AphB Influences Acid Tolerance of Vibrio vulnificus by Activating Expression of the Positive Regulator CadC

Strains, plasmids, and culture media.The strains and plasmids used in this study are listed in Table 1. E. coli strains used for plasmid DNA replication or conjugational transfer of plasmids were grown in Luria-Bertani (LB) broth or on LB broth containing 1.5% (wt/vol) agar. Unless noted otherwise, V. vulnificus strains were grown in LB medium supplemented with 2.0% (wt/vol) NaCl (LBS). When required, cultures of V. vulnificus strains were buffered at pH 5.8 with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma, St. Louis, MO) or pH 7.6 with 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (Sigma).

Cloning of V. vulnificus aphB.A mutant that was more sensitive to low pH was screened from a library of V. vulnificus mutants generated by random transposon mutagenesis using mini-Tn5 lacZ1 (5, 32). A DNA segment flanking the transposon insertion was amplified by PCR as described previously (13). Since a database search for homology to the amino acid sequence deduced from the resulting PCR product singled out V. cholerae AphB, a whole aphB ORF was amplified from the genomic DNA of V. vulnificus by PCR using a pair of oligonucleotide primers, APHB031 and APHB032 (Table 2). The primers were designed using the genomic sequence of V. vulnificus (GenBank accession number VV11998; www.ncbi.nlm.nih.gov ). The amplified 889-bp aphB gene was ligated into pGEM-T Easy (Promega, Madison, WI) to result in pJR0308 (Table 1).

Generation of aphB deletion mutants.To inactivate aphB in vitro, one-third (300 bp) of the aphB ORF in pJR0308 was deleted and the resulting 0.6-kb ΔaphB construct was ligated with SphI/SacI-digested pDM4 (28), forming pJR0325. To generate the ΔaphB mutant by homologous recombination, E. coli SM10 λpir tra (containing pJR0325) was used as a conjugal donor to V. vulnificus ATCC 29307 (27). For the construction of the lacZ aphB double mutant, ΔlacZ, an isogenic mutant of ATCC 29307 which lacks lacZ, was used as the recipient (34). The conjugation and isolation of the transconjugants were conducted using methods previously described (9, 11, 32).

Acid challenges and measurement of lysine decarboxylase activities.Acid tolerance was assessed in 10 mM sodium citrate buffer (pH 4.4) supplemented with 2.0% NaCl (SCBN) and filter sterilized. Cultures of V. vulnificus strains, buffered at pH 5.8, were grown at 30°C under aeration to an optical density at 600 nm (OD₆₀₀) of 0.8 and used to inoculate flasks containing 50 ml of SCBN to achieve a final concentration of ca. 10⁵ CFU ml⁻¹. Following inoculation, the flasks were incubated at 30°C with shaking (150 rpm) for 90 min. The samples then removed were plated on LBS, and the percentages of survivors were calculated, using the CFU ml⁻¹ determined immediately after inoculation as 100%.

Cultures grown to an OD₆₀₀ of 0.8 as described above were used to determine the lysine decarboxylase activity based on procedures described previously (20, 31). A unit of the enzyme activity was defined as an increase in absorbance (1,000 × A ₃₄₀) of the reaction solution per min per cell density (OD₆₀₀) of cultures. Averages and standard errors of the means (SEM) were calculated from at least three independent trials.

Transcript analysis.Total cellular RNA was isolated from the V. vulnificus cultures grown at pH 5.8 by use of a Trizol reagent kit (Invitrogen, Carlsbad, CA). For Northern analyses, reactions were performed according to standard procedures (35) with 20 μg of total RNA. The DNA probe CADAP was prepared by labeling DNA fragments containing the cadA coding region with [α-³²P]dCTP as previously described (34) and used for the hybridizations.

For the primer extension experiments, an end-labeled 30-base primer, CAD0101 (Table 2), complementary to the coding region of cadC, was added to the RNA and then extended with SuperScript II RNase H⁻ reverse transcriptase (Invitrogen) as previously described (3, 9, 10). The cDNA products were purified and resolved on a sequencing gel alongside sequencing ladders generated from pJR990 (Table 1) with the same primer used for the primer extension. Primer extension products and Northern hybridization products were visualized using a phosphorimager (model BAS1500; Fuji Photo Film Co. Ltd., Tokyo, Japan).

