INTRODUCTION

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Vibrio cholerae is a highly motile gram-negative bacterium and is the causative agent of epidemic cholera. Since an aquatic environmental reservoir exists for this intestinal pathogen, it is reasonable to hypothesize that the expression of genes endowing the appropriate physiology and virulence attributes for human infection is up-regulated upon entry of the bacteria into the human host. We know for example, that in El Tor biotype strains, which cause almost all cholera in the world at present (26), it is difficult to detect the expression of genes within the ToxR/ToxT virulence gene regulon during growth in standard laboratory media; however, upon entry into the host intestinal environment, this regulon undergoes significant induction (3, 18). This regulon includes many of the known virulence factors of V. cholerae such as the phage-encoded cholera toxin structural genes and the toxin-coregulated pilus biosynthetic operon (15, 24).

To learn more about the physiology and virulence of V. cholerae during infection, we recently developed a genetic screen that utilized gene fusions to a site-specific DNA recombinase to identify transcription units that were specifically induced during infection in an infant mouse model of cholera (5). Among the transcripts identified was iviVII, whose transcription was silent during growth of the bacteria in a rich medium but was induced during infection. Initial DNA sequence characterization of iviVII revealed an open reading frame (ORF) that lacked amino acid similarity to known proteins. An insertion mutation within this ORF resulted in a slight reduction in colonization ability in an infant-mouse competition assay (5). These initial results suggested that iviVII encoded a polypeptide(s) that was expressed only during infection and that played a role in intestinal colonization.

In the present study, we have extended our characterization of iviVII to include a complete nucleotide sequence analysis and comparisons of the transcriptional activity of this gene during in vitro growth and during infection of the infant-mouse intestinal tract. iviVII was found to be the last gene, herein renamed vieB, of a putative tricistronic operon that encodes a sensor kinase (VieS) and two distinct response regulatory proteins (VieA and VieB). Evidence for differential transcriptional regulation of the vie genes during in vitro growth was obtained. vieB was found to be transcribed exclusively within the intestine and not in vitro. Finally, a recombinase gene fusion to vieB was used to localize its initial transcriptional induction to the proximal and mid-small intestine at an early time in the infectious process.

	MATERIALS AND METHODS

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DNA sequencing and analysis. Using the previously determined DNA sequence of an internal portion of vieB (iviVII in reference 5), inverse PCR (16) was used to amplify adjacent regions for sequence determination (data not shown). Cycle sequencing with fluorescent dideoxynucleotides was performed and analyzed on an ABI 373A automatic sequencer as specified by the manufacturer (PE Applied Biosystems).

Plasmid constructions. All plasmids used are mobilizable suicide plasmids and are listed in Table 1. Transcriptional fusions to vie genes were constructed by integration of pIVET5 (5) derivatives within the vie locus. Plasmid pAC301 is a derivative of pIVET5 used as an intermediate in some plasmid constructions. A 1-kbp kanamycin resistance (Km^r) gene flanked on both sides by both SfiI and BamHI restriction endonuclease recognition sequences was ligated into the unique BglII site in pIVET5 to generate pAC301. pAC301 was digested with SfiI, and the vector backbone DNA fragment was band purified on an agarose gel. This fragment was subsequently used in cloning some vie gene DNA fragments containing SfiI adapters on each end. The SfiI adapter used was 5'-CGTGGCCGCAC-3', with the last three bases at the 3' end unpaired. A 1,105-bp vieS' fragment (bases 1089 to 2193 in Fig. 1 [see below] and in the GenBank submission) was amplified by PCR with primers 5'-GCAACAACAGAGTGGTTTG-3' and 5'-GGCAAAGGGTTTTTCTTCCAT-3' with Pfu DNA polymerase (Stratagene). SfiI adapters were ligated onto the PCR product ends, and the ligation product was purified away from unincorporated primers and adapters and subsequently ligated into the SfiI-digested pAC301 backbone to generate pAC303. This SfiI cloning method has the advantage that intramolecular ligations of vector and insert are prohibited (data not shown). pAC258 is a derivative of pCRII (Invitrogen) containing a 555-bp inverse PCR product of vieA (bases 5637 to 6148 and 6194 to 6236) generated with AflIII-digested AC-V51 chromosomal DNA and the primers 5'-ATGTATGGCGTTGGGCTATCG-3' and 5'-CACCTCATGCTCCGTCATCTC-3'. Plasmid pAC258 was digested with Sau3AI to release a 403-bp internal fragment of vieA (bases 5669 to 6071), which was subsequently ligated into the unique BglII site of pIVET5 to generate pAC298. Plasmid pSL116 is a derivative of pIVET5. A 4,783-bp vieSA' fragment (bases 755 to 5537) was amplified by PCR with the primers 5'-GCTCTAGACGATAACGCTCCGCATTGAT-3' and 5'-GCTCTAGACCATCTGCGGCATTCGAATA-3', which contained the restriction endonuclease recognition site for XbaI within their 5' termini. The PCR-amplified product was digested with XbaI and subsequently ligated into the unique XbaI site of pIVET5.

