Protein-DNA complexes were resolved on 3% or 4% MetaPhor agarose gel. Primers used in gel mobility shift assays are listed in Additional file 2. Results Determination of new H-NS targets involved in the regulation
of glutamate-dependent acid resistance As H-NS strongly represses the glutamate-dependent acid stress response, there is a very low level of survival after acid stress in the FB8 wild-type context [6]. As a consequence, H-NS targets involved in this process are only expressed when hns is removed. To find AG-881 further H-NS-dependent intermediary actors of glutamate-dependent acid resistance, several of the H-NS induced targets, identified PRIMA-1MET in a previous transcriptome analysis [1] and related either to acid stress resistance or to information pathways, were deleted in an
hns-deficient strain. We looked for a decreased glutamate-dependent acid resistance, in comparison to that displayed in the parent hns-deficient strain. Different extent of decrease in resistance to acidic conditions was observed with deletion of several genes known to be related to acid stress response including dps (coding for the Dps protein – DNA-binding protein of starved cells), rpoS (coding for the RNA polymerase sigma-38 factor), yhiM (coding for an inner membrane protein), evgA (coding for a transcriptional activator), ydeP (coding for a putative anaerobic dehydrogenase) and ydeO (coding for a transcriptional regulator, which is a target of sRNA OxyS) (Table 2), suggesting a role in the H-NS-controlled glutamate-dependent acid resistance. Furthermore, a reduced resistance was also observed with genes, not previously associated
with acid stress, such as aslB (coding for an anaerobic sulfatase-maturating enzyme homolog) and hdfR (coding for the H-NS-dependent flhDC regulator) (Table 2). However, the single deletion of several genes including evgA, ydeP, ydeO and aslB in hns 3-Methyladenine cost background only slightly affected Pregnenolone the acid stress survival, suggesting their redundant function in this H-NS-dependent process. Table 2 Glutamate-dependent acid resistance of E. coli strains Strain (relevant genotype) Glutamate-dependent acid resistance (% survival) FB8 (wild-type) 0.1 BE1411 (hns::Sm) 51.7 BE2823 (hns::Sm ΔrcsB) < 0.001 BE2825 (hns::Sm ΔhdfR) 12.5 BE2826 (hns::Sm dps::Km) 20.1 BE2827 (hns::Sm rpoS) 27.5 BE2828 (hns::Sm yhiM::Km) 24.2 BE2829 (hns::Sm ΔevgA) 32.0 BE2831 (hns::Sm ydeP::Km) 35.6 BE2832 (hns::Sm ydeO::Km) 38.2 BE2830 (hns::Sm ΔaslB) 38.6 BE2837 (hns::Sm ΔadiY) 5.4 BE2939 (hns::Sm cadC1::Tn10) 58.1 Data are the mean values of two independent experiments that differed by less than 20%.