of different metabolic conditions and metal speciation on planktonic bacteria growth and biofilm establishment and development under metal stress. Here we show that the nature of bacterial growth (planktonic and biofilm development) is dramatically affected by the interplay between toxic metals, carbon source and media composition. The capacity of a media to bind toxic metals as well as quality of carbon source greatly influences the amount of metal that bacteria can tolerate, depending on both the bacterium and metal. Future studies evaluating metal ion toxicity should consider these effects, as well as their interactions with specific environments into account in order to improve clean-up success.
Metallomics 04/2013; · 3.90 Impact Factor
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ABSTRACT: The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.
Biochemical and Biophysical Research Communications 05/2012; 422(2):316-20. · 2.48 Impact Factor
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ABSTRACT: The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1’ binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.
Biochemical and Biophysical Research Communications 05/2012; · 2.48 Impact Factor
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ABSTRACT: Bitumen extraction from the oil sands of Alberta has resulted in millions of cubic meters of waste stored on-site in tailings ponds. Unique microbial ecology is expected in these ponds, which may be key to their bioremediation potential. We considered that direct culturing of microbes from a tailings sample as biofilms could lead to the recovery of microbial communities that provide good representation of the ecology of the tailings. Culturing of mixed species biofilms in vitro using the Calgary Biofilm Device (CBD) under aerobic, microaerobic, and anaerobic growth conditions was successful both with and without the addition of various growth nutrients. Denaturant gradient gel electrophoresis and 16S rRNA gene pyrotag sequencing revealed that unique mixed biofilm communities were recovered under each incubation condition, with the dominant species belonging to Pseudomonas, Thauera, Hydrogenophaga, Rhodoferax, and Acidovorax. This work used an approach that allowed organisms to grow as a biofilm directly from a sample collected of their environment, and the biofilms cultivated in vitro were representative of the endogenous environmental community. For the first time, representative environmental mixed species biofilms have been isolated and grown under laboratory conditions from an oil sands tailings pond environment and a description of their composition is provided.
FEMS Microbiology Ecology 01/2012; 79(1):240-50. · 3.41 Impact Factor
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ABSTRACT: In this study we report for the first time the comprehensive inhibitor profiling of the Proteus mirabilis metalloprotease virulence factor ZapA (mirabilysin) using a 160 compound focused library of N-alpha mercaptoamide dipeptides, in order to map the S(1)(') and S(2)(') binding site preferences of this important enzyme. This study has revealed a preference for the aromatic residues tyrosine and tryptophan in P(1)(') and aliphatic residues in P(2)('). From this library, six compounds were identified which exhibited sub- to low-micromolar K(i) values. The most potent inactivator, SH-CO(2)-Y-V-NH(2) was capable of preventing ZapA-mediated hydrolysis of heat-denatured IgA, indicating that these inhibitors may be capable of protecting host proteins against ZapA during colonisation and infection.
Biochimie 07/2011; 93(10):1824-7. · 3.02 Impact Factor
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ABSTRACT: Bacterial biofilms are known to withstand the effects of toxic metals better than planktonic cultures of the same species. This phenomenon has been attributed to many features of the sessile lifestyle not present in free-swimming populations, but the contribution of intracellular metabolism has not been previously examined. Here, we use a combined GC-MS and (1)H NMR metabolomic approach to quantify whole-cell metabolism in biofilm and planktonic cultures of the multimetal resistant bacterium Pseudomonas fluorescens exposed to copper ions. Metabolic changes in response to metal exposure were found to be significantly different in biofilms compared to planktonic cultures. Planktonic metabolism indicated an oxidative stress response that was characterized by changes to the TCA cycle, glycolysis, pyruvate and nicotinate and niacotinamide metabolism. Similar metabolic changes were not observed in biofilms, which were instead dominated by shifts in exopolysaccharide related metabolism suggesting that metal stress in biofilms induces a protective response rather than the reactive changes observed for the planktonic cells. From these results, we conclude that differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance. An altered metabolic response to metal toxicity represents a novel addition to a growing list of biofilm-specific mechanisms to resist environmental stress.
Journal of Proteome Research 05/2011; 10(7):3190-9. · 5.11 Impact Factor
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ABSTRACT: Biofilms are microcolonies of microbes adherent to biotic and abiotic surfaces, often responsible for chronic infections and medical device contamination. Escherichia coli is one of the prevalent pathogens involved in uropathogenic infections and contamination of catheters. A biosurfactant produced by Bacillus licheniformis V9T14 was tested alone and in association with various antibiotics against a mature 24-h uropathogenic E. coli CFT073 biofilm. Biofilm was grown on polystyrene pegs of a Calgary Biofilm Device, providing a tool to evaluate the efficacy of antimicrobial agents. Antibiotics tested were ampicillin, cefazolin, ceftriaxone, ciprofloxacin, piperacillin, tobramycin and trimethoprim/sulfamethoxazole (19:1). Biosurfactant alone at the concentrations tested was not able to remove the adherent cells of the pre-formed biofilm. However, the difference between the effect of antibiotic alone and in combination with the biosurfactant was significant and exceeded 1log(10) (90%) reduction in most cases. Results of this study indicate that V9T14 biosurfactant in association with antibiotics leads to a synergistic increase in the efficacy of antibiotics in biofilm killing, and in some combinations leads to total eradication of E. coli CFT073 biofilm.
