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Infection and Immunity, March 2000, p. 1507-1513, Vol. 68, No. 3
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Infectious CTXPhi and the Vibrio Pathogenicity Island Prophage in Vibrio mimicus: Evidence for Recent Horizontal Transfer between V. mimicus and V. cholerae

E. Fidelma Boyd,1 Kathryn E. Moyer,1 Lei Shi,2 and Matthew K. Waldor1,*

Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111,1 and Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan2

Received 12 October 1999/Returned for modification 12 November 1999/Accepted 29 November 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Vibrio mimicus differs from Vibrio cholerae in a number of genotypic and phenotypic traits but like V. cholerae can give rise to diarrheal disease. We examined clinical isolates of V. mimicus for the presence of CTXPhi , the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V. cholerae strains. Four V. mimicus isolates were found to contain complete copies of CTXPhi . Southern blot analyses revealed that V. mimicus strain PT5 contains two CTX prophages integrated at different sites within the V. mimicus genome whereas V. mimicus strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXPhi . We detected the replicative form of CTXPhi , pCTX, in all four of these V. mimicus isolates. The CTX prophage in strain PT5 was found to produce infectious CTXPhi particles. The nucleotide sequences of CTXPhi genes orfU and zot from V. mimicus strain PT5 and V. cholerae strain N16961 were identical, indicating contemporary horizontal transfer of CTXPhi between these two species. The receptor for CTXPhi , the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIPhi , was also present in the CTXPhi -positive V. mimicus isolates. The nucleotide sequences of VPIPhi genes aldA and toxT from V. mimicus strain PT5 and V. cholerae N16961 were identical, suggesting recent horizontal transfer of this phage between V. mimicus and V. cholerae. In V. mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V. cholerae. These results suggest that V. mimicus may be a significant reservoir for both CTXPhi and VPIPhi and may play an important role in the emergence of new toxigenic V. cholerae isolates.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Cholera toxin (CT) is encoded by the ctxAB operon, which resides in the genome of CTXPhi , a filamentous bacteriophage that specifically infects Vibrio cholerae (40). CTXPhi is found in all epidemic V. cholerae isolates but is rarely recovered from non-O1/non-O139 V. cholerae environmental isolates (12). The CTXPhi genome can integrate into the V. cholerae genome to form a stable prophage or it can replicate as a plasmid in isolates lacking an appropriate integration site. Of the nearly 200 recognized serogroups of V. cholerae only the O1 and O139 serogroups are associated with epidemics of cholera (2). In classical biotype strains of V. cholerae serogroup O1 there is a CTX prophage on each of the two V. cholerae chromosomes (24, 38), whereas in El Tor biotype strains of V. cholerae serogroup O1 the CTX prophages are tandemly arranged on the larger of the two chromosomes (26, 30). CTXPhi has a 6.9-kb genome made up of two functionally diverse regions: the core and RS2 regions. The core region encodes CT and contains the genes involved in phage morphogenesis, including genes that are thought to encode the major and minor phage coat proteins and a protein required for CTXPhi assembly (40). The RS2 region contains genes required for replication, integration, and regulation of CTXPhi (20, 43). In El Tor V. cholerae isolates, the CTX prophage genome is often flanked by an additional 2.7-kb region, designated RS1, that is similar to RS2 but that contains an additional gene (15, 26, 43).

Uptake of CTXPhi into V. cholerae is dependent upon the toxin-coregulated pilus (TCP), a bundle-forming pilus that is also an essential intestinal colonization factor (36). Initially, it was shown that the genes encoding TCP resided on a pathogenicity island known as the TCP island or vibrio pathogenicity island (VPI), which is integrated near the ssrA gene (18, 21). However, TCP has recently been proposed to be encoded on a novel filamentous phage named VPIPhi (19). Thus, the filamentous phage VPIPhi encodes TCP, an important virulence factor, and is the receptor for another temperate filamentous phage, CTXPhi , which itself encodes a virulence factor, CT.

