coli and the only abundant protein whose level was altered in response to H2O2, we decided to investigate the influence of flagellin on the survival of the ΔarcA mutant E. coli in the presence of H2O2. To determine if the higher protein levels of flagellin in

the ΔarcA mutant E. coli was due to higher levels of mRNA, we examined the expression of the fliC transcripts by Real-Time Reverse Transcriptase PCR analysis (RT-PCR). RNA was prepared from the wild type and ΔarcA mutant E. coli before and after exposure to H2O2, and subjected to RT-PCR analysis. CP-690550 nmr Similar to protein levels, the ΔarcA mutant E. coli had higher levels of fliC mRNA than the wild type E. coli both TH-302 in vivo constitutively and after exposure to H2O2. In both strains, H2O2exposure reduced the fliC mRNA level progressively (Figure 5). The difference in fliC mRNA levels between the wild

type and ΔarcA mutant E. coli decreased with longer exposure periods and no difference could be detected by 120 minutes of exposure (Figure 5). To determine if ArcA directly regulates fliC expression, we expressed and purified recombinant ArcA from aerobic cultures of E. coli and carried out electrophoretic mobility shift assay of the fliC upstream sequence. No specific binding was detected (data not shown). Figure 5 Expression of fliC messenger RNA is regulated in response to H 2 O 2 exposure. Expression of fliC messenger RNA is regulated in response to H2O2 exposure. The wild type and the ΔarcA mutant E. coli was exposed to H2O2, and the fliC messenger RNA in wild type (diamond) and the ΔarcA mutant E. coli (square) was quantified by Real-time Reverse Transcriptase PCR after various periods of SHP099 concentration exposure. The level of the fliC messenger RNA in the unexposed

wild type E. coli (at 0 hour) learn more was arbitrarily set as 1, and levels of fliC messenger RNA in other samples were expressed as relative expression levels and plotted against the exposure time. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation. Deletion of flagellin increased the survival of the ΔarcA mutant E. coli Flagellin is one of the most abundant proteins in E. coli, and we have shown that its level was higher in the ΔarcA mutant E. coli both constitutively and upon H2O2 exposure (Figure 4 and Table 2). We reasoned that expressing an abundant protein such as flagellin at a higher level might be a burden to the ΔarcA mutant E. coli, especially under stress conditions such as those caused by H2O2. We hypothesize that a deletion of flagellin encoded by fliC may facilitate the survival of the ΔarcA mutant E. coli exposed to H2O2. To test this hypothesis, we generated a non-polar ΔfliC mutant and an ΔarcA/ΔfliC double mutant E. coli. The non-polar deletion of fliC itself had no obvious effect on the survival of E. coli in the presence of H2O2 (Figure 6).