Construction of bacterial strains. The bacterial strains used in this study are listed in Table 1. The transcriptional gene fusion strains AC-V296, AC-V311, and AC-V354 were constructed by mating V. cholerae AC-V66 with E. coli AC-E298, AC-E303, and AC-E355, respectively. Plasmids pAC298, pAC303, and pSL116 were first moved into SM10pir by electroporation with selection on Luria-Bertani (LB) agar supplemented with 50 µg of ampicillin per ml. SM10pir has transfer functions for mobilizing the mobRP4-containing plasmids used in this study (Table 1). Each resultant E. coli donor strain was mixed at approximately a 1:1 ratio with the streptomycin-resistant (Sm^r) recipient strain AC-V66 and mated on LB agar for 4 h at 37°C. Each mating mixture was subsequently streaked onto LB agar supplemented with 50 µg of ampicillin per ml and 100 µg of streptomycin per ml and then incubated overnight at 37°C to select exconjugates in which the suicide plasmid had integrated into the recipient chromosome. Integration occurred by single-crossover homologous recombination between the chromosome and plasmid-containing vie gene sequences. Exconjugates were colony purified and stored at 75°C in 50% glycerol.

In vitro transcription assays. V. cholerae transcriptional fusion strains were grown to stationary phase at 37°C with aeration in LB broth containing 100 µg of streptomycin per ml, 30 µg of ampicillin per ml, and 1 µg of tetracycline per ml. These cultures were diluted 1/2,000 into the following media and grown at 37°C with aeration until stationary phase unless indicated otherwise. For strain AC-V232, dilutions were made into the following: LB broth; M9 minimal medium-0.2% glucose-0.05% (wt/vol) Casamino Acids; LB-0.2% or 0.4% (wt/vol) horse bile (Sigma); LB-1, 10, 20, or 50% (vol/vol) 5-day-old CD-1 mouse (Charles River Breeding Laboratories) intestinal homogenate (made by mechanically shearing one small intestine in 0.8 ml of LB broth); syncase broth (8); AKI broth (11); low-iron LB broth containing 0.1 mM dipyridyl (Sigma); low-magnesium M9 minimal medium containing 0.2% (wt/vol) glucose, 0.05% (wt/vol) Casamino Acids, and 50 µM MgCl₂; low-nitrogen-source M9 minimal medium containing 0.2% glucose and 0.05% Casamino Acids but lacking NH₄Cl; LB broth buffered with HCl to pH 8, pH 7, pH 6, or pH 5.7; and LB broth containing 5 mM glutathione. All other V. cholerae fusion strains were diluted in LB broth. Serial dilutions of the resulting stationary-phase cultures were plated on LB agar containing 100 µg of streptomycin per ml. Strain AC-V232 was also grown extracellularly on two intestinal cell lines as follows. Overnight cultures of AC-V232 were washed with fresh LB broth, and 10⁵ CFU was used to infect semiconfluent monolayers of CaCo-2 (20) and the mucus-producing HCT-8 (25) cell lines (kindly provided by D. W. Acheson) grown in modified Eagle's medium (Sigma) at 37°C in a 5% CO₂ atmosphere. After 30 min, the monolayers were washed with phosphate-buffered saline to remove nonadherent V. cholerae, fresh medium was added, and the incubation was continued for another 2 h at 37°C in 5% CO₂. The monolayers were disrupted with Triton X-100, and serial dilutions were plated on LB agar containing 100 µg of streptomycin per ml. After each of the above treatments, the percentage of colonies that were Tc^s was determined by replica plating colonies onto LB agar containing 2 µg of tetracycline per ml and incubating the replica plates at 37°C for 8 h. Loss of Tc^r resulted from TnpR-mediated excision of the res1-tet-res1 cassette from the chromosome and was a measure of the transcriptional activity of the corresponding vie gene fusion to tnpR-lacZY (4). For some strains, the -galactosidase activity of the vie gene fusion to tnpR-lacZY was measured visually after growth on LB agar containing 50 µg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) per ml.

