Collaborative regulation of Escherichia coli glutamate-dependent acid resistance by two AraC-like regulators, GadX and GadW (YhiW)

Abstract

An important feature of Escherichia coli pathogenesis is an ability to withstand extremely acidic environments of pH 2 or lower. This acid resistance property contributes to the low infectious dose of pathogenic E. coli species. One very efficient E. coli acid resistance system encompasses two isoforms of glutamate decarboxylase (gadA and gadB) and a putative glutamate:gamma-amino butyric acid (GABA) antiporter (gadC). The system is subject to complex controls that vary with growth media, growth phase, and growth pH. Previous work has revealed that the system is controlled by two sigma factors, two negative regulators (cyclic AMP receptor protein [CRP] and H-NS), and an AraC-like regulator called GadX. Earlier evidence suggested that the GadX protein acts both as a positive and negative regulator of the gadA and gadBC genes depending on environmental conditions. New data clarify this finding, revealing a collaborative regulation between GadX and another AraC-like regulator called GadW (previously YhiW). GadX and GadW are DNA binding proteins that form homodimers in vivo and are 42% homologous to each other. GadX activates expression of gadA and gadBC at any pH, while GadW inhibits GadX-dependent activation. Regulation of gadA and gadBC by either regulator requires an upstream, 20-bp GAD box sequence. Northern blot analysis further indicates that GadW represses expression of gadX. The results suggest a control circuit whereby GadW interacts with both the gadA and gadX promoters. GadW clearly represses gadX and, in situations where GadX is missing, activates gadA and gadBC. GadX, however, activates only gadA and gadBC expression. CRP also represses gadX expression. It does this primarily by repressing production of sigma S, the sigma factor responsible for gadX expression. In fact, the acid induction of gadA and gadBC observed when rich-medium cultures enter stationary phase corresponds to the acid induction of sigma S production. These complex control circuits impose tight rein over expression of the gadA and gadBC system yet provide flexibility for inducing acid resistance under many conditions that presage acid stress.

FIG. 1.

RT-PCR analysis of gadX and gadW transcripts. Inset, left, represents the gadX and -W genes and the location of primers used for RT-PCR. gadX forward, oligo-340; gadX reverse, oligo-341; gadW forward, oligo-414; gadW reverse, oligo-415; intergenic forward, oligo-458; and intergenic reverse, oligo-459. PCRs with and without RT (Reverse Tntp′ase) were run, and products were separated on 2% agarose gels. Lane 1, HindIII-cut lambda DNA; lane 2, 100-bp ladder; conditions for lanes 3 to 12 are shown above the gel. RNA used was from log-phase (pH 5.5), LB-grown EK227 cells. Similar results were obtained using RNA from cells grown at pH 8 and minimal-glucose-grown cells (data not shown).

FIG. 2.

Effects of GadX and GadW on pH-regulated gadA and gadBC expression in exponential- and stationary-phase cells grown in LB. Western blot analysis of extracts prepared from cultures grown to mid-log (OD₆₀₀ = 0.4) (A) or stationary (Stat) (OD₆₀₀ = 3.8) (B) phase in LB medium buffered to the pH values indicated. Strains and genotypes are indicated in the figure. Whole-cell proteins were extracted by boiling in SDS, and 5 μg of protein was loaded per sample on SDS-10% PAGE gels. Blots were probed with polyclonal anti-GadA and -B antibody.

FIG. 3.

Northern blot analysis of gadW effects on gadA and gadBC message. Cells were grown to log phase in buffered LB medium. RNA was extracted, and 5 μg was electrophoresed in formaldahyde-agarose gels and probed with a 1.4-kb gadA and -B probe that can detect gadAX, gadA, and gadBC transcripts. Strains and relevant genotypes are indicated in the figure.

FIG. 4.

Northern blot analysis of GadW effects on gadX message. Cells were grown to log phase in buffered LB medium. RNA was extracted; 5 μg was electrophoresed in formaldahyde-agarose gels and probed with an 827-bp radiolabled gadX probe that can detect gadAX and gadX transcripts. Strains and relevant genotypes are indicated in the figure. pH is given immediately over the blot.

FIG. 5.

GadW binds to the gadA and gadB promoters. EMSA conditions were described in Materials and Methods. Radiolabeled promoter fragments (5,000 cpm) were incubated with purified Trx-GadW-His₆ protein for 30 min at 25°C and were electrophoresed through a 5% nondenaturing polyacrylamide gel.

FIG. 6.

Effects of promoter deletions on GadX and GadW regulation of gadA. Cells were grown to exponential phase in LB medium buffered to pH 8 with 100 mM MOPS or to pH 5.5 with 100 mM MES and were examined for β-galactosidase activity (Miller units). (A) gadA-lacZ fusions; (B) gadA-lacZ fusions; and (C) gadA-lacZ fusions. The length of the gadA promoter region (in nucleotides) is given along top of panels. Wild type (WT), EF833, EF932, and EF931; ΔX, EF839, EF949, and EF948; ΔXW, EF933, EF935, and EF934; and ΔW, EF921, EF923, and EF922.

FIG. 7.

Western blot analysis of crp and rpoS effects on GAD, RpoS, and GadX protein levels. Cells were grown in LB medium with MES, pH 5.5, or LB medium with MOPS, pH 8, to log phase (A to C) or to stationary phase (D). Strains used and relevant genotypes are indicated in the figure.

FIG. 8.

Low-pH and growth phase induction of glutamate decarboxylase and RpoS in rich media. EK227 was grown to different optical densities in LB media buffered to pH 5.5 or 8. The inset illustrates the growth under both conditions measured as OD₆₀₀. Extracts were probed by Western blot analysis as described in the Fig. 2 legend. (A) Detection of RpoS; (B) detection of glutamate decarboxylase (GAD).

FIG. 9.

Effects of H-NS on gadA and gadBC expression. Cells were grown in complex, buffered LB media to exponential phase. Western blot analysis using anti-GadA and -B antibody was performed as described in the Fig. 2 legend. For panel A, all cells were gadA and gadBC⁺ gadX⁺ gadW⁺. For panel B, all cells lacked GadX, GadW, and CRP (hns⁺, lanes 1 and 3, EF865; hns mutant, lanes 2 and 4, EF929).

FIG. 10.

Model of the GadX and -W and CRP-RpoS control circuit. GadX activates expression from PgadA, while GadW inhibits expression (in the presence of GadX) from PgadA and PgadX. Transcription of the activator gene gadX is largely dependent on the RpoS sigma factor, tying expression of the system to various stress conditions that increase RpoS levels. Expression is also influenced by growth on carbohydrates through CRP, which dampens expression of RpoS, an effect more evident in LB-grown cells, where cAMP levels are high, than in cells grown on glucose, where cAMP levels are low. H-NS is also reported to inhibit gadX expression but appears capable of inhibiting gadA and gadBC expression independently of gadX control.