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). Table 1 Bacterial strains and plasmids
used in this study Strain or plasmid Genotype or description Reference or source Strains JD21162 KP7600 (F- lacIQ lacZdeltaM15 galK2 galT22 lambda- in (rrnD-rrnE)1, W3110 derivative) ydeP ::Km [19] JD24946 KP7600 (F- lacIQ lacZdeltaM15 galK2 galT22 lambda- in (rrnD-rrnE)1, W3110 derivative) yhiM ::Km [19] JD25275 KP7600 (F- lacIQ lacZdeltaM15 galK2 galT22 lambda- in (rrnD-rrnE)1, W3110 derivative) hdeA ::Km [19] JD26576 KP7600 (F- lacIQ lacZdeltaM15 galK2 galT22 lambda- in (rrnD-rrnE)1, W3110 S63845 molecular weight derivative) ydeO::Km [19] JD27509 KP7600 (F- lacIQ lacZdeltaM15 galK2 galT22 lambda- in (rrnD-rrnE)1, W3110 derivative) dps ::Km [19] JW5594 BW25113 (rrnB ΔlacZ4787 HsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1) ΔaslB ::Km [18] JW2366 BW25113 (rrnB ΔlacZ4787 HsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1) ΔevgA ::Km [18] EP247 W3110 cadC1::Tn10 [41] FB8 Wild type [42] BE1411 FB8 hns::Sm [43] BE2823 FB8 hns::Sm ΔrcsB ::Km [6] BE2825 FB8 hns::Sm ΔhdfR ::Tet This study
BE2826 FB8 hns::Sm dps ::Km FB8 hns::Sm × P1 JD27509 BE2827 FB8 hns::Sm rpoS 359 click here ::Km This study BE2828 FB8 hns::Sm yhiM ::Km FB8 hns::Sm × P1 JD24946 BE2829 FB8 hns::Sm ΔevgA ::Km FB8 hns::Sm × P1 JW2366 BE2830 FB8 hns::Sm ΔaslB ::Km FB8 hns::Sm × P1 JW3772 BE2831 FB8 hns::Sm ydeP ::Km FB8 hns::Sm × P1 JD21162 BE2832 FB8 hns::Sm ydeO ::Km FB8 hns::Sm × P1 JD26576 BE2836 FB8 hns::Sm ΔhdeA ::Km FB8 hns::Sm × P1 JD25275 BE2837 FB8 hns::Sm ΔadiY ::Tet This study BE2939 FB8 hns::Sm cadC1::Tn10
FB8 hns::Sm × P1 EP247 Plasmids pDIA640 pet22b ::hdfR with C terminal His tag This study pDIA642 pet16b ::rcsB D56E with N terminal His tag [6] pDIA645 pet22b ::gadE with C terminal His Phosphatidylinositol diacylglycerol-lyase tag [6] pDIA646 pet16b ::adiY with N terminal strep tag This study 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 selleck kinase inhibitor phosphorylated and activated RcsB), GadE-His6 and Strep-AdiY proteins as previously described [6, 10].