In vivo transcription assays. V. cholerae transcriptional fusion strains were grown overnight to stationary phase at 30°C with aeration in LB broth containing 100 µg of streptomycin per ml, 30 µg of ampicillin per ml, and 1 µg of tetracycline per ml. Approximately 10⁶ CFU of each overnight culture diluted in 50 µl of LB broth was used to intragastrically inoculate 5-day-old CD-1 mice as previously described (4). After 24 h, the bacteria were recovered from the small intestines by homogenization as previously described (4) and approximately 200 CFU was grown on LB agar containing 100 µg of streptomycin per ml. In addition, the overnight cultures used as inocula were serially diluted and approximately 200 CFU was grown on LB agar containing 100 µg of streptomycin per ml. The percent Tc^s CFU was determined for both inocula and intestinally grown bacteria by replica plating as described above.

Competition assays of mutant strains. Each Lac⁺ V. cholerae mutant test strain and the Lac derivative of wild-type strain C6709-1 (AC-V66) were grown to stationary phase at 30°C in LB broth containing 100 µg of streptomycin per ml and then mixed at a 1:1 ratio and diluted 1/2,000 in LB broth. Approximately 10⁶ CFU of each mixture was used to intragastrically inoculate eight 5-day-old CD-1 mice, and the infections were allowed to proceed for 24 h. Each in vivo competition was accompanied by an in vitro competition assay with the same inoculum. The in vitro competition assay was done by diluting a portion of the original inoculum 1/100 in LB broth and incubating it for 24 h at 37°C with aeration. Finally, the precise ratio of test strain (Lac⁺) to virulent strain (Lac) was determined for each inoculum, in vitro competition, and in vivo competition by plating serial dilutions on LB agar containing 100 µg of streptomycin per ml and 50 µg of X-Gal per ml as previously described (5). The plates were incubated overnight at 37°C, and the numbers of Lac⁺ and Lac colonies were counted. The ratios for in vitro and in vivo competitions were corrected for deviations in the inoculum ratio from a value of 1:1.

Nucleotide sequence accession number. The vieSAB genes and flanking sequences have been deposited in GenBank under accession no. AF031552.

	RESULTS

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Sequence analysis of the V. cholerae vieSAB locus. In an earlier study, the sequence of a portion of vieB (originally referred to as iviVII) was determined (5). The deduced amino acid sequence had no similarity to known proteins. To determine the complete amino acid sequence of VieB and to identify flanking genes which might be cotranscribed with vieB during infection, we determined the nucleotide sequence upstream and downstream of vieB on both strands. vieB was found to lie downstream of and in the same transcriptional orientation as two genes designated vieS and vieA (Fig. 1). The absence of identifiable factor-independent transcriptional terminators within or between the three vie genes, as well as the presence of nearly overlapping stop and start codons, suggested that these three genes may be cotranscribed as a tricistronic operon. Upstream of vieSAB was a divergently transcribed ORF designated mgtE, and downstream was a convergently transcribed ORF designated vcc (Fig. 1 and see below).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the V. cholerae vie genes. The vieSAB genes are flanked by a partially sequenced, divergently transcribed ORF designated mgtE and a partially sequenced, convergently transcribed ORF designated vcc. The sites of the transcriptional fusions in strains AC-V311, AC-V336, AC-V354, AC-V296, and AC-V232 are shown at the top. The flanking sequences used to construct in-frame deletion mutations are shown at the bottom along with the corresponding deleted regions, designated by the thin angled lines.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Schematic representations of VieS, VieA, and VieB, and multiple alignments of conserved domains characteristic of two-component signal transduction proteins. Identical residues are indicated by black squares, and functionally similar residues are indicated by gray squares. Conserved phosphorylated histidine and aspartate residues are indicated below by an asterisk. Protein domains in the alignments are numbered and are designated by the abbreviated species name joined to the gene name. Abbreviations: Vc, V. cholerae; Ec, E. coli; Bp, B. pertussis; Ps, Pseudomonas syringae; Bs, Bacillus subtilis; St, Salmonella typhimurium.