The species Vibrio mimicus was first proposed to encompass biochemically atypical non-O1 V. cholerae isolates. V. mimicus is phenotypically and genotypically distinct from V. cholerae in several respects, and it can be readily differentiated from V. cholerae on the basis of a number of biochemical reactions (10). Thus, unlike V. cholerae, V. mimicus is negative in sucrose, Voges-Proskauer, corn oil, and Jordan tartrate reactions (10). Sequence analysis of the mdh (malate dehydrogenase) gene from a V. mimicus strain revealed that the average pairwise divergence between typical V. cholerae isolates and V. mimicus was 10.5% (6). The natural habitat of V. mimicus, like that of V. cholerae, is the aquatic ecosystem, where it has been found both as a free-living bacterium and in association with phytoplankton and crustaceans (1, 7). Consumption of V. mimicus-contaminated shellfish has been linked to the development of gastroenteritis (1). However, unlike V. cholerae, V. mimicus has not been associated with epidemics of diarrhea. The virulence determinants of V. mimicus have not been well characterized. The ability of V. mimicus strains to produce various toxins has been studied (7, 8, 31, 33-35). Several V. mimicus strains isolated from clinical and environmental sources were shown to produce multiple toxins, including a hemolysin, zonula occludens toxin, and a heat-stable enterotoxin, as well as a CT-like toxin (7, 8, 31, 33-35). To date, the presence of CTXPhi in V. mimicus has not been reported.

The distribution of CTXPhi and VPIPhi outside of V. cholerae is unknown, as is the extent to which V. mimicus and V. cholerae share pools of bacteriophages. We therefore examined clinical isolates of V. mimicus to determine if this species contains the genes composing CTXPhi and/or VPIPhi and consequently whether V. mimicus can serve as a reservoir for these important bacteriophages. We found V. mimicus isolates that contained both the CTX and VPI prophages. Nucleotide sequence identity of genes from both these prophages with the respective V. cholerae prophages suggests contemporary horizontal transfer of bacteriophages between these two Vibrio species.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Bacterial strains. The bacterial strains used in this study are shown in Table 1. Bacterial strains were stored at -70°C in Luria-Bertani (LB) broth containing 20% glycerol. The antibiotic streptomycin was used at 200 µg/ml.

                              
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  		TABLE 1.   Bacterial strains used in this study

Southern hybridization analyses. V. mimicus DNA was extracted and purified using the G-nome DNA isolation kit from BIO101 (Vista, Calif.). DNA was digested with several restriction enzymes (New England Biolabs, Beverley, Mass.), and the fragments were separated by electrophoresis in 0.6% agarose. The fragments were then transferred to nylon membranes for hybridization. Three nonradioactive DNA probes to detect the presence of CTXPhi were produced by PCR amplification using V. cholerae strain SM115 (15) as a template (Table 2 and Fig. 1). DNA probes were labeled with fluorescein-conjugated nucleotides and, after hybridization, were detected by the ECL system according to the manufacturer's protocol (Amersham; Arlington Heights, Ill.). The rstA probe (5) assays for the presence of the RS2 region, whereas the core (5) and ctx probes assay for the presence of the core region of the CTXPhi genome. Southern blot analyses to detect the presence of DNA sequences that flank the CTX prophage in El Tor V. cholerae, the TLC element and an RTX cluster, in V. mimicus were performed using the DNA probes tlc (5) and rtx (5).

                              
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  		TABLE 2.   Primers and probes used in this study



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  		FIG. 1.   (A) Schematic representation of the CTX prophage (~7 kb) and surrounding sequences in V. cholerae. The open reading frames (ORFs) are shown as arrows. Black arrowheads, locations of the PCR primers listed in Table 2; vertical lines, restriction enzyme sites; horizontal bars, positions of the three DNA probes used for hybridization. (B) Schematic representation of the VPI prophage (~40 kb) in V. cholerae. The ORFs and gene clusters are shown as dotted arrows. Black arrowheads, locations of the PCR primers listed in Table 2; horizontal bars, locations of the four DNA probes used for hybridization.

To determine the distribution of VPIPhi among V. mimicus isolates, four DNA probes which span the genome of VPIPhi , ald, tcpA, toxT, and acf (Fig. 1 and Table 2), were utilized. The ald probe includes the aldA gene found in the 5' region of the VPI prophage. The tcpA probe encompasses the tcpA gene in the 5' region of the tcp operon, whereas the toxT probe hybridizes to the 3' region of this operon. The acf probe is limited to the acfB gene of the acf gene cluster in the 3' region of the VPI prophage. All probes were made by PCR amplification from V. cholerae strain SM115 and purified using the Qiaquick PCR purification kit (Qiagen, Valencia, Calif.).

