ABSTRACT: Comparison of Pseudomonas aeruginosa planktonic cultures and bacterial biofilms grown under variations in growth media to demonstrate carbon catabolite repression phenomena using bottom-up proteomics
Project description:P. aeruginosa isolates were grown in LB broth media. The bacterial media was then digested after incubation for 24 hours and analyzed to identify bacterial proteins related to beta-lactam drug resistance. Bottom-up proteomics analysis was performed.
Project description:GCC was used to determine the response of P. aeruginosa nucleoid structure to different environmental conditions. Overall design: Pseudomonas aeruginosa strains were stored (-80°C) and cultured (37°C) on/in mineral salts medium (MM) with either 0.05% (w/v) or 0.2% (w/v) NH4Cl. NH4Cl was used as nitrogen source. The cells were cultivated with 2% (w/v) sodium gluconate. For GCC analyses, P. aeruginosa strains were recovered from -80°C on LB-plates for 24 hours prior to starter culture inoculation. Starter cultures were cultured (37°C, 160rpm, O/N) in MM medium (gluconate 2% w/v) with or without nitrogen, as indicated. Test cultures were inoculated, from the starter cultures, in MM media gluconate (2% w/v) with or without nitrogen, grown (37°C, 160rpm) and harvested at an OD600 = 0.6-8 (log phase) or OD600 = 4-5 (stationary phase).
Project description:The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). We identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. Overall design: mvfR mutants of PA14 and control PA14 cells were grown to OD 2.5, a point where QS is turned on, and their expression profiles were compared. AA analogs 4CABA and 6CABA were added directly to the culture media of PA14 cells.
Project description:As production of virulence factors by Pseudomonas aeruginosa is influenced by the host environment, we examined the effect of 10% adult bovine serum (ABS) on the global transcription with P. aeruginoas PAO1 at different phases of growth. At early exponential phase (4 h), serum significantly enhanced expression of 138 genes, most of which are repressed by iron and carry binding sequences for the ferric uptake regulator or the Fur-regulated extracytoplasmic function sigma factor PvdS. The expression of another 40 genes was reduced at 4h. However, the expression of only a few genes was significantly altered (reduced) at the early stationary phase of growth (8 h). Transcriptional fusion analyses confirmed serum enhances the expression of toxA, regA, and their regulatory genes regA and pvdS, yet does not interfere with the repression of these genes by iron. The P. aeruginosa strain PAO1 was grown in the iron-limited medium TSB-DC with or without 10% (v/v) ABS. The presence of 10% ABS reduced PAO1 growth, but despite this reduction, the cultures reached early exponential phase at 4 h and early stationary phase at 8 h in both media. Accordingly, we determined the PAO1 transcriptome in both media at these two specific time points. To standardize the comprison, we adjusted the cultures to the same optical density at 600 nm (biomass) by diluting with fresh TSB-DC PAO1 grown in TSB-DC to the same OD as PAO1 grown in TSB-DC/ABS. We obtained the transcriptome profile of 5,552 genes representing the complete P. aeruginosa PAO1 genome.
Project description:This SuperSeries is composed of the following subset Series: GSE33241: Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa [BSM] GSE33244: Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa [LB] Refer to individual Series
Project description:In this experiment the transcriptional response of the opportunistic human pathogen Pseudomonas aeruginosa to sublethal concentrations of NaClO was investigated. To this aim, four independent cultures of P. aeruginosa PAO1 grown in minimal medium BM2 were treated with NaClO (2 ug/ml) for 1 h at 37 C followed by RNA extraction and microarray analysis. Untreated cultures served as controls.
Project description:Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further, this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo, and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat, Pseudomonas aeruginosa. Overall, P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium, Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA), involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed, among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth, in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments, in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions, were hypothesized to be at the origin of these observations. Collectively, our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen, results that are of importance for infectious disease risk assessment and prevention, both during spaceflight missions and in the clinic. Overall design: This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end, P. aeruginosa cultures were grown in space, and the expression profile was compared with ground control samples (both in biological triplicate). Two RWV samples also examined (did not re-analyze them, only compared the outputs).
Project description:A knockout of nrtR gene (PA4916) of Pseudomonas aeruginosa leads to the global changes in metabolic profile of the pathogen to its complete avirulence. Bacterial cells of TBCF10839 and TBCF10839nrtR::Tn5 were grown in LB broth to late exponential phase at 37 oC
Project description:Oberhardt2008 - Genome-scale metabolic
network of Pseudomonas aeruginosa (iMO1056)
This model is described in the article:
network analysis of the opportunistic pathogen Pseudomonas
Oberhardt MA, Puchałka J, Fryer
KE, Martins dos Santos VA, Papin JA.
J. Bacteriol. 2008 Apr; 190(8):
Pseudomonas aeruginosa is a major life-threatening
opportunistic pathogen that commonly infects immunocompromised
patients. This bacterium owes its success as a pathogen largely
to its metabolic versatility and flexibility. A thorough
understanding of P. aeruginosa's metabolism is thus pivotal for
the design of effective intervention strategies. Here we aim to
provide, through systems analysis, a basis for the
characterization of the genome-scale properties of this
pathogen's versatile metabolic network. To this end, we
reconstructed a genome-scale metabolic network of Pseudomonas
aeruginosa PAO1. This reconstruction accounts for 1,056 genes
(19% of the genome), 1,030 proteins, and 883 reactions. Flux
balance analysis was used to identify key features of P.
aeruginosa metabolism, such as growth yield, under defined
conditions and with defined knowledge gaps within the network.
BIOLOG substrate oxidation data were used in model expansion,
and a genome-scale transposon knockout set was compared against
in silico knockout predictions to validate the model.
Ultimately, this genome-scale model provides a basic modeling
framework with which to explore the metabolism of P. aeruginosa
in the context of its environmental and genetic constraints,
thereby contributing to a more thorough understanding of the
genotype-phenotype relationships in this resourceful and
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Project description:To investigate the gene expression profile of pellicle cells of Pseudomonas aeruginosa, microarray analysis was performed. Transcriptome profiles of pellicle cells and planktonic cells grown in LB medium were determined by Affymetrix GeneChip. Gene expression pattern that is specific to pellicle cells was evaluated by comparing the data set with that of planktonic cells. Overall design: Pseudomonas aeruginosa wild type (PAO1ut) strain was cultivated aerobically in LB in Erlenmeyer flasks under static or shaking conditions, and total RNAs were extracted at 24 hours (static culture) and early stationary phase (OD600 = 1.4, shaking culture). The experiment was performed in duplicate independent cultures.