Transcriptional activity of vie genes in vitro and during infection. vieB was previously shown to be transcriptionally induced during infection in an infant-mouse model of cholera (5). To test whether vieS and vieA, which may form a tricistronic operon with vieB, were also induced during infection, we constructed transcriptional fusions of each gene to the promoterless synthetic operon tnpR-lacZY. tnpR codes for the site-specific DNA recombinase enzyme, resolvase, from Tn (17). When tnpR is expressed in a genetic background containing the artificial substrate cassette res1-tet-res1, excision of the tet gene occurs, resulting in a Tc^s phenotype of daughter cells (4). Loss of Tc^r in this system has been shown to be a sensitive ex post facto measure of transcription of the gene fusion (4). The sites of the vie gene fusions to tnpR-lacZY are shown at the top in Fig. 1. Plasmids containing 5' fragments of vieS, vieA, or vieSA fused to tnpR-lacZY were transferred by conjugation into a V. cholerae strain which contained the res1-tet-res1 cassette integrated into the endogenous lacZ gene. These suicide plasmids integrated into the V. cholerae vie locus by homologous recombination (data not shown), resulting in generation of the merodiploid transcriptional fusion strains AC-V311, AC-V296, and AC-V354, respectively. The plasmid containing a 5' fragment of vieS was also integrated into a lacZ::res1-tet-res1 strain in which the vieAB genes were deleted, to generate strain AC-V336. The vieB::tnpR-lacZY transcriptional fusion strain AC-V232 was constructed previously (5). The genotype and the predicted phenotype (based on polarity effects of the integrated plasmid) of each fusion strain are listed in Table 2.

tnpR-lacZY vieAB

Transcription assays of vieB under various in vitro growth conditions. Thus far, transcriptional induction of vieB has been observed only during infection of the infant-mouse intestinal tract. To better understand the intestinal signal(s) necessary for induction of vieB transcription and to facilitate promoter mapping and Northern blot analysis of vieB transcripts, we sought to identify in vitro growth conditions that would induce vieB transcription. First, we tested whether simple manipulations of in vitro growth conditions might induce the transcription of vieB. Strain AC-V232 (vieB::tnpR-lacZY) was grown at 37°C in a minimal medium and under growth-limiting conditions for iron, magnesium, and nitrogen source. Limiting magnesium was chosen as a growth condition because of the close linkage of the vie operon to a putative magnesium transporter (mgtE in Fig. 1). None of these growth conditions induced transcription of the vieB::tnpR-lacZY fusion, as shown by no observed loss of Tc^r (Table 3). Note that in prior studies we have shown that the sensitivity of this recombination assay is exquisite compared to that of traditional transcriptional fusion reporters such as phoA (4) or lacZ (unpublished results). Next, we tested a series of growth conditions thought to mimic one or more intestinal parameters. Strain AC-V232 was grown microaerophilically or aerobically in LB broth supplemented with bile salts, mouse intestinal homogenate, or glutathione (a reducing condition). None of the conditions described resulted in loss of Tc^r (Table 3). To mimic the acid shock that V. cholerae may experience within the host stomach before passage into the small intestine, AC-V232 was grown in LB broth at growth-limiting acidic pH (Table 3). V. cholerae is acid sensitive and does not grow below a pH of 5.7 unless preshocked with acid (data not shown). These conditions also failed to induce vieB transcription. To ascertain whether ToxR/ToxT, the major known virulence regulators in V. cholerae (6), play a role in the transcriptional induction of vieB, strain AC-V232 was grown microaerophilically in AKI medium, a growth condition known to activate the ToxR/ToxT regulon in El Tor biotype strains (11). We observed no loss of Tc^r, suggesting that vieB transcription is not induced by growth conditions known to induce the ToxR/ToxT regulon. Finally, AC-V232 was used to infect two intestinal cell lines, CaCo-2 and the mucus-producing cell line HCT-8. Although V. cholerae adhered well to both cell lines and multiplied extensively (data not shown), neither growth condition resulted in loss of Tc^r (Table 3). The above results suggest that the parameter(s) which induce transcription of vieB is specific to the host intestinal environment.