International journal of antimicrobial agents 02/2011; 37(4):324-31. · 3.03 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS(-) strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS(-) did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS(-) parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.
Microbiology 12/2010; 156(Pt 12):3699-709. · 3.06 Impact Factor
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ABSTRACT: Peritoneal dialysis (PD)-related peritonitis is a common and morbid complication of PD. Bacteria are able to create a biofilm on the PD catheter, which can be a source of recurrent infection. Biofilms undergo a phenotypic change resulting in increased antibiotic resistance. ♢
21 clinical isolates of different patients with PD peritonitis secondary to Staphylococcus aureus were collected. They were analyzed for their antibiotic susceptibility in the planktonic form using the standard minimum inhibitory concentration (MIC) and in a biofilm using minimum biofilm eradication concentration (MBEC). Chi-square was used to compare the sensitivity results. ♢
The isolates were susceptible to all the antibiotics tested using MIC. Every antibiotic except gentamicin lost its efficacy when the bacteria were grown in a biofilm (p > 0.05). The change in susceptibility was statistically significant to a level of p < 0.001 for all antibiotics tested. ♢ Discussion: In PD peritonitis that is long standing, recurrent, or not responsive to therapy, MBEC testing should be considered as a biofilm may be present. Gentamicin should be strongly considered over other agents for empiric gram-negative coverage as it may be providing synergy in the setting of Staphylococcus aureus. Also, the newer anti-staphylococcal drugs should be tested for their performance in a biofilm using the MBEC method.
Peritoneal dialysis international : journal of the International Society for Peritoneal Dialysis. 11/2010; 30(6):652-6.
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ABSTRACT: Pseudomonas pseudoalcaligenes KF707 is a polychlorinated biphenyls (PCBs) degrader, also tolerant to several toxic metals and metalloids. The work presented here examines for the first time the chemotactic response of P. pseudoalcaligenes KF707 to biphenyl and intermediates of the PCB biodegradation pathway in the presence and absence of metals. Chemotaxis analyses showed that biphenyl, benzoic acid and chlorobenzoic acids acted as chemoattractants for KF707 cells and that metal cations such as Ni(2+) and Cu(2+) strongly affected the chemotactic response. Toxicity profiles of various metals on KF707 cells grown on succinate or biphenyl as planktonic and biofilm were determined both in the presence and in the absence of PCBs. Notably, KF707 cells from both biofilms and planktonic cultures were tolerant to high amounts (up to 0.5 g L(-1)) of Aroclor 1242, a commercial mixture of PCBs. Together, the data show that KF707 cells are chemotactic and can form a biofilm in the presence of Aroclor 1242 and specific metals. These findings provide new perspectives on the effectiveness of using PCB-degrading bacterial strains in bioremediation strategies of metal-co-contaminated sites.
FEMS Microbiology Ecology 11/2010; 74(2):291-301. · 3.41 Impact Factor
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ABSTRACT: Batch culture of biofilms on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibility. In this paper, we describe a core protocol and a set of parameters (surface composition, the rate of rocking or orbital motion, temperature, cultivation time, inoculum size, atmospheric gases and nutritional medium) that can be adjusted to grow single- or multispecies biofilms on peg surfaces. Mature biofilms formed on peg lids can then be fitted into microtiter plates containing test agents. After a suitable exposure time, biofilm cells are disrupted into a recovery medium using sonication. Microbicidal endpoints can be determined qualitatively using optical density measurements or quantitatively using viable cell counting. Once equipment is calibrated and growth conditions are at an optimum, the procedure requires approximately 5 h of work over 4-6 d. This efficient method allows antimicrobial agents and exposure conditions to be tested against biofilms on a high-throughput scale.
Nature Protocol 07/2010; 5(7):1236-54. · 8.36 Impact Factor
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ABSTRACT: While antibiotic resistance has grabbed the headlines and the attention of the pharmaceutical industry, the lack of susceptibility of biofilms formed both on animate and inanimate surfaces deserve greater attention from the industry, medical practitioners and regulators. The current literature tells us that the inherent tolerance to antibiotics demonstrated by antibiotic-sensitive organisms when grown as a biofilm clearly identifies a major disconnect between our current practices in antimicrobial development, diagnostics and efficacy in patient treatment. A paradigm shift is required in the way we utilize conventional antimicrobials and in the way we screen for next-generation antibiotics with efficacy to treat biofilms associated with chronic, recurrent and device related infections. This paradigm shift must not only take place in industry but also in how drugs are brought to the marketplace for acceptance.
