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Journal of Bacteriology, November 2005, p. 7870-7875, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7870-7875.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

CadC Activates pH-Dependent Expression of the Vibrio vulnificus cadBA Operon at a Distance through Direct Binding to an Upstream Region

Jee Eun Rhee,1 Kun-Soo Kim,2 and Sang Ho Choi1*

Department of Food Science and Technology, School of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742,1 Department of Life Science, Sogang University, Seoul 121-742, South Korea2

Received 12 June 2005/ Accepted 5 September 2005


   ABSTRACT
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The Vibrio vulnificus cadBA genes were transcribed as a transcriptional operon by a single promoter, PcadBA, which was activated by CadC in a pH-dependent manner. A direct interaction between CadC and the PcadBA DNA was demonstrated, and a CadC binding site centered at –233.5 was mapped by deletion analyses of PcadBA and confirmed by a DNase I protection assay.


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Bacteria have developed elaborate protection systems to allow survival and/or growth during exposure to acidic environments (2, 6). Among such bacterial acid protection systems, acid pH neutralization mechanisms are based on the production of cytoplasmic amino acid decarboxylases (8, 12). Of the several amino acid decarboxylases known to be present in Escherichia coli, the cadBA genes encode a lysine/cadaverine antiporter and a lysine decarboxylase, whose combined activity leads to the synthesis and excretion of cadaverine to counteract external acidification (12, 13). Previous studies have noted that the lysine decarboxylase of E. coli is induced at an acidic pH (16, 24). Mutational analyses have proposed that the expression of the E. coli cadBA operon is regulated by CadC, an activator, and LysP, a repressor (17, 24).

The pathogenic marine bacterium Vibrio vulnificus occurs in raw seafood and has been identified as a causative agent of food-borne diseases (7, 11). We have recently cloned a 4.3-kb DNA fragment of V. vulnificus containing the cadBA genes (20). It was demonstrated that gene products of cadBA contribute to the acid tolerance of V. vulnificus and that their contribution is dependent on prior exposure of cells to a moderately acidic pH (20). Recently, an open reading frame, cadC, was also identified upstream of cadBA, and it has been proposed that CadC is essential for the survival of V. vulnificus upon exposure to an acidic pH and acts as a positive regulator for the expression of cadBA (21). However, the question of whether CadC directly or indirectly affects cadBA expression has not yet been addressed. Neither the promoter(s) of the cadBA genes activated by CadC nor the sequences upstream of the cadBA required for activation by CadC have been previously identified. Accordingly, here we extend our efforts to elucidate the regulation of the cadBA expression at a molecular level.

The strains and plasmids used in this study are listed in Table 1. Unless noted otherwise, V. vulnificus strains were grown in Luria-Bertani medium supplemented with 2.0% (wt/vol) NaCl (LBS). The lysine decarboxylase activity in the cells and cadaverine excretion in the supernatants were determined according to procedures previously described (19, 20). For the statistical analysis, Student's t test was used to evaluate the differences between the enzyme activities for the various strains (SAS software; SAS Institute Inc., Cary, NC). The averages and standard errors of the means (SEM) were calculated from at least three independent trials.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
Transcription of cadBA as a single transcriptional unit. Total RNA was isolated from the cultures of the wild-type and JR202 strains grown to an optical density at 600 nm (OD600) of 0.8 with LBS buffered to pH 5.8 and used for Northern blot analyses (22). The DNA probes CADBP and CADAP were prepared by labeling DNA fragments containing the coding region of either cadB or cadA (Fig. 1A), respectively, with [{alpha}-32P]dCTP and used for the hybridizations (4, 10).



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  		FIG. 1. Schematic representation of the V. vulnificus cad genes and Northern blot analysis of the cadBA operon. (A) The arrows represent the transcriptional directions and the coding regions of cad genes. The DNA probes, CADBP and CADAP, used for the Northern blot analyses are depicted by shaded bars. (B) For Northern blot analyses, total RNA from wild type (WT) and JR202 (cadB) was separated and hybridized to a DNA probe as indicated. The molecular size markers (Invitrogen, California) and the cad transcript are shown in kilobases.