Purification of GST-CadCN fusion protein and Western blot analysis of CadC.The DNA fragment encoding the N-terminal 169 amino acids of CadC (CadCN) was amplified by PCR using the primers CAD0113 and CAD0114 (Table 2). The DNA fragment had BamHI and EcoRI sites at the ends that were used to make an in-frame gene fusion between the truncated cadC gene and the 3′ end of the gst gene encoding glutathione S-transferase (GST) in pGEX2T (Amersham, Uppsala, Sweden). The GST-CadCN protein was expressed and purified by affinity chromatography according to the manufacturer's procedures (Amersham). For Western blot analyses, the purified GST-CadCN was used to raise the anti-CadC antibody and immunoblotting was performed according to a procedure previously described by Jeong et al. (10).

Construction of set of cadC-lacZ transcriptional fusions.The primer CAD0414 (Table 2), with a BamHI restriction site followed by bases corresponding to the 5′ end of the cadC coding region, was used in conjunction with one of the following primers to amplify the DNA upstream of cadC: CAD0530 (for pJR0530), CAD0531 (for pJR0531), CAD0533 (for pJR0533), CAD0534 (for pJR0534), or CAD0535 (for pJR0535) (Table 2). The primers were designed to amplify the P_cadC promoter region extended up to −152, −82, −37, −13, and +49, respectively (see Fig. 5A). A PstI restriction site was added to these primers to facilitate cloning of the DNA fragments. The PCR products were digested with BamHI and PstI and inserted into pRKΩlacZ, which carries the promoterless lacZ β-galactosidase gene (1), to create five cadC-lacZ reporter constructs. All constructions were confirmed by DNA sequencing. The CHCl₃-sodium dodecyl sulfate method described by Miller (26) was used to measure the β-galactosidase activity.

Overexpression and purification of V. vulnificus AphB.The aphB coding region was amplified by PCR using the primers APHB041 and APHB042-1 (Table 2) and then subcloned into a His₆ tag expression vector, pET22b(+) (Novagen, Darmstadt, Germany). The resulting plasmid, pHG0503, encoded AphB with a His₆ tag at the amino terminus. The His-tagged AphB protein was then expressed in E. coli BL21(DE3), and the protein was purified by affinity chromatography according to the manufacturer's procedure (QIAGEN, Valencia, CA).

Gel mobility shift assay and DNase I footprinting.For gel mobility shift assays, the 228-bp upstream region of cadC, extending from residues −82 to +146, was amplified by PCR using [γ-³²P]ATP-labeled CAD0101 and unlabeled CAD0531 as the primers (Table 2). The labeled 228-bp DNA (7 nM) fragment was incubated with various concentrations of purified His-tagged AphB protein for 30 min at 30°C in a 20-μl reaction mixture containing 1× binding buffer [50 mM HEPES (pH 7.6), 30 mM KCl, 1 mM EDTA, 10 mM (NH₄)₂SO₄, 1 mM dithiothreitol, 0.2% Tween 20] and 200 ng of poly(dI-dC) (Sigma). Following the incubation, the samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel (3). For competition analyses, various amounts of competitor DNA, the same but unlabeled 228-bp DNA, were added to the reaction mixture containing 7 nM of the labeled DNA prior to the addition of 80 nM of AphB.

For the DNase I protection assays, a 298-bp fragment of the cadC promoter region was generated by PCR amplification using a combination of [γ-³²P]ATP-labeled and unlabeled primers, CAD0530 and CAD0101, respectively (Table 2). The protein-DNA binding reactions with AphB were carried out as described above, except that 20 μl of 10 mM MgCl₂ and 5 mM CaCl₂ mix was added to the reaction mixtures, along with 1 μl of a DNase I solution (10 ng/μl; Sigma) (19). After incubation for 1 min at 25°C, the DNase I digestion was stopped by the addition of 80 μl of stop solution (35), and the DNA products were purified by ethanol precipitation. The purified DNA products were resolved on a sequencing gel alongside sequencing ladders of pJR990 generated using CAD0530 as a primer. Gels were processed as described for the primer extension analyses.

