Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK1
Author for correspondence: J. M. Ketley. Tel: +44 116 252 3434. Fax: +44 116 252 3378. e-mail: ket{at}leicester.ac.uk
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ABSTRACT |
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Keywords: Vibrio cholerae, PhoBR, two-component, regulation, colonization
Abbreviations: CT, cholera toxin; HK, histidine kinase; RILAT, rabbit ileal loop anastomosis test; RR, response regulator; TCR, two-component regulator; TGLP, Tris/glucose low phosphate medium; TGHP, Tris/glucose high phosphate medium
The GenBank accession number for the sequence reported in this paper is AF043352.
a Present address: Laboratorio de Fisiologia Celular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro, Brazil.
b Present address: Deparment of Veterinary Pathology, Glasgow University, Bearsden, Glasgow G61 1QH, UK.
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INTRODUCTION |
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Limitation of inorganic phosphate leads bacterial cells to synthesize a number of proteins, many being involved in the acquisition of phosphate (Wanner, 1996 ). Phosphate limitation can also play a role in microbial virulence. In Pseudomonas aeruginosa, the expression of an extracellular heat-labile haemolysin is induced under phosphate-limiting conditions and is required for virulence (Ostroff et al., 1989
). In Agrobacterium tumefaciens (Aoyama et al., 1991
; Winans, 1990
) and Salmonella typhimurium (Libby et al., 1990
), low phosphate levels induce synthesis of virulence factors. Furthermore, mutations in entero-invasive Escherichia coli genes involved in phosphate acquisition gave a hyper-invasive phenotype (Sinai & Bavoil, 1993
), and in an E. coli strain pathogenic to pigs, mutation of a gene of the Pho regulon (Lee et al., 1989
) produced an avirulent strain (Daigle et al., 1995
).
The expression of proteins regulated by phosphate concentration requires PhoR and PhoB, members of a TCR system, where PhoR is the HK and PhoB the RR (Stock et al., 1989 ). The set of genes whose expression depends on PhoB constitutes the Pho regulon (Lee et al., 1989
). Pho regulon promoters contain one or more copies of a conserved consensus sequence, the Pho box, that functions as a PhoB-binding site (Makino et al., 1986a
).
In this report, we describe the cloning and sequencing of the V. cholerae phoR and phoB genes (designed phoRvc and phoBvc, respectively). Thus, this is the first description of a member of the OmpR subfamily of TCRs in V. cholerae, as ToxRs similarity with OmpR is limited to the DNA-binding motif (Miller et al., 1987 ) and the proteins are otherwise structurally and functionally different. Our analysis of phoBvc mutants indicates that V. cholerae responds to phosphate limitation in a way similar to E. coli (Wanner, 1996
). In addition, mutation of the phoBvc gene reduced colonization ability, suggesting a role for the Pho regulon in adaptation of V. cholerae to the intestinal environment.
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METHODS |
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Preparation of E. coli competent cells and electroporation were carried out as described by Sambrook et al. (1989 ). V. cholerae competent cells were prepared and electroporated as described by Marcus et al. (1990
). Briefly, cells were grown in LB to OD600 0·40·6 and centrifuged; the pellet was washed three times with 10% (v/v) glycerol containing 272 mM sucrose and finally resuspended in 1/100 of the original culture volume. Electroporations were carried out with a Bio-Rad Gene Pulser with the Pulse controller set at 25 µF, 2·5 kV and 200
. After electroporation, the suspension was diluted in 1 ml SOC (Sambrook et al., 1989
), incubated in a shaker for 1 h at 37 °C and plated on appropriate selective media.
Southern blots were obtained by transferring DNA from agarose gels to nylon membranes (Hybond-N, Amersham) using 10xSSC (Sambrook et al., 1989 ). Labelling of DNA probes with digoxigenin-11-dUTP (DIG) by random priming, hybridization and detection were carried out according to the labelling and detection kit instructions (Boehringer Mannheim). Prehybridization and hybridization were at 68 °C; membrane washings were under stringent conditions: two washes with 2xSSC containing 0·1% SDS at room temperature and two washes with 0·1xSSC containing 0·1% SDS at 68 °C.
