Article Figures & Data
Figures
- FIG. 1.
Genetic organization of the wb* regions. (a) Agarose gel electrophoresis of XL-PCR amplification products of the O-antigen biosynthesis regions (wb*) from different serogroup strains. The line at the top is a schematic diagram of the wb* region with gmhD and rjg at the ends; the primers used in XL-PCR (indicated by the small bars below the line) are situated at the 5′ ends of the gmhD and rjg genes. Lane S contained a 1-kb ladder, and lane L contained a HindIII digest of λ DNA. The positions (in kilobases) of the markers are indicated on the left. (b) PFGE and Southern analysis of the NotI fragments of O1, O139, and O105 serogroup strains. The blot was hybridized with a gmhD probe. The asterisk indicates the position of unexplained recombination intermediates or partial digestion products. Lanes 1, 3, and 5 contained parent strains without the NotI site at the left junction, and lanes 2, 4, and 6 contained the strains with the NotI sites flanking the wb* regions. Lanes 2, 4, and 6 also contained partially digested parent fragments of the wb* region in addition to the newly generated wb* fragments. (c) Southern blot analysis of the gmhD and rjg junction fragments in various strains. The genomic DNAs were digested with EcoRI and were simultaneously hybridized with the gmhD and rjg probes. The upper and lower bands in each lane are the rjg- and gmhD-specific fragments, respectively.
- FIG. 2.
Comparison of the O37 wb* region with the O1 wbe and O139 wbf regions. The genetic organization of the wb* region of the O37 strain is compared to the genetic organizations reported previously (49) for the O1 and O139 strains. The O139, O1, and O37 regions are 35, 22, and 27 kb long, respectively. The ORFs and the directions of transcription are indicated by the arrows, and the common ORFs in the three serogroups are indicated by the same types of arrows. The common junction genes (gmhD and rjg) of the three serogroups are indicated by solid arrows. While the left junction is at gmhD in all three serogroups, the right junction in O37 is different from the right junction in O139. The O1 right junction genes, wbeV through rjg, are conserved in the O37 wb* region.
- FIG. 3.
Analyses of the left junctions (LJ) and the right junctions (RJ) of O1, O139, and O37 wb* regions performed using the ClustalW program (http://www.ebi.ac.uk/index.html). The asterisks indicate identical bases in the sequences compared. (A) Left junctions of the O1, O37, and O139 wb* regions. The solid arrowhead and its orientation indicate the gmhD ORF start codon and the direction of transcription, respectively. The homology breakpoints between O1 and O37 are ca. 120 bp upstream of the start codon of gmhD since the sequences diverge near this position. The homology breakpoints of the O1 and O139 sequences are at the start codon of the gmhD ORF, since the sequences after gmhD completely diverge. (B) Right junctions of the O1 and O37 sequences. The homology breakpoint is in the wbeV ORF, which is preceded by wbeU in O1 and by orf-18 in O37. The stop codon of wbeV (indicated by the arrowhead pointing upward) overlaps with the start codon of orf-18 in O37. The stop codon of wbeV of O1 is indicated by the arrowhead pointing downward. (C) Right junctions of the O1 and O139 sequences published previously (14, 45). The homology breakpoint is at the start of the rjg ORF. The rjg genes of O1 and O139 strains have different N terminus and start codons. In all these cases, the actual recombination crossover sites could be anywhere within homologous segments far away from the homology breakpoints and not necessarily at the junctions.
- FIG. 4.
IS1004 fingerprinting and RFLP analyses of the various serogroup strains. (a) IS1004 fingerprints of HpaII-digested chromosomal DNAs of various V. cholerae strains. Since HpaII does not cut within IS1004, each band represents one copy of the element. (b) RFLP analysis of the smt region of chromosome II of various V. cholerae strains. The genomic DNAs were digested with SphI and hybridized simultaneously with multiple probes (smtrgn 1 to 5). The identities of the bands were deduced from their predicted sizes, based on the published V. cholerae genome sequence (22), and they were confirmed by separately hybridizing the same blot with individual probes. The sizes of the molecular weight markers (in kilobases) are indicated on the left. (c) Blot used in panel b rehybridized with an smt probe (primers M 668 and M 669). Lanes O144, O9, and O27 were omitted since they did not show any hybridizing band with this probe.
- FIG. 5.
