Project description:Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyper swarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. Swarming inhibition did not mediate rhamnolipid production, which regulates swarming motility in P. aeruginosa. Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and pyochelin, flagellar, and pili synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.
Project description:Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyper swarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. Swarming inhibition did not mediate rhamnolipid production, which regulates swarming motility in P. aeruginosa. Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and pyochelin, flagellar, and pili synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa. In total 4 samples: gene expressions of P. aeruginosa with (2 samples) or without (2 samples) 1-naphthol
Project description:Abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen found ubiquitously in the environment. Responsible for considerable human morbidity and mortality, particularly in nosocomial infections and individuals with cystic fibrosis, P. aeruginosa can adapt to surface growth by undergoing swarming motility. In P. aeruginosa, swarming motility is a rapid multicellular movement that occurs on soft surfaces of appropriate viscosity with amino acids as a nitrogen source. Here we tested the small synthetic host defense peptide, innate defense regulator 1018, and found that it inhibited swarming motility at concentrations as low as 0.75 μg/ml, well below the MIC for planktonic cells. A screen of the PA14 transposon insertion mutant library revealed twenty-nine mutants that demonstrated partial tolerance to 1018 under swarming conditions. Two of these mutants, in the genes anr and rhlB (a regulator of anaerobic metabolism and protein involved in rhamnolipid production, respectively), were complemented to restore susceptibility to 1018. RNA-Seq of peptide-treated cells under swarming conditions revealed the dysregulation of 1,190 genes compared to the untreated swarm front, and 67% of these genes were similarly dysregulated at the untreated swarm centre. In contrast, expression of 70 Anr-regulated genes was upregulated by peptide treatment, and 45 genes showed differential or opposite regulation of expression in peptide-treated and swarm centre cells. Many transcriptional regulators required for swarming were dysregulated in peptide-treated cells, indicative of a mechanism by which 1018 may inhibit swarming motility. Overall, this study illustrates a use for peptide 1018 in inhibiting swarming surface motility, an important bacterial adaptation.
Project description:Purpose: Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date post-transcriptional regulation has not been extensively investigated. Small non-coding RNAs (sRNA) are hypothesized to play post-transcriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. Overexpressing these revealed one sRNA, PA0805.1, that when cloned, transformed into PAO1 WT and overexpressed led to broad phenotypic changes. Methods: The sRNA PA0805.1 was cloned and overexpressed in PAO1 WT. Phenotypic screens including motility, cytotoxicity and adherence assays were performed. RNA-Seq and proteomics were performed under swarming conditions compared to the empty vector strain to discover which genes were influenced by the overexpression of PA0805.1 Results: Phenotypic screens revealed that PA0805.1 resulted in reduced swarming, swimming and twitching motility, as well as increased adherence, cytotoxicity, and tobramycin resistance. A deletion mutant ∆PA0805.1 was supersusceptible to tobramycin under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty vector strain by RNA-Seq and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles were revealed, encompassing 1121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD. Conclusions: The sRNA PA0805.1 appears to regulate diverse bacterial lifestyles, most likely through a regulatory cascade.
Project description:Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues and is required for growth in many organisms. Here we show that Bacillus subtilis EF-P also alleviated ribosome pausing at sequences encoding tandem prolines, and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.
Project description:Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues and is required for growth in many organisms. Here we show that Bacillus subtilis EF-P also alleviated ribosome pausing at sequences encoding tandem prolines, and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.
Project description:Swarming motility by the urinary tract pathogen Proteus mirabilis has been a long-studied, but little understood phenomenon. On agar, a P. mirabilis colony grows outward in a bullseye pattern formed by consecutive waves of rapid swarming followed by consolidation into shorter cells. To examine differential gene expression in these growth phases, a microarray, constructed based on the completed genome sequence and annotation, was undertaken. RNA from 1) broth-cultured, or 2) swarming cells was extracted to assess transcription during each of these growth states.