PCR amplifications were performed in an automatic thermal cycler (Ominigene) using standard conditions (Innis & Gelfand, 1990 ). Oligonucleotide primers for PCR were as follows. For amplifications across the multiple cloning site of pUC19 and derivatives, P1L (5'-GGGTTTTCCCAGTCACGACGTTGT-3') and P2L (5'-TATGTTGTGTGGAATTGTGAGCGG-3') were used. For construction of the mutation in phoBvc, JK3 (5'-GAAGATCTTGATGCCACTACCACCA-3') and JK4 (5'-GAAGATCTGAAGCGGGAAGAGATGA-3') were used. The oligonucleotides JK3 and JK4 span nt 11361119 and 11511168, respectively, in the 4·48 kb insertion of pWK1 (Table 1
), and each incorporates a BglII restriction site extension. For the construction of clones for the complementation analysis, WK9 (5'-CGGAATTCATCCATCCCACCACAAC-3'), which incorporates an EcoRI site extension, was used.
DNA was sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). To sequence the 4·48 kb fragment, the nested-deletion strategy with exonuclease III and S1 nuclease (Pharmacia) was used to generate a series of deletion clones that were then sequenced using the M13/pUC reverse and forward sequencing primers (Gibco-BRL). A primer-walking strategy was also used and primers, based on a reliable sequence obtained in a previous cycle, were designed to provide complete sequence data for both strands. Sequence reaction products were analysed with an ABI automated DNA sequencer (Protein and Nucleic Acids Chemistry Laboratory, Leicester University). Comparison of the nucleotides and derived polypeptides with sequences in the GenBank and EMBL databases were carried out by the use of fasta (Pearson & Lipman, 1988 ) and blast (Altschul et al., 1990
). The nucleotide sequence of the 4·48 kb fragment can be found in GenBank under the accession number AF043352.
Cloning procedures.
A 300 bp fragment containing a DNA sequence with homology to the phoB gene of E. coli was obtained by PCR amplification of chromosomal DNA of V. cholerae strain CVD103 (S. Humphreys & J. M. Ketley, unpublished results) using degenerate primers (PCRDOP; Wren et al., 1992 ). The fragment was cloned into pUC19 to form pSH4. CVD103 chromosomal DNA was digested with several restriction enzymes and analysed by Southern hybridization using the 300 bp insert of pSH4 as a probe. The probe hybridized to single chromosomal fragments cut with EcoRI (4·48 kb) and BglII (6·5 kb). A library was constructed of size-selected (3·55·5 kb) CVD103 genomic DNA digested with EcoRI and cloned into the EcoRI site of pUC19. A clone was isolated that contained a plasmid with a 4·48 kb insert that hybridized with the probe. The plasmid was named pWK1.
For the complementation analysis, P2L and WK9 were used with pWK1 to amplify a fragment of about 1·75 kb that contained the phoBvc gene plus the regulatory region of the phoBRvc operon. Oligonucleotide WK9 spans nt 17231739 in the 4·48 kb insertion of pWK1 and has an EcoRI site at its 5' end. The amplified fragment was digested with EcoRI and cloned into pUC19 and pLG339, both digested with the same enzyme. Complementation tests were also done using pLG339 derivatives containing the 3·15 kb PstI fragment from pWK1 (nt 13150).
Mutant construction.
The 3·15 kb PstI fragment from pWK1 was cloned into the PstI site of pUC19 to form pWK5. The primer pairs P1L/JK4 and P2L/JK3 were used with pWK5 to amplify by PCR two fragments of 2·2 kb and 0·95 kb, respectively. Each fragment contained part of the gene with a new BglII restriction site at one end and was subcloned via several steps into pUC19 (pWK5ac; Table 1). The resulting plasmid, pWK6, contains the phoBvc gene with an internal deletion of 14 bp and a 26 bp insertion from pUC19 containing sites for the enzymes SmaI, BamHI and XbaI. Plasmid pWK6 was then digested with BamHI and a 1·5 kb kanamycin-resistance cassette, derived from pUC-4K, was cloned into the phoBvc gene to form pWK7.