 
When hybridized with the CADBP DNA probe, only a single transcript, approximately 3.7 kb, was detected in the RNA of the wild-type strain (Fig. 1B). Based on the DNA sequence of cadBA, it was anticipated that the cadB mRNA would be approximately 1.3 kb in length. The cotranscription of cadB and cadA was predicted to produce a 3.7-kb transcript. CADAP also hybridized to 3.7-kb RNA (Fig. 1B), thereby demonstrating that the cadBA genes are transcribed as a transcriptional operon rather than as two independent genes. Strain JR202 is a null mutant in which the chromosomal cadB is replaced with cadB:nptI as previously described (20). Neither the cadA transcript nor the cadB transcript was apparent in the RNA prepared from JR202 (Fig. 1B). This lack of the cadA transcript in JR202 indicates that the insertional mutation of cadB has a polar effect on cadA, thus supporting the present assumption that cadBA is one transcriptional unit.

Effect of a cadC mutation on lysine decarboxylase activity and cadaverine excretion. To inactivate cadC in vitro, 1.2-kb nptI DNA conferring resistance to kanamycin (18) was inserted into a unique SalI site present within the cadC open reading frame to result in pJR0011 (Table 1). The cadC mutants (Table 1) were constructed by allelic exchanges and double crossovers, in which each wild-type cadC gene on the chromosome was replaced with the cadC:nptI allele on pJR0011 using previously described methods (4, 10).

For the wild-type strain grown at pH 5.8, lysine decarboxylase was produced and reached a maximum of 2.0 units (Fig. 2A). The disruption of cadC in the cadC mutant JR201 resulted in a reduced production of lysine decarboxylase activity (P < 0.05). The residual level of lysine decarboxylase activity in JR201 corresponded to less than 1/10 of that of the wild type. As to the cadaverine excretion in the cadC mutant, it was significantly decreased with a trend similar to that for the lysine decarboxylase (Fig. 2B). When pJR0012 carrying recombinant cadC (Table 1) was reintroduced, the lysine decarboxylase activity and excretion of cadaverine in JR201 (pJR0012) were restored to levels comparable to the wild-type levels (Fig. 2A and B). Therefore, the decreased lysine decarboxylase activity and excretion of cadaverine in JR201 were proved to result from the functional inactivation of cadC rather than any polar effects on genes downstream of cadC. These results suggested that CadC acts as a positive regulator in the expression of cadA and cadB of V. vulnificus.



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  		FIG. 2. Effect of a cadC mutation on lysine decarboxylase activity and cadaverine excretion. For both panels, cultures of the wild type (WT) and JR201 (cadC mutant) were grown at a pH of 5.8. Samples were removed at an OD600 of 0.8 and analyzed for lysine decarboxylase activity (A) and for cadaverine excretion (B). Complementations of the cadC mutant with functional cadC (pJR0012) are also presented as indicated. The error bars represent the SEM.

 
Identification of a transcription start site of the cadBA operon. For the primer extension experiments, RNA was prepared from the wild type and the cadC mutant JR201 grown at pH 7.6 and 5.8, respectively. An end-labeled 24-base primer, CAD9902, complementary to the coding region of cadB was added to the RNA and then extended with SuperScript II RNase H– reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) as previously described (4, 9). A reverse transcript was identified from the RNA isolated from the wild-type cells grown at pH 5.8 (Fig. 3). In contrast, no detectable reverse transcripts were apparent with the RNA from JR201 grown at pH 5.8, suggesting that the CadC-dependent variation in the lysine/cadaverine antiporter and lysine decarboxylase activity (Fig. 2A and B) resulted from changes in the level of cadBA transcription. Primer extension analyses performed with the RNA prepared from cells of the wild type as well as the cadC mutant grown at pH 7.6 failed to produce a visible product (Fig. 3). Thus, it was apparent that CadC functioned as a positive regulator only when cells were grown at pH 5.8, and hence that the effect of CadC on the transcription of cadBA is dependent on the preexposure of the cells to an acidic pH.



