However, in the human intestine, low oxygen tension permits E. coli to grow by fermentation or respiration using an alternative

electron acceptor. As nitrate is readily available in the human intestine (14 μmol/kg [36]) and can be readily utilized by intestinal bacterial flora including E. coli [37, 38] we examined succinate selection using this alternate electron receptor. Interestingly, host nitrate synthesis can be stimulated in response to infections caused by gastroenteric pathogens [38]. To test if selection for loss of RpoS can occur under low oxygen conditions, cultures were grown in anaerobic jars (see Methods). GS-1101 cell line We first compared the anaerobic growth of wild type and aerobically-selected Suc++ www.selleckchem.com/products/rg-7112.html mutants on glucose and succinate plates. Wild type EDL933 grew as well as an isogenic rpoS knockout Y27632 mutant and derivative Suc++ mutants on glucose, while the rpoS and Suc++ mutants grew much better than wild type on succinate under both aerobic and anaerobic conditions (Figure 2). The growth of Suc++ mutants was similar to that of the

control rpoS null mutant under all conditions tested. Figure 2 Growth of EDL933 and derivative Suc ++ mutants on M9 glucose (Glu) and succinate (Suc) media. Colony size (diameter) was determined under a light microscope at 40× magnification. All VTEC strains were then tested for selection on succinate under anaerobic conditions. As under aerobic conditions, Suc++ mutants could be selected from all tested strains, except for CL3, R82F2 and N99-4390. Most (87%) of the Suc++ had

reduced catalase activity. We sequenced the rpoS region of 15 Suc++ mutants isolated Aspartate from EDL933 and found mutations in rpoS, resulting in impaired RpoS function, in 13 mutants while the rpoS gene in the other two Suc++ mutants remained unchanged (data not shown). Expression of virulence-related traits, RDAR and cell adherence Mutations in rpoS may affect virulence factor expression in pathogenic strains [39, 40]. To test this, we examined two virulence-related traits, the RDAR morphotype and cell adherence. Extracellular components, such as curli fimbriae and cellulose, are correlated with biofilm formation and virulence in Salmonella sp. and E. coli strains [41–43]. The expression of curli and cellulose can be visualized by staining with Congo Red dye to produce a red, dry and rough morphotype (RDAR) [43, 44]. Biosynthesis of both curli and cellulose is positively regulated by RpoS through a transcriptional regulator CsgD in E. coli K12 [45, 46]. However, to our knowledge, the role of RpoS in expression of RDAR has not been previously tested in pathogenic E. coli isolates. Wild type EDL933 exhibited a more pronounced RDAR morphotype than an isogenic rpoS null deletion mutant and Suc++ mutants (Figure 3A), suggesting that RpoS is important for RDAR development.