A derivative of the suicide plasmid pGP704 was used to introduce the mutation into the chromosomal phoBvc gene. The 2·89 kb EcoRIEcoRV fragment containing the mutated phoBvc gene from pWK7 was blunt-end ligated into the EcoRV site of pGP704, resulting in pWK8. To facilitate the selection of the mutant, the sacB gene of Bacillus subtilis was cloned into the construct. pJG9 was digested with SmaI and XhoI and the 1·9 kb fragment containing the sacB gene was blunt-ended and ligated into the blunt-ended EcoRI site of pWK8 to form pWK9.
pWK9 was transformed into E. coli SY327 pir and then to SM10
pir for mobilization into streptomycin-resistant derivatives of V. cholerae strains CVD103 and 569B. Merodiploid transconjugants, the result of a single recombination event at the site of V. cholerae homologous sequences, were selected on medium containing ampicillin, streptomycin and kanamycin. Recombinants that had undergone a second recombination event were selected by growing the merodiploids firstly in LB with streptomycin and then in the same medium supplemented with 5% sucrose for several generations. The cells were plated on LA plates supplemented with streptomycin and sucrose and the colonies tested for ampicillin sensitivity and kanamycin resistance. Ampicillin-sensitive kanamycin-resistant cells were expected to possess the mutated copy of the putative phoBvc gene, whereas the ampicillin-sensitive kanamycin-sensitive cells were expected to be wild-type, generated by an alternative resolution of the merodiploids. Kanamycin-resistant insertion mutants in the phoBvc gene constructed in strains CVD103SR and 569BSR were named WK1 and WK3, respectively. The formation of the merodiploid, the insertion of the kanamycin cassette in the phoBvc gene and the recovery of the wild-type genotype were confirmed by Southern hybridization in which the 3·15 kb PstI fragment from pWK5 was used as the probe.
Mutants not containing the kanamycin-resistance cassette in the V. cholerae phoBvc ORF were also constructed. Firstly, the 2·89 kb EcoRIEcoRV fragment from pWK6 containing the mutated phoBvc gene was cloned into pGP704 digested with the same enzymes, forming pWK10. The sacB gene on the 1·9 kb BamHIEcoRV fragment from pJG9 was then cloned into pWK10 digested with EcoRV and BglII, resulting in pWK11. This plasmid was used to construct new mutants in the V. cholerae strains CVD103SR and 569BSR as described above for WK1 and WK3. The BamHI site introduced during the mutation of the phoBvc gene was used to differentiate between the mutant and wild-type genotypes. The formation of the merodiploids and of the putative phoBvc mutants, and the recovery of the wild-type genotype, were confirmed by Southern hybridization. The kanamycin-sensitive phoBvc mutants in CVD103SR and 569BSR were named WK2 and WK4, respectively.
Alkaline phosphatase assay.
Levels of alkaline phosphatase activity were determined by a variation of the permeabilized whole-cell assay (Gutierrez et al., 1987 ). The optical density of the culture at 600 nm was measured and a determined volume of the culture was centrifuged. The pellet was resuspended in 1 ml 1 M Tris/HCl, pH 8·0, and the cells were permeabilized by addition of hexadecyltrimethylammonium bromide (CTAB; final concentration, 0·025%) followed by 10 s vortexing. The reaction was carried out at 37 °C by the addition of ONPG (Sigma; final concentration, 0·04%) and stopped with K2HPO4 at 0·1 M. The units of alkaline phosphatase were calculated using the formula 103x[A420-(1·75xA550)]/txOD600xV, where A420 and A550 are the absorbancies of the reaction mix after an incubation time t (min), OD600 is the optical density of the culture and V is the volume (ml) of the culture used in the assay. The activity measured was expressed in Miller units (Miller, 1972
).
Protein analysis.
Proteins from whole-cell lysates were analysed by electrophoresis through 11% polyacrylamide (T=10%, C=1·6%) SDS gels as previously described (Laemmli, 1970 ). Outer-membrane proteins prepared by differential solubilization of cell envelopes with Triton X-100 (Schnaitman, 1974
) were analysed by SDS-PAGE with 10% running gels. Periplasmic proteins were obtained by centrifugation of a suspension of spheroplasts prepared as described by Roy et al. (1982a
). The resulting supernatant was precipitated and the periplasmic proteins were analysed by SDS-PAGE with 11% running gels as above. Gels were stained with Coomassie brilliant blue R 250 (Bio-Rad). Protein concentration was estimated by the method of Bradford (1976
).