PCR analyses. To determine whether the chromosomal DNA sequences flanking the 5' and 3' ends of the CTX prophage in V. mimicus were identical to those of El Tor V. cholerae, we carried out a PCR assay with primer pairs rig1 and tlc3 and ctxB3 and rtxA2 (Table 2; Fig. 1A), which are located within the CTX prophage and the known flanking sequences of V. cholerae El Tor strain N16961.

PCR assays were also used to determine whether the chromosomal regions flanking the 5' and 3' ends of the VPI prophage in V. mimicus were identical to those in V. cholerae. Primer pairs VPI5 and VPI8 and VPI9 and VPI10, designed from the 5' and 3' junction sequences of the VPI prophage from V. cholerae strain N16961, were used for these assays. Primer VPI5 is derived from a sequence in the 5' chromosomal flanking region of the VPI prophage integration site in V. cholerae, and primer VPI8 is located within the 5' region of the VPI prophage genome. To amplify the VPI prophage 3' chromosomal junction, primer VPI9, which lies within the putative integrase gene of VPIPhi , and primer VPI10, which is located within the 3' flanking chromosomal DNA of V. cholerae strain N16961, were used (Table 2, Fig. 1B).

Detection of the replicative form of CTXPhi . To determine whether the V. mimicus strains that were found to contain the CTX prophage also harbored the replicative form of CTXPhi , Qiagen plasmid spin kits were used to isolate plasmid DNA from 5-ml overnight cultures. These plasmid DNA preparations were tested for the presence of the ~7-kb pCTX plasmid band by Southern blot analysis using the CTXPhi core region DNA probe (Table 2, Fig. 1A).

Recovery of infectious CTXPhi particles. We assayed filter-sterilized supernatants from each of the CTXPhi -positive V. mimicus strains for the presence of infectious CTXPhi particles as previously described (9, 22). Briefly, 1.5 ml of sterile supernatant from 2-ml overnight cultures was mixed with 1 µl of agglutinated classical strain O395 for 20 min. After addition of 1.5 ml of LB broth, the cultures were incubated overnight at 30°C. Qiagen plasmid spin kits were then used to isolate plasmid DNA from these overnight cultures. These plasmid DNA preparations were digested with SphI, which linearizes the CTXPhi genome, separated on agarose gels, and assayed for the presence of pCTX DNA by Southern analysis.

Nucleotide sequencing and analysis. The orfU and zot genes from the CTXPhi in V. mimicus strain PT5 were sequenced directly from the products of PCR. PCR primers were designed from the published sequences of these genes in V. cholerae (14, 37) (Table 2). The aldA and toxT genes of VPIPhi from PT5 were also sequenced directly from PCR products with primers designed from the published V. cholerae sequences (18, 21) (Table 2 and Fig. 1). The mdh gene sequence was also determined for V. mimicus strain PT5 using primers derived from a V. cholerae El Tor strain N16961 sequence (http://www.tigr.org/tigr_home/tdb/mdb/mdb.html). Sequencing was performed with an Applied Biosystems model 373A automated DNA sequencing system at the Tufts University School of Medicine Core Facility using a DyeDeoxy terminator cycle sequencing kit. The sequences were determined in both orientations with additional internal primers, and the overlapping sequences were assembled and edited with the MacVector program. Comparisons of the V. mimicus mdh, orfU, aldA, and toxT gene sequences with the respective V. cholerae sequences were carried out using MacVector.

Mouse colonization assay. A competition assay between either O395 or a lacZ deletion mutant of O395, LAC-1 (42), and spontaneous Smr derivatives of three of the four VPIPhi -positive V. mimicus strains was done essentially as described previously (42). PT5 is lacZ positive, whereas 9583 and 523-80 are lacZ negative. The LAC-1 strain has been shown to colonize the suckling mouse intestine as well as O395 does. In this assay LAC-1 and O395 served as standards for comparative analysis of the intestinal colonization properties of the different test strains. Three- to five-day-old suckling mice (Charles River) were inoculated orally with a 1:1 mixture of V. cholerae O395 or LAC-1 and a V. mimicus test strain and then sacrificed 24 h later. Viable-cell counts were obtained by plating dilutions of the intestinal homogenates on LB agar containing streptomycin and 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-Gal) (40 µg/ml). Three animals were used per group. The relative colonization efficiencies of the two strains were calculated by comparing the ratios of blue (lacZ+) colonies to white (Delta lacZ) colonies in the intestinal homogenates and the inocula.

