59 research outputs found
Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics
Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic suscepti bility, we previously ident ified 3 genes or operons of P. aeruginosa UCBPP-PA14 ( ndvB , PA1875–1877 and tssC1 ) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents
A structure/function analysis of the interaction of the Escherichia coli NusA protein with RNA polymerase, the phage lambda N protein, and nut site RNA
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University of TorontoNusA is an 'E. coli' protein that controls transcription elongation, termination and antitermination. In this thesis, I show that the functions of NusA in transcription are facilitated by interactions with RNA polymerase, RNA and the [lambda] N protein. Use of a series of deletion constructs of NusA allowed me to identify specific regions of NusA involved in specific interactions and in various aspects of NusA function. Genetic evidence suggested that NusA may interact with the 'boxA ' portion of the N-utilization site ('nut' site= ' boxA, interbox,' and 'boxB'). By constructing multiple nucleotide substitutions in the 'nut' site, I showed that the identities of certain nucleotides at the 3' end of ' boxA' and in the 'interbox ' were important for NusA to associate with an N-'nut' site complex. NusA association with RNA in the presence of N is presumably facilitated by its S1 and KH homology regions, two types of RNA-binding domains in NusA. Elimination or mutation of the S1 homology region prevented the association of NusA with an N-' nut' site complex. Using affinity chromatography experiments, I found that RNA polymerase bound equally well to an amino-terminal RNA polymerase-binding region in amino acids 1-137 and a carboxy-terminal RNA polymerase-binding region in amino acids 232-495 of NusA. By contrast, the à subunit of RNA polymerase only bound to the carboxy-terminal RNA polymerase-binding region of NusA. N protein also bound to a carboxy-terminal region of NusA, and both N and à allowed NusA to associate with RNA in a gel mobility shift assay. When the carboxy-terminal region of NusA was deleted in NusA (1-348), the loss of N-binding and Ã-binding ability did not abolish NusA function in termination and antitermination assays. This minimal functional NusA protein retained the KH and S1 homology regions and the amino-terminal RNA polymerase-binding region. Unlike full length NusA (1-495), NusA (1-416) could bind RNA on its own. These observations suggest that the carboxy-terminal region of NusA inhibits RNA binding and that this inhibition can be relieved by interaction with the [lambda] N protein or the à subunit of RNA polymerase.Ph.D
Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)
Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibioticsâ–¿
Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics
Loss of the Two-Component System TctD-TctE in Pseudomonas aeruginosa Affects Biofilm Formation and Aminoglycoside Susceptibility in Response to Citric Acid
Nutrient availability is an important contributor to the ability of bacteria to establish successful infections in a host. Pseudomonas aeruginosa is an opportunistic pathogen in humans causing infections that are difficult to treat. In part, its success is attributable to a high degree of metabolic versatility. P. aeruginosa is able to sense and respond to varied and limited nutrient stress in the host environment. Two-component systems are important sensors-regulators of cellular responses to environmental stresses, such as those encountered in the host. This work demonstrates that the response by the two-component system TctD-TctE to the presence of citric acid has a role in biofilm formation, aminoglycoside susceptibility, and growth in P. aeruginosa.The two-component system TctD-TctE is important for regulating the uptake of tricarboxylic acids in Pseudomonas aeruginosa. TctD-TctE accomplishes this through derepression of the gene opdH, which encodes a tricarboxylic acid-specific porin. Previous work from our lab revealed that TctD-TctE in P. aeruginosa also has a role in resistance to aminoglycoside antibiotics. The aim of this study was to further characterize the role of TctD-TctE in P. aeruginosa in the presence of citric acid. Here it was found that deletion of P. aeruginosa PA14 TctD-TctE (ΔtctED) resulted in a 4-fold decrease in the biofilm bactericidal concentrations of the aminoglycosides tobramycin and gentamicin when citric acid was present in nutrient media. Tobramycin accumulation assays demonstrated that deletion of TctD-TctE resulted in an increase in the amount of tobramycin retained in biofilm cells. The PA14 wild type responded to increasing concentrations of citric acid by producing less biofilm. In contrast, the amount of ΔtctED mutant biofilm formation remained constant or enhanced. Furthermore, the ΔtctED strain was incapable of growing on citric acid as a sole carbon source and was highly reduced in its ability to grow in the presence of citric acid even when an additional carbon source was available. Use of phenotypic and genetic microarrays found that this growth deficiency of the ΔtctED mutant is unique to citric acid and that multiple metabolic genes are dysregulated. This work demonstrates that TctD-TctE in P. aeruginosa has a role in biofilm development that is dependent on citric acid and that is separate from the previously characterized involvement in resistance to antibiotics
Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in E. coli.
Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK
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