Two-dimensional polyacrylamide gel electrophoresis was performed with a Multiphor II horizontal unit (Pharmacia) on an immobilized pH gradient 310 (Immobiline DryStrip pH310L) for the first dimension and SDS-PAGE on a 1214% gradient gel (ExcelGel XL) for the second dimension. Samples were prepared as recommended by the supplier by resuspending cells in lysis buffer (8·99M urea, 0·02% Triton X-100, 0·13 M DTT, 0·02%, v/v, Pharmalyte 3-10, 8 mM PMSF) for 2 h at room temperature, followed by ultracentrifugation (250000 g). A volume containing approximately 20 µg proteins in sample buffer was loaded onto the strip and the proteins were focussed at 1 mA for 20·5 h at 20 °C. The gel strip was then loaded onto the polyacrylamide-SDS slab gel and electrophoresed at 600 V for 3·5 h. Gels were then fixed and silver stained.
CT ELISA assay.
V. cholerae strains were grown in TGHP and TGLP, pH 6·5, at 30 °C with aeration for approximately 18 h (DiRita et al., 1990 ). The cells were centrifuged and the toxin in the supernatant was assayed by GM1-ELISA (Holmgren, 1973
).
Conjugation.
V. cholerae strains CVD103SR and 569BSR were mated with E. coli SM10 pir harbouring either pWK9 or pWK11 by mixing an equal number of exponential-phase cells. The mating mixture was spotted onto an LA plate and incubated overnight at 37 °C. Dilutions of the mixture were plated on LA containing ampicillin, kanamycin, and streptomycin or ampicillin and streptomycin for the selection of the transconjugants derived from pWK9 or pWK11, respectively. Transconjugants were confirmed as V. cholerae by a positive Kovac oxidase reaction (Kay et al., 1994
).
Rabbit ileal loop anastomosis test (RILAT).
A competitive colonization assay (Ketley et al., 1993 ) was carried out using RILAT (Ketley et al., 1987
) with the following modifications. The peritoneal cavity was opened and the washed intestine was clamped with intestinal clamps and resected 10 cm proximal to the ileo-caecal junction. Moving proximal to the resection, the length of intestine required for the construction of ligated loops was measured and the intestine clamped and resected; the isolated segment of intestine was temporarily replaced into the peritoneal cavity. The clamped, resected ends of the intestine were anastamosed, joint integrity and blockage were assessed and the intact intestine was replaced into the peritoneal cavity. The resected intestinal segment was removed from the peritoneal cavity, the ends closed by suturing and ligated sample loops and spacer loops constructed and inoculated as before. After closing the laparotomy, the rabbit was allowed to recover before termination of the procedure 18 h after loop inoculation.
The samples inoculated into ligated loops were prepared as follows. V. cholerae strains were grown to mid-exponential phase in LB at 37 °C, the cultures were then centrifuged and the pellets resuspended in saline. The OD600 of the suspensions was obtained and they were diluted (based on previously determined growth curves) to give the required number of cells to be inoculated in 500 µl saline. Cells were also plated for viable counts. The two test strains were inoculated separately or equal numbers of cells were combined to the same total count and inoculated to assess the ability to colonize competitively. Three inoculum sizes were used: 2x102, 2x103 and 2x106 cells in 500 µl saline. In some experiments, the cells were resuspended in saline supplemented with KH2PO4 at a final concentration of 6·5 mM. The samples were injected into 5 cm intestinal loops. Saline was used as a negative control and a loop containing CT (0·5 µg) was included to check the physiological response of the tissue. On termination of the procedure, a small piece of tissue from each loop was removed, weighed, washed three times with saline, transferred to 2 ml saline and homogenized. Appropriate dilutions were plated onto LA and LA containing streptomycin to determine the total number of vibrios and to allow differentiation of the strains. The ratios of the antibiotic-sensitive (parental strain) to -resistant colonies (mutants) per gram of tissue were determined.
