Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of E. coli subjected to random mutagenesis and subsequent analysis of SdiA variants affecting biofilm formation

ABSTRACT: The Escherichia coli quorum-sensing regulator, SdiA, belongs to the LuxR family of transcriptional regulators and is responsible for detecting signals from other bacteria. Previously we found that SdiA is necessary for E. coli to control its biofilm formation with indole just as SdiA is necessary for E. coli to alter its biofilm formation in the presence of N-acylhomoserine lactones (AHLs). Here we engineered SdiA by random mutagenesis to further control biofilm formation in the presence of indole and AHLs. After screening of 477?? mutants with indole and two AHLs (N-butyryl-DL-homoserine lactone, and N-(3-oxooctanoyl)-L-homoserine lactone, C6HSL), five SdiA variants were obtained that altered biofilm with and without signals of indole and AHLs. Two truncation variants (1E11 and 14C3) lacking the C-terminal DNA-binding domain of SdiA showed the reduction of biofilm formation by 5-fold and 10-fold in LB and LB glu, respectively. DNA microarrays show that the evolved SdiA (1E11) compared to wild-type SdiA influences indole synthesis genes, AI-2 uptake genes, acid-resistance genes, motility related genes, cold-shock protein genes, and several regulatory protein genes. Corroborating DNA microarrays, SdiA variants produced various amounts of indole which led to different survivals in low pH and influenced swimming motility and final cell density. Also, an AHL sensitive variant (2D10) 2-fold increased biofilm formation in the presence of C6HSL, while another variant (6B12) lowered biofilm formation in the presence of C6HSL. Hence, the results clearly showed that mutation of SdiA itself directly controls biofilm formation and SdiA variants could be further recognized by the inter-species signal AHLs. This is the first random protein engineering to control biofilm formation. Experiment Overall Design: For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL LB ( 1 mM IPTG + Cm30) in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures diluted that were 1:100. The cells were shaken at 250 rpm and 30C for 12 hours to form biofilms on the glass wool, and RNA was isolated from the the biofilm cells.

ORGANISM(S): Escherichia coli

SUBMITTER: Jintae Lee

PROVIDER: E-GEOD-11779 | 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).

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Transcription profiling of E. coli subjected to random mutagenesis and subsequent analysis of SdiA variants affecting biofilm formation

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