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  		FIG. 3. Primer extension analysis of cadBA transcripts. The transcription start site was determined by the primer extension of the RNA derived from the wild type (WT) and JR201 (cadC mutant), grown at pH 5.8 or pH 7.6 as indicated. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pJR991. The asterisk indicates the site of the transcription start.

 
The 5' end of the cadBA transcript was located 54 bp upstream of the translational initiation codon of cadB and subsequently designated +1 (Fig. 3). The putative promoter constituting this transcription start site was named PcadBA to represent the cadBA promoter. Despite several attempts, no other transcription start sites were identified by primer extension analyses using different sets of primers hybridizing to the coding region of cadA as well as cadB (data not shown). This indicated that a single promoter, PcadBA, is used for the transcription of both cadB and cadA genes and that cadBA is expressed as a transcriptional operon.

CadC directly binds to the cadBA promoter. To determine whether CadC binds to the cadBA promoter in vivo, the cross-linked chromatin from the wild-type and cadC mutant JR201 cells was immunoprecipitated using the anti-CadC antibody (Fig. 4). The chromatin immunoprecipitation experiments were performed using formaldehyde cross-linking as described by Shang et al. (23). As positive controls, the input chromatin from both the wild type and JR201 appeared to carry the cadBA promoter DNA (Fig. 4). After the cross-links were reversed, the cadBA promoter fragment was detected in thechromatin precipitate from the wild type, induced with the anti-CadC antibody, based on a PCR using the primers CAD0305 and CAD0306 (Table 2). The primers were designed to specifically amplify the cadBA promoter DNA, which is 360 bp in length. The presence of the cadBA promoter DNA in the precipitated chromatin was caused by the specific binding of the CadC protein to the DNA, since no cadBA promoter DNA was detected in the precipitate induced in the absence of the anti-CadC antibody. Consistent with this, no detectable level of the cadBA promoter fragment was detected in the anti-CadC immunoprecipitate of the cadC mutant (Fig. 4), indicating that the CadC protein directly binds to the cadBA promoter in V. vulnificus.



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  		FIG. 4. Analysis of CadC binding to the upstream region of cadBA. The cells were cross-linked, washed, and then sonicated to produce sheared chromatin as described elsewhere (23). The DNA was purified from the sheared chromatins before precipitation (input, positive control) and after precipitation with protein A-Sepharose in the presence (+) or absence (–) of the anti-CadC antibody. The DNA was then amplified by a PCR using primers specific to the cadBA promoter. WT, wild type; cadC, cadC mutant.

 

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  		TABLE 2. Oligonucleotides used in this study

 
Deletion analysis of cadBA promoter region. To delineate the cis DNA sequences in the PcadBA promoter region required for CadC activation, transcriptional fusions of the putative cadBA regulatory region were made to the reporter gene lacZ. The primer CAD0112-1 (Table 2) was used in conjunction with one of the following primers for PCR amplification of the DNA upstream of cadBA: CAD0515 (for pJR0515), CAD0516 (for pJR0516), CAD0517 (for pJR0517), CAD0518 (for pJR0518), and CAD0519 (for pJR0519) (Table 2). The primers were designed to amplify the PcadBA promoter region extending up to –270, –252, –202, –152, and +49, respectively (Fig. 5A). The PCR products were inserted into pRK{Omega}lacZ, which carries the promoterless lacZ ß-galactosidase gene (1), to create five cad-lacZ reporter constructs (Fig. 5A). The reporter constructs were transferred into the Vv{Delta}lacZ strain and lacZ cadC double mutant JR243, and the ß-galactosidase activities were used to quantify the ability of each cadBA upstream fragment to activate the transcription of the cadBA operon (Fig. 5B).