Spatiotemporal studies of vieB transcription during infection. Our inability to induce vieB transcription during in vitro growth, coupled with our lack of knowledge of where the vieB promoter lies, greatly limits the experimental tools we can use to further characterize this gene. However, the resolvase gene fusion reporter system facilitated the further characterization of vieB expression during intestinal infection. Specifically, this reporter system allowed us to determine the spatial and temporal transcriptional induction patterns of vieB during the course of infection. To determine the earliest time of vieB transcriptional induction, we inoculated strain AC-V232 into several infant mice. Then, at various times after inoculation a small intestine was harvested, bacteria were recovered, and loss of Tc^r was measured by replica plating. Both the total CFU and percent Tc^s CFU were determined at each time point (Fig. 3). Transcriptional induction of the vieB::tnpR-lacZY fusion occurred as early as 3 h postinoculation and the percent Tc^s CFU began to level off at approximately 80% after 5 h. The total CFU in the small intestine decreased approximately 20-fold by 5 h but then increased ~15-fold over the next 5 h. These results demonstrate that only ~5% of the inoculum was able to colonize the small intestine and initiate growth. In addition, these results suggest that the majority of the colonizing bacteria experienced the microenvironment(s) necessary to signal transcriptional induction of vieB.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Kinetics of transcriptional induction of vieB during infection. V. cholerae AC-V232 (vieB::tnpR-lacZY) was used to intragastrically inoculate infant CD-1 mice. At the postinoculation times indicated on the x axis, the small intestines were removed and homogenized. Total CFU per intestine, shown on the left axis, was determined by plating serial dilutions on agar medium. The percent Tcs CFU per intestine, shown on the right axis, was determined by replica plating. Each time point was investigated in triplicate, i.e., three animals were used per time point, and the means and standard deviations are shown.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Localization of the intestinal segment where vieB transcription is induced. V. cholerae AC-V232 (vieB::tnpR-lacZY) was used to intragastrically inoculate three infant CD-1 mice. At 3.5 h postinoculation, the small intestines and cecum were removed and dissected into 10 segments of equal length. The total CFU, shown on the right axis, and percent Tcs CFU (bars), shown on the left axis, were determined for each segment. The total CFU for segments 1 to 6 are shown in parentheses above each data point. The data in this figure are from one animal and are representative of the results found with the other two animals.