Expert Opinion on Pharmacotherapy 04/2010; 11(8):1233-7. · 3.20 Impact Factor
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ABSTRACT: Biological contamination of surfaces, both in industry and in health care, plays an important role as a potential vector of disease transmission. Metals have been described to be effective antibiofilm agents, and the efficacy of silver ions as a disinfectant has been known for centuries. The activity of AgNO3 combined with the lipopeptide biosurfactant V9T14 has been studied against a preformed Escherichia coli biofilm on the Calgary Biofilm Device. Results indicated that the activity of silver can be synergistically enhanced by the presence of V9T14, both allowing for a reduction in the quantity of silver used and for greater antimicrobial activity. The concentration of silver needed to obtain this reduction in the silver-biosurfactant solution was from 129- to 258-fold less than the concentration of silver alone. To our knowledge, this is the first time that a synergistic interaction between a lipopeptide biosurfactant and silver has been observed.
Canadian Journal of Microbiology 03/2010; 56(3):272-8. · 1.36 Impact Factor
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ABSTRACT: Colony morphology variants isolated from natural and laboratory-grown biofilms represent subpopulations of biofilm cells that may be important for multiple aspects of the sessile lifestyle, from surface colonization to stress resistance. There are many genetic and environmental factors that determine the frequency at which colony morphology variants are recovered from biofilms. One of these factors involves an increased selection for variants in biofilms of Pseudomonas species bearing inactivating mutations in the global activator of cyanide biosynthesis/regulator of secondary metabolism (gac/rsm) signal transduction pathway. Here we characterize two distinct colony morphology variants isolated from biofilms of Pseudomonas fluorescens missing the gacS sensor kinase. These variants produced more biofilm cell mass, and in one case, this was likely due to overproduction of the exopolysaccharide cellulose. Nuclear magnetic resonance (NMR) metabolomics revealed distinct metabolic changes for each of the two phenotypic variants, and these changes involved amino acids and metabolites produced through glutathione biochemistry. Some of these metabolites are hypothesized to play a role in redox and metal homeostasis, and corresponding to this, we show that biofilm populations grown from each of these variants had a different ability to survive when exposed to toxic doses of metal ions. These data suggest that colony morphology variants that evolve during growth of P. fluorescens as a biofilm may have distinct metabolic capacities that contribute to their individual abilities to withstand environmental stress.
Environmental Microbiology 03/2010; 12(6):1565-77. · 5.84 Impact Factor
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ABSTRACT: Microbiological metal toxicity involves redox reactions between metal species and cellular molecules, and therefore, we hypothesized that antioxidant systems might be chromosomal determinants affecting the susceptibility of bacteria to metal toxicity. Here, survival was quantified in metal ion-exposed planktonic cultures of several Escherichia coli strains, each bearing a mutation in a gene important for redox homeostasis. This characterized approximately 250 gene-metal combinations and identified that sodA, sodB, gor, trxA, gshA, grxA and marR have distinct roles in safeguarding or sensitizing cells to different toxic metal ions (Cr(2)O(7)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), AsO(2)(-), SeO(3)(2-) or TeO(3)(2-)). To shed light on these observations, fluorescent sensors for reactive oxygen species (ROS) and reduced thiol (RSH) quantification were used to ascertain that different metal ions exert oxidative toxicity through disparate modes-of-action. These oxidative mechanisms of metal toxicity were categorized as involving ROS and thiol-disulfide chemistry together (AsO(2)(-), SeO(3)(2-)), ROS predominantly (Cu(2+), Cr(2)O(7)(2-)) or thiol-disulfide chemistry predominantly (Ag(+), Co(2+), Zn(2+), TeO(3)(2-)). Corresponding to this, promoter-luxCDABE fusions showed that toxic doses of different metal ions up- or downregulate the transcription of gene sets marking distinct pathways of cellular oxidative stress. Altogether, our findings suggest that different metal ions are lethal to cells through discrete pathways of oxidative biochemistry, and moreover, indicate that chromosomally encoded antioxidant systems may have metal ion-specific physiological roles as determinants of bacterial metal tolerance.
Environmental Microbiology 07/2009; 11(10):2491-509. · 5.84 Impact Factor
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ABSTRACT: The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3,841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28 : 0), also termed the very long chain fatty acid (VLCFA), attached at the 2' position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFA-containing lipid A is critical to cellular traits that are important for survival in the rhizosphere.
Microbiology 06/2009; 155(Pt 9):3055-69. · 3.06 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.
Microbiology 06/2009; 155(Pt 8):2612-9. · 3.06 Impact Factor
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ABSTRACT: Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a Delta yafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the Delta yafQ strain had up to a approximately 2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a approximately 10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.
Antimicrobial Agents and Chemotherapy 04/2009; 53(6):2253-8. · 4.84 Impact Factor
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ABSTRACT: The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many other aspects of bacterial physiology. Although it is known that the sensor kinases RetS and LadS feed into the GacS/GacA system, the mechanism through which this occurs is unknown, as are the protein-protein interactions in this system. To characterize and define these interactions, we utilized a bacterial two-hybrid system to study the interactions of GacS and GacA from Pseudomonas fluorescens CHA0. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in vivo was investigated. We found that the entire GacA molecule is required for GacA to interact with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. In addition, homologs of Pseudomonas aeruginosa hybrid sensor kinases, RetS and LadS, were identified in P. fluorescens, and shown to interact with GacS, but not GacA.
FEMS Microbiology Letters 02/2009; 292(1):50-6. · 2.04 Impact Factor