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RESULTS |
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Nucleotide sequence analysis of the insert in pWK1 (accession number AF043352) revealed the presence of four ORFs. One of them, 0·69 kb long, contained the same nucleotide sequence as the 300 bp fragment used as a probe. The deduced amino acid sequence resulted in a 229 residue protein that is the same length and bears approximately 72% identity with PhoB of E. coli (Makino et al., 1986a ). The V. cholerae PhoB sequence (here designated as PhoBvc) contains conserved amino acid residues and two blocks of four hydrophobic residues found in all the OmpR-like subfamily sequences (Stock et al., 1989
). The corresponding gene, phoBvc, is preceded by a ShineDalgarno sequence (AGG; Shine & Dalgarno, 1975
), a Pribnow box and a short sequence with extensive sequence identity to the phosphate box (Pho box; Makino et al., 1986a
) in the regulatory region of the E. coli phoB gene (Fig. 1a
).
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Mutation of the phoBvc gene
A unique BamHI site engineered into the phoBvc gene (pWK6; Fig. 1b) was used to insert a kanamycin-resistance cassette (pWK7). The mutated fragment was then cloned into the suicide vector pGP704 containing a sacB gene (pWK9). After introduction of pWK9 into V. cholerae, a phoBvc merodiploid was isolated by selection for kanamycin resistance and the loss of the sacB gene, and used to obtain a mutant containing the resistance cassette in phoBvc. Kanamycin-resistant mutants were constructed in V. cholerae strains CVD103SR and 569BSR, and designated WK1 and WK3, respectively. The insertion of the kanamycin-resistance cassette into phoBvc, and absence of vector sequences in WK1 and WK3, was confirmed by Southern hybridization (data not shown).
In-frame deletion mutants in phoBvc that did not contain the kanamycin-resistance cassette were constructed in strains CVD103SR and 569BSR and named WK2 and WK4, respectively. The phoBvc mutated fragment (Fig. 1b) from pWK6 was subcloned directly into pGP704 containing sacB (pWK11). Following the introduction of pWK11 into V. cholerae, phoBvc merodiploids were isolated and used to obtain new phoBvc mutants by selection for the loss of the sacB gene. The insertion of the BamHI site associated with the in-frame mutation and absence of vector sequences in WK2 and WK4 was confirmed by Southern hybridization (data not shown).
Growth and properties of the phoBvc mutants
A mutation in the phoBvc gene would be expected to have physiological consequences with respect to phosphate metabolism. Accordingly, growth ability at 37 °C was examined in liquid cultures in LB and in TGHP or TGLP media at pH 8·0 (Fig. 2). No significant reduction in growth rates or final cell-culture densities was seen for the four mutants in comparison to the parent strains when they were grown in LB (Fig. 2a
and data not shown) or in TGHP (Fig. 2b
and data not shown). The growth rate in LB was greater than that in TGHP and the cultures reached higher cell densities at stationary phase. In contrast, when the mutant and parent strains were grown under phosphate-limited conditions (TGLP) all strains showed a reduction in both growth rate and cell density at stationary growth phase in comparison to growth in LB or TGHP. More importantly, in TGLP a marked difference was observed in the growth rate and cell density of mutants WK2 and WK4 (Fig. 2b
) and WK1 and WK3 (data not shown) when compared to the parental strains. No difference was observed between the phoBvc mutants in the same V. cholerae strain constructed by different strategies.
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PhoBvc has a role in intestinal colonization
Colonization of the small intestine by V. cholerae strains CVD103 and 569B and their mutants, WK2 and WK4 respectively, was studied in the adult rabbit using ligated ileal loops. The results are summarized in Table 4. Comparison of CVD103 and its streptomycin-resistant derivative (the parental strain of WK2) was carried out by inoculating an equal number of cells in intestinal loops of the rabbit. After 18 h, the number of c.f.u. associated with intestinal tissue and the ratio of the two inoculated strains were determined. As previously observed (Ketley et al., 1993
), strain CVD103SR colonizes the small intestine of the rabbit slightly less efficiently than the parent strain CVD103. In a competitive assay between CVD103 and the phoBvc mutant WK2, CVD103 colonized about 10-fold better than the mutant when the cells were inoculated in saline. However, when the cells were in saline supplemented with phosphate (6·5 mM KH2PO4), the colonizing ability of the mutant in comparison to CVD103 improved markedly. In a similar competitive assay, the colonization abilities of the strain 569B and of the phoBvc deletion mutant WK4 were compared. When inoculated in saline, strain 569B colonized the small intestine of the rabbit 27-fold better than the mutant. As with the CVD103/WK2 comparison, in the presence of high phosphate levels, the colonization ratio dropped. These results indicate that the mutation of the phoBvc gene affects the colonization ability of V. cholerae. This reduction in colonization ability can be partly reversed by increasing the lumenal concentration of inorganic phosphate.