Identification of aphB gene and construction of aphB mutants of V. vulnificus.The amino acid sequence deduced from the aphB nucleotide sequence revealed a protein, a LysR homologue, AphB, composed of 291 amino acids with a theoretical molecular mass of 33,132 Da and a pI of 6.76. The amino acid sequence of V. vulnificus AphB was 80% identical to that of AphB of V. cholerae (data not shown; http://www.ebi.ac.uk/clustalw ). The predicted profile of the hydrophobicity (http://www.expasy.ch ) was significantly similar to that of AphB of V. cholerae and is consistent with the fact that the AphB protein is a cytosolic soluble protein (data not shown). To examine the role of AphB, V. vulnificus aphB isogenic mutants (Table 1) were constructed by allelic exchanges. Double crossovers, in which each wild-type aphB gene on the chromosome was replaced with the ΔaphB allele, were confirmed using previously described methods (10, 32) (data not shown). The mutants chosen for further analysis were named JR312 for the aphB mutant and JR313 for the lacZ aphB double mutant (Table 1).

Acid tolerance of the aphB mutant.The survival of the wild type (ATCC 29307) was significantly greater (P < 0.05) than that of the aphB mutant JR312 when challenged in SCBN adjusted to pH 4.4 (Fig. 1). The wild type decreased ca. 1.0 log₁₀ CFU/ml (90%) whereas the JR312 strain decreased ca. 3.0 log₁₀ CFU/ml (99.99%) after a 90-min acid challenge. This indicated that the aphB mutant is more sensitive to acid than the wild type and that AphB plays an important role in the acid tolerance of V. vulnificus. For the complementation of JR312, plasmid pJR0309 was constructed by subcloning the aphB coding region into pRK415 and under an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter (12). Complementation of the aphB gene in JR312 with a functional aphB gene (pJR0309) restored acid tolerance to a level equivalent to that of the wild type (Fig. 1). Therefore, the decreased acid tolerance of JR312 apparently resulted from the inactivation of functional aphB rather than any polar effects on the genes downstream of aphB.

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FIG. 1.

Acid tolerance of ATCC 29307 and isogenic aphB mutant. Cultures grown in LBS adjusted to a pH of 5.8 were used for the acid challenge as described in Materials and Methods. The percentages of survivors were calculated, using the CFU/ml of ATCC 29307 (wild type [WT]) that survived immediately after the challenge as 100%. Survivors of the mutant complemented with a functional aphB gene (pJR0309) are also presented. All bars represent the mean numbers of survivors from three independent trials, and the error bars represent the SEM. aphB, aphB mutant.

Effect of mutation in aphB gene on lysine decarboxylase activity.For the wild type grown at pH 5.8, lysine decarboxylase was produced and reached a maximum of 2.0 U (Fig. 2A). The disruption of aphB in the mutant JR312 resulted in a reduced production of lysine decarboxylase activity (P < 0.05). The residual level of lysine decarboxylase activity in JR312 corresponded to less than 1/10 of that of the wild type (Fig. 2A). The cadA transcript was also decreased in the aphB mutant (Fig. 2B), indicating that the lysine decarboxylase expression was positively regulated by AphB at transcription level. Similarly to results from the acid tolerance tests, complementation of ΔaphB in JR312 with a functional aphB gene (pJR0309) restored the lysine decarboxylase activity and the cadA transcript to levels comparable to those of the wild type (Fig. 2A and B). Therefore, these results indicated that the reduced level of lysine decarboxylase could be the major, if not sole, cause for the decreased acid tolerance of the V. vulnificus aphB mutant.

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FIG. 2.

Effect of mutations in aphB gene on lysine decarboxylase activity and cadBA transcription. Cultures were grown as described in the legend for Fig. 1, and then samples removed at an OD₆₀₀ of 0.8 were analyzed for lysine decarboxylase activity (A) and cadA transcript (B). Complementation of the mutant with a functional aphB gene (pJR0309) is also presented. Relative activities of lysine decarboxylase were calculated as described in the text. Error bars represent the SEM. WT, wild type; aphB, aphB mutant.

Effect of AphB on cadA expression is mediated through CadC.There are several possible ways for AphB to affect cadA expression. One is by binding directly to the cadA promoter, P_cadBA (34). However, when examined by a gel mobility shift assay, AphB was not able to specifically bind to the cadA promoter region even after several attempts (data not shown). The other possibility is that AphB influences the accumulation of another regulator(s), which in turn is directly responsible for the activation of P_cadBA.