Role of the vieSAB genes in colonization. We found previously that a V. cholerae strain in which vieB was disrupted by a plasmid insertion exhibited a slight but reproducible reduction in colonization ability as assessed by an infant-mouse competition assay (5). The possibility of negative effects on virulence associated with plasmid sequences being present in the V. cholerae chromosome was controlled for in those experiments by using a virulent isogenic competing strain that contained the same plasmid inserted into the endogenous lacZ gene. These results indicated a possible role for vieB in colonization. To test this possibility more rigorously, we constructed a nonpolar in-frame deletion mutation in vieB, as well as in vieAB and vieSAB, and then tested each mutant strain in the infant-mouse competition assay. In each case, allelic exchange was used to delete the majority of the coding region of each gene (Fig. 1). For the triple and double gene deletion mutations, a fusion of the remaining coding regions of vieS and vieB and of vieA and vieB, respectively, was generated as a result. Each Lac⁺ mutant strain was competed against a fully virulent isogenic Lac strain, both in vitro in LB broth and in vivo in infant mice. Then bacteria recovered from the small intestines and from the in vitro competitions were plated on LB agar supplemented with X-Gal to allow enumeration of each strain. A ratio of test strain to virulent strain (the competitive index) that is less than 1 indicates a decreased colonization ability of the former. All three mutant strains had competitive indices of approximately 1 for both the in vitro and mouse competitions (Table 4). These results show that the vieSAB genes do not play a detectable role in growth in LB broth or in colonization of the infant-mouse small intestine by this competition assay. These results further suggest that the colonization defect observed in the original vieB mutant strain, which contained a plasmid insertion within vieB, may have been due to production of a truncated form of VieB during infection, since amino acids 1 to 350 of VieB is expected to be produced by that strain (data not shown).

	DISCUSSION

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In this study, we have determined the complete nucleotide sequence of the vieSAB genes from Vibrio cholerae. The deduced amino acid sequences revealed that vieSAB codes for three proteins which are predicted to be members of the two-component signal transduction family. This represents the first such system described for this intestinal pathogen. VieS belongs to a subclass of complex sensor kinases such as BvgS of Bordetella pertussis and ArcB of E. coli, which contain two transmitter domains surrounding a central receiver domain. A recent study (9) suggests an intramolecular phosphorelay model for this class of sensor kinases, whereby appropriate stimulation of the sensor domain results in autophosphorylation at the N-terminal transmitter domain followed by transphosphorylation of the medial receiver domain and then of the C-terminal transmitter domain; the last of these can then transfer the phosphate to its cognate response regulator, i.e., BvgA or ArcA in the examples above. In the case of the V. cholerae VieS sensor kinase, an additional level of complexity is introduced by the presence of two distinct response regulators, VieA and VieB, which are encoded by genes in an apparent operon with vieS. VieA and VieB both contain highly conserved N-terminal phosphoreceiver domains, but only VieA appears to have a C-terminal DNA-binding domain. Thus, the role of VieB is not clear. It may modulate the phosphorylation state of VieA indirectly by competing for phosphate from VieS. Alternatively, but not exclusively, VieB may serve as an effector protein having an activity unrelated to regulation of transcription.

In a previous study, we showed that vieB was an infection-induced gene that was transcriptionally silent during in vitro growth (5). In this study, transcriptional fusions of vieS and vieA to reporter genes encoding the site-specific DNA recombinase, resolvase, and the E. coli LacZY proteins revealed that vieS is transcribed during in vitro growth in a VieAB-independent manner. In contrast, vieA was transcriptionally active during in vitro growth only in the presence of VieA. Because vieA was found to be transcriptionally silent during infection in a vieA mutant background, vieA is also autoregulatory during infection or is simply not transcribed in the host. These data, coupled with our finding that transcription of vieB occurs only during infection and is VieB independent, lead to several potential regulatory schemes for vieS, vieA, and vieB. A likely scenario is that each of the three vie genes has its own distinct promoter, where that for vieS is constitutively active, that for vieA is autoregulatory both in vitro and during infection, and that for vieB is active only during infection and may rely on VieS and VieA for its induction. A second possibility is that vieA and vieB are cotranscribed via a single promoter which is VieA dependent and VieB independent but that transcription terminates prior to reaching vieB during in vitro growth. Because vieB is transcribed during infection, this latter scenario requires either antitermination during infection or a sufficient level of readthrough if vieA transcription was fully induced. Experiments are in progress to distinguish between these and other possible regulatory schemes. The differential transcription patterns of vieSAB observed thus far reveal that the vie genes are not cotranscribed as a tricistronic operon during in vitro growth.

