70 adiC 11.62 nd Nd nd 1.41 Lysine-dependent specific pathway cadC 4.62 5.77 6.38 nd nd General acid stress resistance pathway hdeA 1 32.37 nd Nd 41.20 6.55 hdeD 18.96 nd Nd 17.57 5.89 adiY 5.08 5.00 5.00 nd nd nd: non-determined. 1: Since several genes are organized in operon and/or are highly homologous to each other, results obtained with gadA also corresponds to gadBC; with gltD to gltB; with hdeA to hdeB; with dctR to slp. Quantitative RT-PCR were performed on total RNA isolated from exponential growth phase cultures. Standard deviations were less than 20% of the mean. Identification of the target genes for major regulators To decipher the

regulatory GPCR Compound Library hierarchy in acid stress resistance involving several new H-NS controlled regulators, the mRNA level of target genes was buy Ulixertinib compared between wild-type and hns, hns rcsB, hns gadE, hns hdfR, hns adiY mutant strains, using real-time quantitative RT-PCR (Table 4). In particular, we compared the expression ratio between a double mutant and

the wild-type strain with that for hns-deficient and the wild-type strain. H-NS having negative effect on target genes, these genes are strongly derepressed in hns mutant in comparison with wild-type strain. If this strong H-NS repressive effect is abolished in the absence of a regulator negatively controlled by H-NS, we can conclude that this deleted regulator has positive effect on target gene expression and may be an intermediary actor in H-NS-dependent control for this target, as previously shown [6]. It was found that RcsB and GadE upregulate, at the similar level, newly identified genes involved in acid stress resistance pathways dependent on glutamate (yhiM and aslB), but these two regulators did not affect the expression of regulatory genes, cadC and adiY (Table 4). Neither RcsB nor GadE controlled hdfR regulatory gene expression (data not shown), suggesting that the hdfR is not

the target of RcsB-P/GadE complex. We found that HdfR controlled only the expression of aslB and gltBD in the glutamate-dependent acid stress resistance regulon (Table 4). As expected, AdiY strongly affected adiA and adiC expression, and also the expression of some genes related to the glutamate specific pathway (aslB, gadA, gadBC, gltBD, and slp-dctR) and to general acid resistance (hdeAB and hdeD) (Table 4). These results demonstrated a multiple control of several target genes involving 2-hydroxyphytanoyl-CoA lyase different regulators acting independently from each other. Identification of the new targets directly controlled by RcsB-P/GadE complex Gel mobility shift assays were performed with a mixture of purified RcsBD56E and GadE proteins to know whether the regulatory complex directly controlled yhiM and aslB. It was established that the RcsBD56E/GadE regulatory complex binds to the promoter regions of the two genes (Figure 1A), demonstrating the direct control by the RcsB-P/GadE complex. Figure 1 Gel mobility shift assays with GadE/RcsB D56E complex, HdfR and AdiY. A.