Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE

Abstract

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the -10 element of gadA promoter and is involved in repression of this operon.

Figure 1.

Synergistic regulation of in vitro transcription initiation at the gadA promoter by GadE and RcsB. The gadA promoter-containing linear template was pre-incubated as indicated with RNA polymerase alone (lane 1), plus 760 nM MBP-GadE (lane 2), plus 3, 30, 300 or 3000 nM (His)₆-RcsB (lanes 3–6, respectively), or plus MBP-GadE and (His)₆-RcsB at the same concentrations (lanes 7–10, respectively). The figure shows a representative autoradiogram.

Figure 2.

Identification of MBP-GadE and (His)₆-RcsB binding sites in the gadA regulatory region. (A) Nucleotide sequences of the gadA and gadB promoter regions. Putative –35 and –10 elements of the gad promoters are boxed. The GAD and putative RcsB boxes are shaded. +1 denotes the transcription start point and numbers below indicate distance relative to this point. (B) DNase I footprinting assays were performed with 20 µM of MBP-GadE and/or 40 µM of (His)₆-RcsB on the non-template strand. Protected regions and corresponding sequences are indicated above the autoradiogram. A DNase I hypersensitive site is indicated by an asterisk. The positions relative to the +1 start of transcription are shown below. No protein (lane 1), 20 µM MBP-GadE (lane 2), 40 µM (His)₆-RcsB (lane 3), 20 µM MBP-GadE and 40 µM (His)₆-RcsB (lane 4).

Figure 3.

Binding of MalE-GadE and (His)₆-RcsB to the gadA promoter region. (A) EMSA with gadA promoter region-templates (from –101 to +24 relative to the +1start of transcription). No protein (lane 1), 10 µM MBP-GadE (lane 2), 0.1, 1 and 10 µM (His)₆-RcsB plus 0.1 µM MBP-GadE (lanes 6–8) or plus 1 µM MBP-GadE (lanes 9–11), plus 10 µM MBP-GadE (lanes 12–14). (B) EMSA using protein from wt or rcs mutants with gadA promoter region templates. Proteins were purified from either a wild-type strain (lanes 3 and 4) or a ΔrcsA ΔrcsCDB ΔgadE strain (lanes 2, 5 and 6). No protein (lane 1), 1 µM (His)₆-RcsB (lane 2), 3 µM MBP-GadE (lanes 3 and 5), 3 µM MBP-GadE and 1 µM (His)₆-RcsB (lanes 4 and 6).

Figure 4.

RcsB and GadE bind GAD box as a heterodimer. EMSA was performed as described in Figure 3 using the following proteins at 1 µM: (His)₆-RcsB, MBP-RcsB, MBP-GadE and (His)₆-GadE. Schematic representations of complexes II obtained are presented on the left-hand side.

Figure 5.

Effects of GAD or RcsB box mutations on RcsB and GadE binding. (A) Point mutations introduced into the GAD and RcsB boxes. Conserved bases are shown in bold. (B) EMSA of gadA promoter fragment mutants in the presence of (His)₆-RcsB (5 µM), and/or MBP-GadE (4 µM). NS: non-specific DNA.

Figure 6.

Effects of RcsB or GAD box mutations on the transcriptional regulation of gadA in vivo. (A) β-Galactosidase activities of transcriptional gadA-lacZ fusions (either wild-type or M1 mutant promoter) with or without overexpressed (His)₆-GadE or (His)₆-RcsB. Strains carrying gadAp-lacZ and either pHGadE or pHRcsB plasmids with (white bars) or without (gray bars) 0.5 mM IPTG added for 1 h. (B) Same experiment as in A for gadAp_wt-lacZ and gadAp_M3-lacZ fusions with or without induction of RcsB synthesis. Activities were measured 30, 60 and 120 min after IPTG addition.

Figure 7.

Regulatory model of gadA transcription. Schematic representation of gadA regulatory region with the GadXW (33), GAD and RcsB boxes (gray boxes). The H-NS binding sites responsible for gadA repression are represented as dotted line (37). Direct regulation is shown as a continuous arrow. Repression of gadE transcription by RcsB, in which a direct effect has not been confirmed, is shown as a dotted arrow. This putative feed-forward loop could coordinate gadA expression.