In the course of infection, V. cholerae cells must encounter a variety of biochemical and nutritional parameters that constitute the microenvironments of the small intestine. Since vieB is an infection-induced gene, its transcription must be modulated by one or more of these parameters. Our attempts to induce transcription of vieB by mimicking some of these parameters in vitro were wholly unsuccessful. Transcription of vieB was not modulated by growth in a minimal medium, limiting divalent cations or nitrogen source, acid shock, or reducing conditions. Transcription of vieB was also not induced during growth under conditions known to induce genes within the ToxR/ToxT virulence gene regulon. We were also unable to induce transcription of vieB by introducing factors endogenous to the small intestine such as bile salts and intestinal homogenate. Growth of V. cholerae on two intestinal cell lines, CaCo-2 and the mucus-producing cell line HCT-8, also failed to induce transcription of vieB. These results suggest that the host intestinal parameter(s) which induces the transcription of vieB may be complex, i.e., requiring multiple signals for induction, and/or cryptic, i.e., requiring an unknown or simply untested parameter(s) for induction. To our knowledge, vieB represents the first bacterial gene characterized for which an in vitro growth condition capable of inducing its transcription has not been identified. Indeed, the identification and characterization of such a gene would be exceedingly difficult without a reporter system like that used in the present study (resolvase gene fusions), which provides a means to assay transcription during bacterial growth in complex environments such as the intestinal tract of an intact animal.

In this and in a previous study (5), vieB transcription was measured during infection by using a resolvase gene fusion reporter. In this report, we have expanded the use of this reporter technology to include anatomic and temporal determinations of vieB transcription during a bona fide infection. First, we determined that vieB transcription was induced sharply at an early time of infection (3 h). Then we made use of this result to determine the spatial pattern of vieB transcription at 3.5 h postinoculation, and we found that the proximal and mid-small intestine (duodenum and jejunum) contain an inducing environment for vieB.

A second result of these experiments was the finding that only 5% of the initial infecting population of V. cholerae colonizes the small intestine and then multiplies. This is in contrast to the result previously reported by Skorupski and Taylor (22), who found no drop in the number of bacteria recoverable from the small intestine at any time during the infection. This discrepancy may be due to the use in that study of classical biotype strains of V. cholerae, which may colonize the small intestine more efficiently than the El Tor biotype strains like that used in the present study. Thus, in our experiments, it is unclear what happens to the other 95% of the bacteria. Given that the number of bacteria increases greatly toward the distal small intestine and cecum (at 3.5 h postinoculation), it is likely that these "missing" bacteria simply passed through the lumen of the small intestine to the large intestine. Interestingly, induction of vieB transcription at this early time of infection occurred predominantly among the minor population of bacteria that colonized the proximal and mid-small intestine. We predict from these results that transcriptional induction of vieB occurs soon after colonization of the small intestine and that the majority of bacteria inoculated do not colonize this site and are thus not exposed to the inducing environment. Our data do not preclude the existence of other vieB-inducing environments further down the intestinal tract or at later times during infection. Our identification of the time and site of vieB transcriptional induction within the intestinal tract may aid in the identification of the exact host inducing parameter(s). This may require further dissection of the inducing environment, for example by recovering bacteria from different locations within the inducing region (lumen, mucus layer, epithelial surface) and assaying vieB expression with the resolvase or other reporter system.

There are several possible ramifications for the transcriptional induction of an infection-specific gene in relation to colonization ability of V. cholerae. One is the requirement of the gene product to assist attachment to host surfaces, and a second is to support survival or growth within the host. For example, mutations disrupting the V. cholerae toxin-coregulated pilus major subunit gene tcpA have been shown to cause a severe reduction in colonization (3, 18), presumably because the bacteria can no longer attach to mucosal surfaces in the small intestine. A plasmid insertion mutation in vieB caused a reduction in colonization but had no effect on in vitro growth (5). However, deletion mutations in vieB as well as in vieAB and vieSAB do not cause a detectable reduction in colonization by the same assay. The discrepancy between these two results may be due to production (during infection) of a truncated peptide of VieB in the plasmid insertion strain that is somewhat toxic to the bacteria. These results lead to yet a third ramification for the transcriptional induction of an infection-induced gene in V. cholerae, which is that such a gene, as is the case for the vieSAB genes, may not play a detectable, or any, role during colonization. Such a result may be due to a limitation(s) of the animal model used or may be due to the presence of redundant regulatory and/or effector functions.