FIG. 1.

P1 is the Na⁺-sensitive promoter of nhaA. (A) Primer extension assay of nhaA transcription was conducted, as described in Materials and Methods, with total RNA (15 μg) isolated from cells grown exponentially for 2 h in LBK-BTP broth at pH 7.5, either in the presence or absence of 300 mM NaCl for induction. The same primer was used for the sequencing ladder, and the reaction mixtures were run on an 8% DNA sequencing gel. The cells used were TA15 (wild type) grown without addition of sodium (lane 1); TA15, sodium induced (lane 2); W3313–2S (nhaR1) cells grown without sodium (lane 3); RK20 (ΔnhaA), sodium added (lane 4); and OR100 (ΔnhaR), sodium added (lane 5). (B) Primer extension assay conducted as for panel A with RNA isolated from OR200 (ΔnhaA ΔnhaR) transformed with plasmid pGM42 (lane1) or pGM433 (lane 2). (C) Promoter elements of nhaA. Start sites (arrows), −10 and −35 sequences (underlined) are marked on the DNA sequence of nhaA. The putative third promoter elements of nhaA are indicated with question marks. Boxed area, binding site of NhaR (6). Shaded areas, nhaA sequences protected by NhaR from DNase I digestion (6).