Nucleotide sequence accession numbers. The nucleotide sequences obtained during this study have been deposited in GenBank under accession no. AF207856 to AF207858.


    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References

Presence of CTXPhi genes in V. mimicus. A recent report by Shi et al. (33) demonstrated the presence of ctxA-related sequences in four V. mimicus clinical isolates. Since we previously found that the ctxAB genes are part of the genome of CTXPhi , we investigated whether these four strains contained a CTX prophage(s). We also tested five additional V. mimicus clinical isolates that were previously found not to contain the ctxA gene for CTXPhi -related sequences. We found by PCR and DNA hybridization analyses that the four V. mimicus isolates previously reported to contain ctxA-related sequences, PT5, PT48, 523-80, and 9583, each contained a complete CTXPhi (Table 3). Remarkably, although these four V. mimicus strains all belonged to the same serogroup (O115), they were recovered at different times and from different continents (Table 1). The five additional V. mimicus isolates examined did not show homology to any of the CTXPhi probes, indicating the absence of CTXPhi in these isolates.

                              
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  		TABLE 3.   Distribution of CTXPhi and VPIPhi genes in V. mimicus strains

We further analyzed the CTXPhi genes in the four strains containing ctxAB by Southern blotting, to determine if the CTXPhi genome was integrated into the V. mimicus chromosome (as is observed in V. cholerae) and to assess the copy number and arrangement of the CTX prophages. Southern hybridization analyses were carried out on chromosomal DNA prepared from each V. mimicus strain digested with restriction enzymes that cut either at no sites (EcoRI) or at one site (SphI, BglII) in the CTX prophage genomes in classical and El Tor V. cholerae strains (30). Similar to analyses of classical strains of V. cholerae, these analyses of strain PT5 indicate the presence of two CTX prophages at different locations in the genome (Fig. 2). Thus, when strain PT5 chromosomal DNA was digested with EcoRI and hybridized with either the core region or rstA probes, two bands were observed (Fig. 3A and B). The presence of two unlinked CTX prophages in PT5 was confirmed in blots of SphI- and BglII-digested DNA. In these digests, with either the core or rstA probes, two bands were also observed (Fig. 3).


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  		FIG. 2.   Chromosomal arrangement of CTX prophages in V. cholerae strains SM115 and O395 and in V. mimicus strains PT5, PT48, 523-80, and 9583 as determined by Southern blot and PCR analyses. Vertical lines indicate restriction enzyme sites. Boxes with question marks adjacent to the CTX prophages represent those cases where the phage-chromosome junction sequences are unknown.


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  		FIG. 3.   Southern blot analyses of the integrated copies of CTXPhi in V. mimicus strains PT5, PT48, 583-80, and 9583 and V. cholerae classical strain O395 and El Tor strain SM115. Equal amounts of chromosomal DNAs from the indicated strains were digested with EcoRI, SphI, and BglII, electrophoresed, transferred to nitrocellulose, and probed with CTXPhi core (A) and rstA (B) probes. R and L, right and left junction fragments, respectively; T, tandem arrangements of integrated CTXPhi .

In V. mimicus strain PT48, a clinical isolate from Bangladesh, Southern blot analyses demonstrated the presence of tandemly arranged CTX prophages, similar to the arrangement present in El Tor V. cholerae strains (Fig. 2). Southern hybridization of EcoRI-digested PT48 DNA probed with the core or rstA probes revealed only a single hybridizing band >12 kb (Fig. 3A and B). However, PT48 DNA digested with SphI and hybridized with the core probe revealed two hybridization species: a 5-kb band and a 7-kb band (Fig. 3A). The 5-kb band represents the right junction fragment of CTXPhi in the chromosome, and the 7-kb band corresponds to the tandemly integrated copies of CTXPhi . Hybridization with the rstA probe also identified two bands, a 7-kb tandem band and an 8-kb left junction fragment (Fig. 3B). The fact that the size of the tandem band in the SphI digests of PT48 probed with the core and rstA probes is 7 kb and not ~10 kb, as seen with the El Tor strain SM115, suggests that there is no intervening RS1 sequence between the CTX prophages in PT48. BglII digests of PT48 DNA hybridized with the core and rstA probes revealed two bands, confirming the tandem arrangement of CTX prophages in PT48.

