The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis

Abstract

AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

FIG. 1.

(A) Binding of purified AphB to various promoter fragments. For each promoter, the first fragment contains both the left (L) and right (R) dyad arms of the LysR motif shown below in panel B whereas the second fragment has only the right dyad arm. The first lane in each set has no protein added, the second lane has 160 nM (100 ng) AphB. The sequence of the AphB binding sites at the various promoters is shown in panel B compared to the site identified at the tcpPH promoter (23). Mismatches are shown in red.

FIG. 2.

Influence of a ΔaphB mutation on the expression of various promoter-lacZ fusions in V. cholerae. Strains were grown in AKI medium lacking bicarbonate statically for 3.5 h. The strains are as follows, from left to right: KSK2896 (cadC-lacZ), KSK2900 (ΔaphB), WL344 (VC0770-lacZ), WL346 (ΔaphB), GK1232 (nhaB-lacZ), GK1234 (ΔaphB), WL558 (cah-lacZ), WL562 (ΔaphB), GK1222 (clc-lacZ), and GK1224 (ΔaphB). wt, wild type.

FIG. 3.

Influence of AphB on the expression of a cadC-lacZ fusion in E. coli. Strains were grown aerobically in LB medium buffered to pH 7.0 with 100 mM Trizma, aerobically in LB medium buffered to pH 5.5 with 100 mM MES, or anaerobically in LB medium buffered to pH 7.0 with 100 mM Trizma. The first three bars represent the values obtained with empty vector pMF3, and the second three bars represent the values obtained with the AphB plasmid (pGKK263).

FIG. 4.

Influence of Fnr and ArcAB on the expression of a cadC-lacZ fusion in V. cholerae. Strains (WL547 [ΔcadC-lacZ] and WL972 [Δfnr ΔarcA]) were grown in LB medium either aerobically or anaerobically.

FIG. 5.

Activation of cadC-lacZ promoter deletions in V. cholerae. (Left graph) Strains were grown aerobically in LB medium buffered to pH 5.5 with 100 mM MES. (Right graph) Strains were grown anaerobically in LB medium buffered to pH 7.0 with 100 mM Trizma. The strains are as follows, from left to right: KSK2896 (cadC-lacZ), WL737 (−102), WL762 (−91), WL786 (−78), KSK2917 (−63), and KSK2900 (ΔaphB [ΔB]).

FIG. 6.

Inorganic acid tolerance assays. (A) Strains were exposed to LB challenge medium at pH 4.0 for 20 min without adaptation. (B) Strains were adapted for 60 min to LB medium at pH 5.7 and then exposed to LB challenge medium at pH 4.0 for 60 min. The strains are as follows, from left to right: C6706 (wt), WL361 (ΔVC0770), WL540 (ΔcadC), WL564 (Δcah), GK954 (ΔaphB), GK1240 (ΔnhaB), and GK1242 (Δclc).

FIG. 7.

Organic acid tolerance assays. (A) Strains were exposed to LB challenge medium containing organic acids at pH 4.5 for 20 min without adaptation. (B) Strains were adapted for 60 min to LB medium containing organic acids at pH 5.7 and then exposed to LB challenge medium containing organic acids at pH 4.5 for 60 min. The strains are as follows, from left to right: C6706 (wt), WL361 (ΔVC0770), WL540 (ΔcadC), WL564 (Δcah), GK954 (ΔaphB), GK1240 (ΔnhaB), and GK1242 (Δclc).