Since it has already been reported that CadC, a transmembrane protein, activates cadBA expression by directly binding to the upstream sequences of P_cadBA (34), the cellular levels of CadC were determined in the same amount of total protein isolated from the wild type and from the aphB mutant JR312 (Fig. 3A). Western blot analysis revealed that the cellular level of CadC in the aphB mutant was almost undetectable and significantly lower than that in the wild type, indicating that AphB influences the accumulation of CadC. From this result, it is likely that AphB indirectly activates the activity of P_cadBA by increasing the cellular level of CadC, which is required for P_cadBA activity.

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FIG. 3.

Effects of aphB mutation on cellular level of CadC and P_cadC activity. Cultures were grown as described in the legend for Fig. 1 and then were analyzed for their CadC levels (A) and P_cadC activities (B). Complementations of the mutant with a functional aphB gene (pJR0309) are also presented. (A) CadC protein levels were determined by Western blot analyses using the immunoglobulin G fraction of rat anti-V. vulnificus CadC serum. (B) P_cadC activities were determined by primer extension of the RNA derived from each strain. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pJR990. The asterisk indicates the site of the transcription start. WT, wild type; aphB, aphB mutant.

In order to characterize the effect of AphB on the level of CadC in more detail, the levels of cadC transcription in the wild type and the aphB mutant were compared by primer extension analyses. A reverse transcript was identified from the RNA isolated from the wild-type cells (Fig. 3B). In contrast, no detectable reverse transcripts were apparent with the RNA from the aphB mutant, suggesting that AphB-dependent variation in the CadC level (Fig. 3A) resulted from changes in the level of cadC transcription. Determined based on the intensity of the bands of the reverse transcripts, the decreased cadC transcription in the aphB mutant was restored by the introduction of recombinant aphB (pJR0309) (Fig. 3B). Consequently, it appeared that AphB and CadC functioned sequentially in a regulatory cascade to activate P_cadBA activity.

The 5′ end of the cadC transcript was located 62 bp upstream of the translational initiation codon of CadC and subsequently designated +1 (Fig. 3B) (also see Fig. 6). The putative promoter constituting this transcription start site was named P_cadC to represent the cadC promoter.

AphB activates cadC expression by directly binding to P_cadC.The 228-bp DNA fragment encompassing the cadC regulatory region was incubated with increasing amounts of AphB and then subjected to electrophoresis. As seen in Fig. 4A, the addition of AphB at a concentration of 10 nM resulted in a shift of the 228-bp DNA fragment to a single band with a slower mobility. The binding of AphB was also specific, because assays were performed in the presence of 200 ng of poly(dI-dC) as a nonspecific competitor. In a second gel mobility shift assay, the same but unlabeled 228-bp DNA fragment was used as a self-competitor to confirm the specific binding of AphB to P_cadC (Fig. 4B). The unlabeled 228-bp DNA competed for the binding of AphB in a dose-dependent manner (Fig. 4B), confirming that AphB binds specifically to the cadC regulatory region.

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FIG. 4.

Gel mobility shift assay for binding of AphB to cadC regulatory region. A 228-bp DNA fragment of the upstream region of P_cadC was radioactively labeled and then used as a probe DNA. The radiolabeled fragments (7 nM) were mixed with increasing amounts of AphB. (A) Lanes 1 to 5 show 0, 10, 20, 40, and 80 nM of AphB, respectively. (B) For competition analysis, the same but unlabeled DNA fragment was used as the competitor. Prior to the addition of AphB, various amounts of the competitor DNA were added to a reaction mixture containing 7 nM labeled DNA. Lanes 1 to 4, probe DNA incubated with 80 nM of AphB and 0, 7, 14, and 28 nM of the competitor DNA, respectively.

Deletion analysis of cadC promoter region.To delineate the cis DNA sequences in the P_cadC promoter region required for AphB activation, transcriptional fusions of the putative cadC regulatory region were made to the reporter gene lacZ. The pJR-reporter fusions are shown in Fig. 5A. The reporter constructs were transferred into the ΔlacZ strain and lacZ aphB double mutant JR313. β-Galactosidase activities were used to quantify the capacity of each cadC upstream fragment to activate P_cadC.