V. mimicus clinical isolates 523-80 and 9583, which were recovered from patients in the United States, also contained tandemly arranged CTX prophages (Fig. 2). The strains gave identical banding patterns when digested with EcoRI, SphI, or BglII, and probed with the core region or the rstA probe. Thus, two fragments of EcoRI-digested 523-80 DNA hybridized with the core probe: a 7-kb band and an 8-kb band (Fig. 3A). An identical pattern was found with the rstA probe, suggesting the presence of two CTX prophages at different map positions. However, Southern blots of SphI-digested DNA probed with the core probe gave two bands: a 7-kb band and a 5-kb band; similarly, hybridization with the rstA probe gave a 7-kb band and an 8-kb band, suggesting a tandem arrangement of CTX prophages similar to the pattern seen with strain PT48. Taken together, these results suggest that the CTX prophages in 523-80 and 9583 contain an EcoRI site not present in the PT5, PT48, or V. cholerae serogroup O1 CTX prophages, explaining the two bands seen with this enzyme (Fig. 3A and B). Digestion of 523-80 and 9583 DNA with BglII and hybridization with core and rstA probes also suggested DNA sequence polymorphisms in the CTX prophages in these strains, as both probes identified a single >12-kb band, indicating the absence of a BglII site in rstR. These analyses of the arrangements of the CTX prophages in these four V. mimicus isolates demonstrate that, as for V. cholerae, the CTX prophages in V. mimicus occur in multiple copies and have alternative arrangements in the genome.

Analysis of CTX prophage flanking DNA. V. cholerae serogroup O1 strains contain tandemly arranged copies of an integrated toxin-linked cryptic plasmid, pTLC, adjacent to the 5' end of the CTX prophages (32). Adjacent to the region 3' of the CTX prophages there is a recently described RTX toxin gene cluster (23). We used PCR analyses to investigate whether the TLC element and the RTX cluster are present in the CTX prophage-positive and -negative V. mimicus strains. The primer pairs for these PCRs, rig1 and tlc3 and ctxB3 and rtxA2 (Fig. 1), were designed to amplify TLC sequences 5' of the CTX prophage and RTX sequences 3' of the CTX prophage. No PCR products were detected in the nine strains tested using these primer pairs (Table 3). To further investigate whether there are sequences homologous to TLC and RTX in these V. mimicus strains, we performed Southern blot analyses with a 1.5-kb tlc probe and a 1.3-kb rtxA probe. No hybridization bands were obtained. Therefore, V. mimicus, unlike V. cholerae, for which previous studies have demonstrated the co-occurrence of the CTX prophage and the TLC element, has no TLC element.

Detection of CTXPhi RF and infectious virions. To determine whether the four V. mimicus strains that harbor the CTX prophage also contained the ~7-kb plasmid replicative form (RF) of CTXPhi , pCTX, plasmid DNA preparations from these strains were probed with a CTXPhi core region probe in a Southern blot. The plasmid preparations from all four strains yielded hybridizing DNA of the appropriate size (data not shown), indicating that these strains harbored the CTXPhi RF in addition to the CTX prophage.

