Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of P. aeruginosa PA14-WT vs pyrF mutant vs pyrF mutant w/uracil

ABSTRACT: Comparative transcriptome analyses of P. aeruginosa PA14 pyrF mutant with PA14 wild type, and with PA14 pyrF mutant with 1 mM uracil supplement and PA14 wild type with 10 mM uracil in biofilm cells. All samples were cultured in LB with glass wool at 37C for 7h. Experiment Overall Design: Strains: P. aeruginosa PA14 wild type, PA14 pyrF mutant Experiment Overall Design: Medium: LB for PA14 wild type and PA14 pyrF mutant, LB with 1 mM uracil for PA14 pyrF mutant, LB with 10 mM uracil for PA14 wild type Experiment Overall Design: Cell type: Biofilm cells grown on glass wool Experiment Overall Design: Time: 7 h Experiment Overall Design: Temperature: 37C

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Akihiro Ueda

PROVIDER: E-GEOD-9592 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have shown the quorum-sensing signals acylhomoserine lactones (AHLs), autoinducer-2 (AI-2), and indole influence the biofilm formation of Escherichia coli. Here, we investigate how the environment, i.e., temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30ï°C (186 genes) than at 37ï°C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25ï°C and 30ï°C than at 37ï°C, and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30ï°C compared to 37ï°C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD, pyrF, and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30ï°C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37ï°C, indole more significantly decreased flagella-related qseB, flhD, and fliA promoter activity, enhanced antibiotic resistance, and inhibited cell division at 30ï°C. In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37ï°C (63 genes) than at 30ï°C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37ï°C (but not at 30ï°C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37oC but not at 30oC. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined. Experiment Overall Design: We performed seven sets of microarray experiments in LB medium (Table 2): (i) biofilm cells of the BW25113 wild-type strain grown for 7 h at 30Â°C vs. 37Â°C to determine the effect of temperature on E. coli biofilm formation (initial absorbance of 0.05), (ii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 30Â°C to determine the effect of temperature on indole signaling in E. coli (initial absorbance of 0.05), (iii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 37Â°C to determine the effect of temperature on indole signaling in E. coli, (iv) suspension cells of the wild-type strain vs. the sdiA mutant at an absorbance of 4.0 at 600 nm at 30Â°C (since production of indole takes place primarily in the stationary phase) to discern the genes that SdiA controls (initial absorbance of 0.05), (v) biofilm cells of the sdiA mutant grown for 7 h with 1 mM indole vs. no indole at 30Â°C to determine gene expression in response to indole in the absence of SdiA, (vi) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 30Â°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli (initial absorbance of 0.5), and (vii) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 37Â°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli. Ten g of glass wool (Corning Glass Works, Corning, N.Y.) was used to form large amounts of biofilms (Ren et al., 2004a) in 250 mL LB in 1 L Erlenmeyer shake flasks. RNA was isolated from the suspension and biofilm cells as described previously (Ren et al., 2004b).

Project description:Pseudomonas aeruginosa causes many biofilm infections, and the rugose small-colony variants (RSCVs) of this bacterium are important for infection. We found here that mutation in glyA2 (PA2444), which encodes a serine hydroxymethyltransferase, leads to the RSCV phenotype. The phenotype is related to increased c-di-GMP concentrations and depends on an active Wsp chemosensory system, including a diguanylate cyclase WspR. By characterizing the PA2444 enzyme both in vivo and in vitro, we determined the physiological function of PA2444 and related the enzyme to S-adenosylmethionine concentrations and the methylation of a membrane bound methyl-accepting chemotaxis protein WspA. A transcriptome analysis revealed the possible relationship between PA2444 and the redox state of the cells. Hence, we provide a mechanism for how an enzyme of central metabolism controls the community behavior of the bacterium. The P. aeruginosa genome array (Affymetrix, P/N 510596) was used to investigate differential gene expression in biofilm cells between PA14 and the PA2444 mutant. Biofilm cells were harvested from 10 g of glass wool after incubation for 7 h in LB with shaking at 250 rpm, and RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) using a bead beater (Biospec, Bartlesville, OK ) with RNAlater buffer (Applied Biosystems, Foster City, CA) to stabilize the RNA. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 13 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%) and when the expression ratio was higher than the standard deviation for the whole microarrays (1.8 for PA14 wild-type and PA2444).

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Transcription profiling of P. aeruginosa PA14-WT vs pyrF mutant vs pyrF mutant w/uracil

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