Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae

Abstract

Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.

FIG. 1

Abbreviated maps of VPI, CTXφ, and RS1, including relative positions of genes that were studied here. These maps are not complete or to scale. Genes indicated above the lines are transcribed left to right; those below the lines are transcribed in the opposite orientation These maps are based on references , , and .

FIG. 2

RAPD profiles of representative V. cholerae environmental isolates generated using primers 1281 and 1283. Lane designations correspond to RAPD types discussed in the text and to SCE isolates defined in Table 1, as follows: 1a, SCE4 and SCE5; 2, SCE226; 3, SCE258; 4, SCE260 and SCE264; 5, SCE263; 6, SCE200; and 7, SCE223. The O139 epidemic strain used is SG24. m, 1-kb ladder (size marker).

FIG. 3

TcpA alignment and phylogenetic tree. (A) Alignment of inferred amino acid sequences of TcpA-cla with the TcpA-env protein found here (from SCE188) and with other TcpA proteins (Nandi, accession no. AF139626 [33]; Novais, accession no. AF030309 [34]; and El Tor, accession no. M33514). Periods indicate residues identical to those found in TcpA-cla; only amino acid differences (relative to TcpA-cla) are specified. ∗, position whose functional importance in TcpA-cla was tested mutationally (27). (B) Inferred phylogenetic relationships between tcpA alleles.

FIG. 4

Alignment of ToxT from strains SCE263 and 569B (classical biotype, epidemic strain) (GenBank accession no. B45247). toxT alleles nearly identical to that of SCE263 were found by sequencing in isolates SCE226 and SCE256.

FIG. 5

TcpF alignments generated with clustal W. Types II, III, IV, and V are from environmental isolates as indicated in Table 1 and in Fig. 6. Reference strain 569B (here designated type I) is a classical biotype O1 serogroup epidemic strain (tcpF sequence from GenBank accession no. L01623).

FIG. 6

Allele content of environmental isolates. Each pattern of shading represents a different highly divergent sequence, as shown in Fig. 3, 4, and 5, and described in the text for tcpA, toxT, and tcpF and the F-T intergenic (ig) sequence.

FIG. 7

Left-junction translocation. The insertion-deletion structure found at the left end of VPI in isolates of RAPD type 1 by PCR and sequencing, as described in the text, is shown. VC and VCA designations refer to genes in V. cholerae chromosomes 1 and 2, respectively,in a V. cholerae reference strain (19). Numbers above the map refer to rearrangement breakpoints, inferred by comparison with the full genome sequence (19).

FIG. 8

Representative results of PCR amplification of rstR and adjacent regions. Lane designations correspond to SCE isolates, except that VC20 and SG24 designate serogroup O1 El Tor and O139 epidemic strains, respectively, used for reference. (Top) Amplification with primers ig-1-F (specific for conserved sequence in the CTXφ intergenic region, just to the left of rstR) and rstA-R. Note that no amplification product was obtained from SCE264 with these primers and that SCE188 yielded two different products, indicating that it is a double lysogen. (Bottom) Amplification with primers ig-1-F and rstC-R. Note that an amplification product was obtained from SCE264 with these primers.

FIG. 9

ORF structures of putative repressor genes (rstR regions) of CTXφ-related prophages inferred from sequences of PCR products. These two new sequences are designated as the fourth and fifth members of a family of CTXφ-related prophage repressor genes and are therefore designated rstR-4 and rstR-5, pending tests for repressor function.