We then tested whether these strains produced infectious CTXPhi particles. To accomplish this, filtered sterile supernatants from overnight cultures of V. mimicus strains were used to infect agglutinated (TCP+) classical strain O395 with CTXPhi (27), according to an established protocol (9, 22, 40). Cell-free supernatant from one strain, PT5, was found to transfer CTXPhi DNA to O395, thus demonstrating the ability of this strain to produce infectious CTX virions (data not shown). This is the first report of the isolation of CTXPhi outside of V. cholerae. This finding indicates that CTXPhi has more than one host species and hence that V. mimicus may represent an important reservoir of CTXPhi in the natural environment. For reasons which are not yet known, we were unable to detect transfer of CTXPhi DNA from supernatants derived from PT48, 523-80, and 9583. It is possible that our inability to detect transfer of CTXPhi from these three V. mimicus strains into O395 is a reflection of phage immunity. We have found that the CTXPhi repressor, RstR, functions as an effector of phage immunity by repressing transcription of rstA, a gene required for CTXPhi replication (9, 20). If rstA in the CTXPhi derived from V. mimicus strains PT48, 523-80, and 9583 is repressed by the RstRclassical repressor in O395, then these three V. mimicus-derived CTXPhi s would not replicate in this strain.

Comparison of V. mimicus and V. cholerae CTXPhi nucleotide sequences. There is a sporadic distribution of CTXPhi in V. cholerae and V. mimicus strains. Among V. cholerae isolates, CTXPhi is confined to epidemic strains and is rarely recovered from non-O1/non-O139 isolates. This sporadic distribution of CTXPhi in V. cholerae and V. mimicus populations is most likely the result of horizontal transfer of CTXPhi between Vibrio species. However, it is also possible that the CTX prophage was present in the most recent common ancestor of V. cholerae and V. mimicus, with subsequent loss of this phage from most isolates of these two species. To distinguish between these two possibilities and to begin to elucidate the evolutionary history of CTXPhi , we sequenced two genes from the core region of the CTXPhi derived from V. mimicus strain PT5 and compared these sequences with sequences of the same region from El Tor V. cholerae strain N16961 from the TIGR database (http://www.tigr.org/tigr_home/tdb/mdb/mdb.html). We also sequenced the chromosomally encoded malate dehydrogenase gene (mdh), which is a basic metabolic housekeeping gene found in all Vibrio species and therefore is ancestral to the species. Comparison of the mdh sequences from V. mimicus and V. cholerae provides a measure of the divergence of the two species. Of the 640 bp of mdh sequenced from V. mimicus strain PT5, there were 64 polymorphic sites, compared with the V. cholerae mdh from strain N16961. This indicates that the level of nucleotide sequence divergence between the two species is approximately 10%, similar to values from previous reports (6). This level of nucleotide sequence divergence is similar to the level of divergence between Escherichia coli and Salmonella enterica (3, 4). In striking contrast, the 992 bp of the orfU gene and the 1,036 bp of the zot gene derived from V. mimicus strain PT5 and V. cholerae strain N16961 contained no polymorphic nucleotide sites. This nucleotide sequence identity of the CTXPhi genes within otherwise divergent chromosomal backgrounds strongly argues against the possibility that CTXPhi was inherited from a common ancestor of V. cholerae and V. mimicus. Rather, the CTXPhi sequence identity between these two species suggests that there was relatively recent horizontal transfer of this phage between these two species. The site of this phage transfer could be their shared ecological niche: the estuarine environment. However, since the V. mimicus isolates examined are clinical isolates, we cannot formally rule out the possibility that CTXPhi transfer between these two species may have occurred in the human host.

Presence of VPIPhi genes in V. mimicus. The receptor for CTXPhi in V. cholerae is TCP. The genes encoding TCP have recently been proposed to be an integral part of the genome of the newly described novel filamentous phage VPIPhi , found in pathogenic isolates of V. cholerae (19). Since TCP plays a critical role in the uptake of CTXPhi into V. cholerae, we tested whether VPI prophage sequences were present in the four toxigenic and five nontoxigenic clinical isolates of V. mimicus. All four CTXPhi + V. mimicus isolates yielded PCR products with four sets of primer pairs that span the VPIPhi genome (Table 3; Fig. 1). In contrast, PCR products were not amplified from the five V. mimicus strains that did not harbor a CTX prophage (Table 3). Southern analyses confirmed that these five strains lacked sequence homology to VPIPhi . In V. cholerae the VPI prophage is found adjacent to the ssrA locus (19). Interestingly, by PCR analyses we found that the VPI prophage was integrated at the identical chromosomal sites in the four VPI prophage-positive V. mimicus isolates (Table 3), and we were able to amplify by PCR the chromosomal junction fragments of the VPI prophage integration site of V. cholerae from all V. mimicus toxigenic isolates.