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DISCUSSION |
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In this work, we report the cloning and sequencing of the phoBRvc operon of V. cholerae. As might be expected, similarities were found with phoBR of E. coli. The phoBRvc regulatory region contains a putative consensus Pho box located at region -35 of the promoter, suggesting that phoBRvc is under control of PhoBvc and therefore, similar to E. coli, is phosphate regulated. The two ORFs, phoBvc and phoRvc, found downstream of the regulatory region are organized in the same way as in E. coli. There seems to be no consensus promoter sequence in the intergenic region and a putative stemloop structure that could function as a transcription terminator was found downstream of the phoRvc coding region. Therefore, it seems likely that the two ORFs are co-transcribed and that the V. cholerae pho operon consists of two cistrons, phoBvc and phoRvc. The stemloop structure identified within phoRvc may function as a transcriptional attenuator. Both the presence of this possible structure and the poor consensus ShineDalgarno sequence that precedes the phoRvc gene suggest that phoRvc is expressed at a lower level than phoBvc. This bears similarity with both E. coli phoBR (Makino et al., 1986b ; Haldimann et al., 1998
) and ompRenvZ (Comeau et al., 1985
) operons, where expression of the HK gene (phoR or envZ) is lower than the cognate RR gene. At the deduced amino acid sequence level, both PhoBvc and PhoRvc bear extensive homologies with their counterparts from E. coli.
In addition to phoBvc and phoRvc, two incomplete ORFs, orf1 and orf4 were identified in the 4·48 kb chromosomal DNA fragment in pWK1. One was found in the EcoRIDraI fragment (nt 1870); it starts with an ATG 236 nt upstream of the phoBvc translation start codon and would be translated in the opposite direction to phoBRvc. The incomplete orf1 would encode a polypeptide 238 residues long which is 57% identical to a hypothetical protein from the EMBL/GenBank databases of 303 residues that maps in the intergenic region araJaroM of E. coli. The other ORF, orf4, is incomplete and is in the 1·2 kb PstIEcoRI fragment (nt 31504480) of pWK1, downstream of the phoRvc gene. This ORF possesses a high level of similarity to the E. coli ppx gene, which is thought to be a member of the Pho regulon (Kornberg, 1994 ). Ppx is an exopolyphosphatase that acts on the ends of polyphosphate chains, progressively removing orthophosphate residues and, thus, provides an alternative source of organic phosphate to ATP.
In E. coli, mutations in phoB can inhibit the transcription of the Pho regulon genes (Bracha & Yagil, 1973 ; Morris et al., 1974
; Yagil et al., 1975
). Defined mutation of phoBvc generated strains that differed markedly from their parents. Under phosphate-limiting conditions they grew very poorly and did not express alkaline phosphatase, an enzyme synthesized in high levels by the parental strains under the same growth conditions. The poor growth of the phoBvc mutants under phosphate limitation correlates with the inability to express alkaline phosphatase and other proteins synthesized by the parental strains that may be involved in the phosphate metabolism. The pattern of protein expression by the phoBvc mutants in limiting-phosphate conditions was also different to that obtained with the parent strain. The 60 kDa protein is likely to be PhoA, as it is periplasmic and has the same apparent molecular mass as the PhoA protein described previously in V. cholerae (Roy et al., 1982a
). The 31 kDa and 51 kDa proteins, by analogy with periplasmic proteins from E. coli (Tommassen & Lugtenberg, 1980
) induced under the same conditions, are probably involved in binding and transport of phosphorus compounds. The outer-membrane protein of 38 kDa is likely to be the homologue of the E. coli porin PhoE (VanBogelen et al., 1996
). The V. cholerae homologue is approximately the same molecular mass and is associated with peptidoglycan (data not shown), a property of proteins that form channels in the outer membrane (Nikaido, 1983
). A major outer-membrane protein of 33·9 kDa with similar characteristics was identified in Vibrio parahaemolyticus also grown under limiting-phosphate conditions (McCarter & Silverman, 1987
). As no function has been assigned to the majority of proteins differentially expressed in the parental strain under low-phosphate conditions, further work is necessary in order to characterize the V. cholerae Pho regulon.