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  		FIG. 5. Localization of the CadC binding site in the PcadBA. (A) PCR fragments carrying the regulatory region of cadBA with deletions were subcloned into pRK{Omega}lacZ (1) to create each pJR reporter. The 5' ends of the deletions are shown on the left as indicated. Shaded blocks, cadBA coding regions; open blocks, lacZ DNA; solid lines, upstream region of cadBA. The wild-type cadBA regulatory region is shown on top with the proposed –10 region, –35 region, and CadC binding site (CadCB). (B) The ß-galactosidase activities were determined in the wild type (solid bars) and cadC isogenic mutant JR201 (open bars) containing each pJR reporter. Cultures grown to an OD600 of 0.5 at pH 5.8 were used to measure ß-galactosidase activities. The error bars represent the SEM.

 
For the Vv{Delta}lacZ strain containing pJR0515, the ß-galactosidase activity was about 1,000 units (15) (Fig. 5B). This level of ß-galactosidase activity was comparable to that of the Vv{Delta}lacZ strain carrying pJR0516; however, the ß-galactosidase produced in the cadC mutant JR243 carrying pJR0516 (or pJR515) was significantly reduced to approximately 10% of the wild-type level (Fig. 5B). These results indicate that the cadBA upstream region deleted up to –252 is sufficient for the CadC activation of PcadBA.

The ß-galactosidase activity was reduced in the strains that carried pJR0517, and the levels of ß-galactosidase activity in the Vv{Delta}lacZ (pJR0517) and JR243 (pJR0517) strains did not significantly differ (Fig. 5B). Similar results were observed when the ß-galactosidase activities were compared between Vv{Delta}lacZ and JR243 cells containing pJR0518 and pJR0519. Therefore, these data indicate that the sequences necessary for the activation of PcadBA by CadC were absent from the cadBA upstream regions present in pJR0517, pJR0518, and pJR0519. Since the cadBA upstream region in pJR0517 was deleted up to –202, it is reasonable to conclude that the cis-acting element important for the activation of PcadBA by CadC ranges from 252 to 202 bp upstream of the PcadBA transcription start site.

CadC binding site for the cadBA promoter. The greatest limitation in determining CadC binding to the cadBA promoter in vitro was the purification of the CadC protein, as several attempts to overexpress and purify CadC in E. coli were unsuccessful (data not shown). Accordingly, we determined the binding site of CadC for the upstream region of cadBA using a crude extract of V. vulnificus as a source of the CadC protein. The total cellular proteins of V. vulnificus were separated into the cytoplasmic and membrane fractions and then analyzed by a Western blot assay using an anti-CadC antibody to determine the cellular location of the CadC protein. For this purpose, the rat polyclonal antibody specific to CadC was raised and purified according to the procedure previously described by Jeong et al. (10). The CadC protein was primarily found in the membrane fraction (data not shown), which was consistent with the prediction that the CadC protein is a membrane-spanning protein, proposed based on the hydrophobicity profile of the protein (21). The transmembrane region of the CadC was searched using protein topology prediction programs (http://www.sacs.ucsf.edu/Links/transmem.html). The search predicted a transmembrane region spanning between 175 and 203 amino acids from the N terminus.