Since TCP is required for CTXPhi infection of V. cholerae, it was suggested that there were two critical sequential steps in the evolution of pathogenic V. cholerae (5, 12, 13, 25, 40, 41). First, strains had to acquire TCP (presumably via infection with VPIPhi ), and second, having acquired the CTXPhi receptor, these TCP+ strains were then infected with and lysogenized by CTXPhi . Our finding that all four V. mimicus isolates that contained CTX prophages also contained VPI prophages supports the notion that TCP is required for acquisition of CTXPhi . The co-occurrence of these two bacteriophages in two different species is striking from an evolutionary perspective, as it would appear that these two unrelated bacteriophages have similar host ranges and that their acquisition and potentially their function are intimately interconnected.

Comparison of V. mimicus and V. cholerae VPI prophage nucleotide sequences. To begin to address the evolutionary history of VPIPhi in V. cholerae and V. mimicus, we sequenced two genes carried by VPIPhi , aldA and toxT from V. mimicus strain PT5, and compared these sequences with the aldA and toxT sequences from V. cholerae strain N16961. One of the sequenced genes, aldA, codes for an aldehyde dehydrogenase (29), and the other, toxT, codes for a transcriptional activator for virulence gene expression (11). The aldA and toxT sequences from PT5 and N16961 were identical. The identity of these VPI prophage sequences from V. mimicus and V. cholerae suggests recent horizontal transfer of VPIPhi between V. mimicus and V. cholerae and lends additional support to the idea that the VPI is a mobile genetic element.

Mouse colonization assay. The identification of V. mimicus strains containing the CTX and VPI prophages, whose genomes encode many of the known virulence factors of V. cholerae, raised the question of whether these V. mimicus isolates are as pathogenic as V. cholerae isolates that contain these prophages. To begin to experimentally assess the virulence properties of three of the V. mimicus strains containing both prophages (PT5, 523-80, and 9583), we performed mouse colonization assays. Twenty four hours after 1:1 mixtures of O395 with each of these three strains were intragastrically inoculated into suckling mice, the ratio of V. mimicus cells to O395 cells in intestinal homogenates was found to be less than 1:1,000 for all three V. mimicus strains. Thus, compared to V. cholerae, these V. mimicus isolates appear to be extremely attenuated for intestinal colonization. The molecular basis for this attenuation is not known. If TCP is properly expressed in vivo by these V. mimicus strains containing the VPI prophage, these data indicate that other colonization factors, such as the serogroup antigen, are critical for intestinal colonization.

Conclusions. The extent to which V. cholerae and V. mimicus share gene pools is unknown, as is the ancestry of bacteriophages in these species. The data presented in this report indicate that V. cholerae is not the only host of both CTXPhi and VPIPhi . The finding of nucleotide sequence identity of genes from both CTXPhi and VPIPhi derived from V. mimicus and V. cholerae strongly suggests that contemporary horizontal transfer of bacteriophages between these species has occurred. However, we cannot conclude from this sequence identity whether these phage genomes were transferred from V. cholerae to V. mimicus or vice versa. Also, although it seems likely that these two phage genomes were transferred between these Vibrio species via transduction, we cannot strictly come to this conclusion from our data. Regardless of this limitation, V. mimicus may represent an important environmental reservoir for both bacteriophages and, therefore, may play a significant role in the emergence of new toxigenic V. cholerae isolates.


    ACKNOWLEDGMENTS

We thank our colleagues Brigid Davis and Bianca Hochhut for critically reading the manuscript. We are most grateful to Anne Kane and the NEMC GRASP Center (grant P30DK-34928) for providing us with culture media.

This work was supported by NIH grant AI-42347 to M.K.W. E.F.B. was supported by NIH training grant T32 AI-07329. M.K.W. is a PEW Scholar of Biomedical Research.


    FOOTNOTES

* Corresponding author. Mailing address: Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Tufts University School of Medicine, 750 Washington St., Boston, MA 02111. Phone: (617) 636-7618. Fax: (617) 636-5292. E-mail: matthew.waldor{at}es.nemc.org.

Editor:   D. L. Burns


    REFERENCES
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Abstract
Introduction
Materials and Methods
Results and Discussion
References

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