. . . . . . . . . . . . . . . . . . . . . . . . . . . . . R82F2 . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . N00-4067 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A CL3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N99-4390 . . . . . . . . G . . . . . C . . . . . C T . .     N00-4859 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EC6-484 . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . A EC2-044 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EC3-377 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The rpoS gene in E. coli K-12 MG1655 strain was Combretastatin A4 nmr used as the reference for comparison. The G-C transition at codon 33 in MG1655 results in a conversion

of glutamate to glutamine, while the G-T transversion in N99-4390 at codon 243 forms a stop codon resulting in a truncated RpoS protein. The other polymorphic sites are synonymous mutations. Selection of Suc++ mutants Our primary goal was to determine if loss of RpoS in VTEC strains can be selected by growing cells on non-preferred carbon sources. Mutants forming large colonies (Suc++) Torin 1 were readily isolated from seven of ten tested strains at a frequency of 10-8 per cell plated on succinate media, consistent with the frequencies obtained for laboratory strains [23]. Interestingly, strains CL3, R82F2 and N99-4390 grew uniformly well on succinate plates, much better than the other wild type strains, thus no Suc++ mutants were obtained. Similar results were obtained by growing cells on fumarate, another TCA cycle intermediate (data not shown), indicating that this selection is not limited to succinate alone. A group of 12 independent representative Suc++ mutants were selected from each strain to test their RpoS status using catalase plate assays [23]. Most of the Suc++ mutants (depending on parental strain background) were impaired in catalase production (Table 1). In E. coli, there are two catalases, HPI (KatG) and HPII (KatE), but only catalase HPII (KatE) is highly RpoS-dependent [23]. To confirm the plate assay results and to differentiate

between the 17-AAG chemical structure expression of KatE and KatG, we tested the catalase activity in the isolated catalase-negative Suc++ mutants from three representative VTEC strains EDL933, CL106, Ergoloid and EC3-377 using native-PAGE gels. As expected, all Suc++ mutants exhibited substantially reduced HPII catalase activity (Figure 1A). The higher expression of HPI in Suc++ mutants (Figure 1A) is not entirely unexpected. Low levels of HPII may lead to higher accumulation of intracellular hydrogen peroxide which can activate OxyR, the main regulator of HPI [32]. Figure 1 Catalase activity and RpoS expression in representative Suc ++ mutants of VTEC strains EDL933, CL106 and EC3-377. (A) Samples were separated by native PAGE and stained for catalase activity. Catalase HPI (KatG) and HPII (KatE) are indicated. (B) Expression of RpoS and RpoS-regulated AppA by Western analysis.