A DNase I footprinting experiment was performed to determine the precise location of the CadC binding site in PcadBA. The 163-bp DNA fragment of the cadBA promoter region, extending from residues –307 to –145, was amplified by PCR using [{gamma}-32P]ATP-labeled CAD0504 and unlabeled CAD0503 (Table 2) as primers and using DNA as a template. Binding of CadC to the labeled DNA and DNase I digestion of the protein-DNA complexes were carried out following the procedures described previously (4, 10). In this experiment, the membrane fraction was used as the protein source for CadC, and the 20-µl reaction mixture contained 1x binding buffer (20 mM morpholineethanesulfonic acid [pH 5.8], 100 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 0.5 mM EDTA, 1 mM dithiothreitol), 10 mM lysine, and 1 µg of poly(dI-dC) (Sigma, St.Louis, MO). The DNase I footprinting revealed a protection pattern in the cadBA upstream region between –214 and –252 (Fig. 6A). Several nucleotides also showed enhanced cleavage, which has been frequently observed in DNase I protection analyses of the binding sites of transcriptional regulatory proteins (10). The CadC binding site was centered 233.5 bp upstream from the transcriptional start site of cadBA (Fig. 6B), confirming that CadC activates PcadBA directly by binding to the cadBA upstream region.



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  		FIG. 6. Identification of CadC binding site using DNase I protection analysis. (A) DNase I protection analysis of CadC binding to wild-type cadBA regulatory region. Lane 1, 10 µg of membrane fraction of cadC mutant; lanes 2 and 3, 5 µg and 10 µg of membrane fraction of wild type, respectively. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pJR991. The hypersensitivity and protection in the presence of CadC are indicated by the thick lines and open boxes, respectively. (B) Sequence analysis of the cadBA upstream region. The transcription start site is indicated by the bent arrow (PcadBA). The regions protected by CadC 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 solid and broken lines, respectively. The possible V. cholerae CadC binding sequences are identified by homology to the V. vulnificus CadC binding sequences, while conserved nucleotides are indicated above the V. vulnificus DNA sequence in uppercase. The assigned sequences for the Lrp binding sites are shaded, and below them is shown the percentage of identity to the consensus sequence YAGHAWATTWTDCTR (5) (Y = C/T, H = "not G," W = A/T, D = "not C," R = A/G). The ATG translation initiation codon and putative ribosome binding site (GGAGA) are indicated in boldface.

 
The expression of the E. coli cadBA operon is regulated by CadC as a function of the pH (16, 24). In a similar way, CadC positively regulates the expression of the cadBA genes in Vibrio cholerae (14). However, until now, the question of whether CadC directly or indirectly affects the expression of cadBA in E. coli and V. cholerae has not been addressed. The data presented here demonstrated a direct interaction between CadC and PcadBA in V. vulnificus (Fig. 4 and 6), and a DNase I protection assay revealed the CadC binding site centered at –233.5. The sequences assigned for the CadC binding site in V. vulnificus are also found in the cadBA upstream region in V. cholerae (Fig. 6B), indicating that the CadC binding site sequences are conserved between these bacteria.

It would seem that CadC binding at –233.5 is unusually distant for the activation of bacterial promoters. For activation of papBA in E. coli, cyclic AMP receptor protein binding at –215.5 is the most distant binding as reported previously (25). However, in this example, the leucine-responsive regulatory protein (Lrp) binds to multiple sites extended between the cyclic AMP receptor protein binding and the RNA polymerase (RNAP) binding. In the present study, the three sequences of the intervening region revealed reasonable homologies to the Lrp binding consensus sequences (Fig. 6B) (5). Lrp is a small nucleoid-structuring protein that binds and bends DNA at specific sequences. Lrp-induced bending (forming a DNA loop) facilitates a protein-protein interaction between an upstream activator and RNAP (3, 5). We have recently identified the Lrp homolog in V. vulnificus and deposited its sequence (GenBank accession number AY160773). However, additional studies are needed to clarify whether these regions act as Lrp recognition sites and whether the Lrp is involved in the activation of PcadBA by forming a DNA loop between CadC and RNAP.


   ACKNOWLEDGMENTS
 
This study was supported by a grant to S.H.C. from the KOSEF (R01-2004-000-10046-0), Republic of Korea.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food Science and Technology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, South Korea. Phone: 82-2-880-4857. Fax: 82-2-873-5095. E-mail: choish{at}snu.ac.kr. Back


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Journal of Bacteriology, November 2005, p. 7870-7875, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7870-7875.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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