Decrypting The H-NS-dependent Regulatory Cascade Of Acid Stress ...

Bacterial strains and plasmids

Bacterial strains and plasmids used in this study are listed in Table 1. Mutants were constructed by replacing the entire gene of interest with an antibiotic cassette using the CF10230 strain, as previously described [17]. These mutations, as well as their Miki and Keio collection counterparts from NBRP (NIG, Japan): E. coli[18, 19] were subsequently transduced into FB8 hns::Sm derivative strains, using P1vir phage. When required, antibiotics were added: ampicillin (100 μg ml-1), streptomycin (10 μg ml-1), kanamycin (40 μg ml-1), tetracycline (15 μg ml-1).

Resistance to low pH

The experiment was performed at least twice, as previously described [6].

RNA preparation and Real-time quantitative RT-PCR

The experiment was performed twice, as previously described [6]. Primers used in real-time quantitative RT-PCR experiments are listed in Additional file 1.

Protein purification

H-NS-His6 was purified as previously described [20]. Recombinant proteins HdfR-His6, His6-RcsBD56E, GadE-His6 and Strep-AdiY were purified as previously described [6].

Gel mobility shift assays

Gel shift assays were performed with 0.1 ng [γ32P]-labelled probe DNA with purified HdfR-His6, His6-RcsBD56E (mimicking phosphorylated and activated RcsB), GadE-His6 and Strep-AdiY proteins as previously described [6, 10]. For competitive gel mobility shift assays with purified H-NS protein 100-200 ng PCR fragments of target promoter regions and 270-200 ng of competitor DNA fragments, obtained by digestion of pBR322 plasmid with Taq I and Ssp I restriction enzymes, were incubated for 15 min at room temperature with H-NS in the previously described reaction mixture [21]. 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.