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. 2009 Aug 3:10:349.
doi: 10.1186/1471-2164-10-349.

Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933

Tao Dong  1 , Herb E Schellhorn
Affiliations

Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933

Tao Dong et al. BMC Genomics. .

Abstract

Background: RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other gamma-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain.

Results: The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (twofold, P<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions.

Conclusion: Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains.

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Figures

Figure 1
Figure 1
Growth of EDL933 in LB media. Cultures were inoculated from overnight cultures to a starting OD600 = 0.0001 and incubated aerobically at 37°C at 200 rpm. RNA samples were isolated at OD600 = 0.3 and 1.5 as indicated. RpoS (ðS) protein levels were tested by Western blot analyses using monoclonal anti-RpoS antiserum as described in Materials and Methods. This experiment was performed in triplicate using independent isolates. Averaged values were used for construction of the growth curve.
Figure 2
Figure 2
Transcriptome profile of WT EDL933 and rpoS mutants. Scatterplot was used to examine the effects of RpoS on gene expression in exponential (A) and stationary (B) phase. Probe sets (including genes and intergenic regions) are outlined by two parallel lines into three different groups: probe sets expressed at least twofold higher in the WT (red), those expressed more than twofold higher in rpoS mutants (green), and those not differentially expressed (black). LI: log2-transformed expression intensity.
Figure 3
Figure 3
Effect of rpoS mutation on survival under stress. Stationary phase cultures were washed and diluted in 0.9% NaCl before exposure to low pH (2.5) (A), H2O2 (15 mM) (B), and heat (55°C) (C). WT, wild type EDL933; rpoS, rpoS mutant.
Figure 4
Figure 4
Effect of RpoS on expression of transporter genes. The mean expression ratio (MER/RpoS-dependence level) is given after each gene. Genes highlighted in red were expressed higher in wild type, those in blue were expressed higher in the rpoS mutant, and those in grey were not found to be significantly different (P > 0.05).
Figure 5
Figure 5
Metabolic pathways that are regulated by RpoS in stationary phase. Genes expressed higher in wild type are colored red and those expressed higher in rpoS mutants are blue. Genes whose differential expression was not significant (P > 0.05) are in black. The mean expression ratio (MER: WT/rpoS) is indicated after each gene.
Figure 6
Figure 6
Western blot analysis of Tir and EspA expression in wild type and rpoS mutants. Cultures were grown aerobically at 37°C in LB media supplemented with 44 mM NaHCO3 to OD600 = 1.5 or in DMEM media in 5% CO2 (two known LEE-induction conditions). Cell pellets were resuspended in SDS loading buffer and boiled for 5 min. Resultant cell extracts were resolved on a 10% SDS-PAGE gel. Proteins were transferred to a PVDF membrane by electrophoresis, followed by incubation of the membrane with anti-Tir or anti-EspA specific antibody. Signals were detected using ECL solution and Hyperfilm-ECL film (Amersham).
Figure 7
Figure 7
RpoS-regulation of genes required for de novo biosynthesis of purine nucleotides pathway I in stationary phase. RpoS-dependence (MER) is indicated in parentheses. A negative value (-) denotes RpoS-negative regulation. The pathway map is adapted from the EcoCyc database. Genes that were significantly differentially expressed (P < 0.05) are highlighted in bold.
Figure 8
Figure 8
Expression of FliA in WT and rpoS mutants of EDL933 in LB. Western blot analyses of the expression of the flagella sigma factor FliA were performed using monoclonal antibody to FliA as described in Material and Methods. To confirm equal protein loading, another protein gel run in parallel was stained by Coomassie blue R250.
Figure 9
Figure 9
Confirmation of microarray data using qPCR. RpoS dependence is represented by the mean expression ratio (WT/rpoS).
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