Both sets of phoBvc mutants could be complemented in trans by the entire phoBRvc operon cloned into a low-copy-number plasmid. The fact that the wild-type phenotype was not restored by in trans complementation with just the phoBvc gene with regulatory region is intriguing. With respect to the mutants WK1 and WK3, one can argue that the insertion of the kanamycin cassette resulted in a polar effect on phoRvc gene expression. In contrast, mutants WK2 and WK4 have small in-frame alterations in the phoBvc DNA sequences, and one should not expect that such mutations would cause a polar effect on phoRvc. The lack of complementation by the wild-type phoBvc gene plus regulatory region suggests that the in-frame mutation in WK2 and WK4 is dominant.
The biosynthesis of CT is not affected by the phoBvc mutation, an indication that its expression is not also under the control of PhoBRvc. It has been known for some time that environmental signals such as temperature, pH, osmolarity and amino acids, among others, cause changes in the expression of CT (Miller & Mekalanos, 1988 ). The diminished expression of CT by the phoBvc mutants and the parent strain in TGLP/amino acids medium was expected. The importance of phosphate in the stimulation of amino acid uptake in minimal medium and, consequently, in growth and expression of CT by V. cholerae has been reported previously (Sagar et al., 1981
).
The mutation of the phoBvc gene affects intestinal colonization of adult rabbit intestine by V. cholerae. The effect was greater on phoBvc mutants derived from the toxigenic strain 569B, probably because CVD103, a ctxA derivative, is less able to colonize the intestine (Levine et al., 1988 ). It is not known which members of the Pho regulon of V. cholerae are involved in intestinal colonization, or how. In P. aeruginosa the phosphate-regulated haemolytic phospholipase C has been shown to be involved in colonization (Ostroff et al., 1989
). Vibrios also produce phospholipases, some of which are induced in phosphate-deficient conditions; however, no involvement of such proteins with pathogenesis has been reported (McCarter & Silverman, 1987
; Fiore et al., 1997
).
The addition of phosphate at a high concentration to the inoculation medium recovered partially the ability of the mutants to colonize the rabbit small intestine. Mutants and parent strain do not grow at the same rate in low phosphate in vitro but there is no difference in their growth rate in high-phosphate conditions (Fig. 2b). Therefore, one could expect that under phosphate-replete conditions, the mutant and parent strain would colonize the rabbit intestinal mucosa with equal ability. The decreased colonization of the mutant in high-phosphate conditions suggests that member(s) of the Phovc regulon may have a role in intestinal colonization. Thus, the control of expression of phoBRvc, or indeed other members of the Pho regulon, in vivo may not be only in response to phosphate levels. It is known that a number of stimuli other than phosphate can induce components of the Pho regulon in a phosphate-dependent and -independent manner (Wanner & McSharry, 1982
; VanBogelen et al., 1996
). Accordingly, in E. coli the expression of several phosphate-regulated promoters can differ depending on their specific molecular controls. A phoB mutation in E. coli K-12 nearly abolishes phoA transcription, but the induction of another phosphate-regulated gene, psiE, is only reduced about tenfold (Wanner, 1986
). Similarly, the mutation in a Pho-regulon gene enhances enteroinvasive E. coli invasion, suggesting that Pho regulon genes are expressed in vivo (Sinai & Bavoil, 1993
). Furthermore, transcription of himA, a phosphate-starvation-induced gene (Wanner, 1986
), has been shown to be selectively expressed by Salmonella cells grown in vivo (Mahan et al., 1993
)
Intestinal colonization is a multistep process involving a hierarchy of events in which the vibrios in the gut overcome the mucus barrier, adhere to the mucosa, increase in number and detach (Benitez, 1997 ). The V. cholerae PhoBR TCR system may play a role in this complex process by regulating the expression of members of the Pho regulon that are involved in adaptive responses required for intestinal colonization. Given the important role of ToxR in V. cholerae pathogenesis, it is possible that there is some overlap between these two regulatory systems.
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ACKNOWLEDGEMENTS |
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Received 28 January 1999;
revised 16 April 1999;
accepted 21 April 1999.