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FIG. 5.

Deletion analysis of the cadC promoter region. (A) Construction of cadC-lacZ fusion pJR plasmids. PCR fragments carrying the regulatory region of cadC with deletions were subcloned into pRKΩlacZ to create each pJR-reporter fusion. Filled blocks, cadC coding regions; open blocks, lacZ DNA; solid lines, upstream region of cadC. The wild-type cadC regulatory region is shown at the top, with the proposed −10 and −35 regions marked. (B) β-Galactosidase activities were determined for the wild type (filled bars) and the aphB mutant (open bars) containing each pJR-reporter fusion as indicated. Cultures grown to an OD₆₀₀ of 0.5 at pH 5.8 were used to measure the β-galactosidase activities. Error bars represent the SEM.

For the ΔlacZ strain containing pJR-reporter fusions with endpoints to −152 (pJR0530) and −82 (pJR0531), the β-galactosidase activities were about 2,200 U (Fig. 5B). However, β-galactosidase produced in JR313 carrying pJR0530 (or pJR0531) was significantly reduced, supporting the hypothesis that the expression of P_cadC is dependent on AphB. β-Galactosidase activities were reduced in the strains that carried reporter fusions with endpoints from −37 to −13 (pJR0533 and pJR0534, respectively), and the residual level was indistinguishable from the basal level (i.e., β-galactosidase activity with pJR0535 deleted up to +49) (Fig. 5A and B). Moreover, the levels of β-galactosidase activity in ΔlacZ (pJR0533) and JR313 (pJR0533) did not significantly differ. Similar results were observed when the β-galactosidase activities of the ΔlacZ and JR313 cells containing pJR0534 were compared (Fig. 5B). Therefore, these data indicate that the sequences necessary for the activation of P_cadC by AphB were absent from the cadC upstream regions carried in pJR0533 and pJR0534. Since the cadC upstream regions in pJR0531 and pJR0533 were deleted up to −82 and −37, respectively, it was reasonable to conclude that the important cis-acting element for the activation of P_cadC by AphB would range from 82 to 37 bp upstream of the P_cadC transcription start.

Identification of AphB binding site using DNase I protection analysis.To determine the precise location of the AphB binding site in the cadC regulatory region, a DNase I footprinting experiment was performed. DNase I footprinting revealed a clear protection pattern in the cadC upstream region between −82 and −41 (Fig. 6A). Several nucleotides also showed enhanced cleavages, which have been observed frequently in DNase I protection analyses of the binding sites of transcriptional regulatory proteins, such as CRP (cyclic AMP receptor protein) (10). The AphB binding site was centered 61.5 bp upstream from the transcriptional start site of cadC (Fig. 6B), confirming that AphB activates P_cadC directly by binding to the cadC upstream region.

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FIG. 6.

Identification of AphB binding site using DNase I protection analysis (A) and sequence analysis of the cadC upstream region (B). (A) The ³²P-labeled 298-bp cadC regulatory region was incubated with increasing amounts of AphB and then digested with DNase I. Lane 1, no AphB added; lanes 2 to 4, AphB at 200, 300, and 400 nM, respectively. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pJR990. The hypersensitivity and protection in the presence of AphB are indicated by thick lines and open boxes, respectively. (B) The transcription start site is indicated by the bent arrow (P_cadC). The regions protected by AphB in a DNase I protection analysis and the −10 and −35 regions assigned on the basis of homology to a consensus sequence from E. coli are underlined with continuous and broken lines, respectively. The possible V. cholerae AphB binding sequences suggested by Kovacikova and Skorupski (16, 17) are indicated above the V. vulnificus DNA sequence in uppercase letters. The ATG translation initiation codon and the putative ribosome binding site (AAGAG) are indicated in boldface type.

In summary, the present study established that a LysR homologue, AphB, contributes to the acid tolerance of V. vulnificus by increasing the cellular level of CadC, which in turn activates the expression of the cadBA genes encoding cadaverine/lysine antiporter and lysine decarboxylase. AphB activates the expression of cadC by binding directly to an AphB binding site centered 61.5 bp upstream of P_cadC. Consequently, it would appear that AphB and CadC function sequentially in a regulatory cascade to activate P_cadBA activity.