RFLP analysis of the virulence regions of the various strains. (a) EcoRI-digested genomic DNAs were probed with ctxAB gene probes (upper panel) and the ctx core probe (lower panel). EcoRI does not cut within the CTXφ genome, and each hybridizing band therefore represents one copy of CTX present in a strain. (b) SphI-digested genomic DNAs of the strains were probed with five probes spanning a 45-kb region surrounding the CTXφ genome integrated on chromosome I. The probes and the sizes of the fragments and the corresponding bands are as follows: ctxrgn 1, 1,106 and 13,818 bp (bands a and j); ctxrgn 2, 2,862 and 13,387 bp (bands e and b); ctxrgn 3, 3,255 and 1,365 bp (bands d and i); ctxrgn 4, 2,651 and 2,180 bp (bands f and h); and ctxrgn 5, 2,651 and 11,400 bp (bands g and c). (c) Genomic DNAs digested with XmnI and probed simultaneously with five different probes from the VPI region. The identities of the bands were confirmed by separate hybridization with individual probes. The probes and the sizes of the fragments and the corresponding bands are as follows: ald, 11,064 bp (band b); tagD, 3,994 bp (band e) and 837 bp (band h); tcpAdn, 3,273 bp (band f) and 1,070 bp (band g); toxT, 12,332 bp (band a); and vpi0845, 3,976 bp (band d) and 7,685 bp (band c).
- FIG. 6.
Model for emergence of the non-O1 and non-O139 strains with epidemic potential by O-antigen switching. A dendrogram of the genetic relatedness of the various serogroups, based on IS1004 fingerprinting analysis, is shown on the left. The O37 strain appears to have been derived from an O1 preclassical strain (classical VPI cluster) by O-antigen shifting, divergence, and subsequent acquisition of an El Tor CTXφ. The El Tor strains diverged from classical strains, acquired a distinct CTXφ, and probably acquired a tcpA allele by recombination (30). The O139 strain appears to have been derived from an El Tor strain by O-antigen switching, and the O27 strain appears to have been derived from an El Tor progenitor and to have acquired a distinct CTXφ and a distinct VPI. It is also probable that O27 emerged from an El Tor strain by O-antigen switching and subsequently changed its rstR and tcpA genes by allelic exchange. The O53 and O65 strains have an El Tor VPI cluster and are identical to each other. They have genetic backbones resembling those of both classical and El Tor strains, and hence, they probably emerged from an O1 progenitor, diverged, and subsequently acquired a classical pre-CTXφ and and an El Tor VPI. These two strains probably underwent O-antigen switching events independently or sequentially, from O1 to O53 and O65. Cl, classical; ET, El Tor; NT, novel type (38).
Tables
- TABLE 1.
Characterization of non-O1 and non-O139 V. cholerae strains with pathogenic potentiala
Strain Country Year Source Sakazaki serogroup CTXφ VPI ctxA ctxB rstR Clusterb tcpA 395 India 1966 Diarrhea O1 cla WT cla cla cla cla NIH35A3 India 1941 Diarrhea O1 cla WT cla cla cla cla 5011 Unknown H. Smith collection O1 cla (O333)c WT cla cla cla cla N16961 Bangladesh 1975 Diarrhea O1 ET WT ET ET ET ET E7946 Bahrain 1978 Diarrhea O1 ET WT ET ET ET ET 365-96 Japan 1996 Prawn, import from Thailand O27 WT NT rstR-4∗∗d NT NTe 1322-69 India 1969 Diarrhea O37 NTf NT ET cla cla 8585 Iraq 1966 Diarrhea O53 (O340)c cla ET ET 981-75 India 1975 Diarrhea O65 cla ET ET 63-93 (MO45) India 1992 Diarrhea O139 WT ET ET ET ET AM2 India 1995 Diarrhea O9 AM107 India 1996 Diarrhea O144 NRT36-S Japan 1990 Diarrhea O31 ↵a Abbreviations: wt, wild type; cla, classical; ET, El Tor; NT, novel type.
↵b Presence of the entire VPI cluster based on restriction mapping and hybridization.
↵c O333 and O340 are Smith serogroups.
↵d rstR-4** = SCE223 (38).
↵e Differs from the tcpA-env allele described by Mukhopadhyay et al. (38) at one position (O27V9D).
↵f There is a single amino acid substitution (wtS46NO37).
- TABLE 2.
List of primers
Primer Gene Sequence Reference(s) J 101 gmhD1 5′-GCCATCCCACTCTGTGGTCGCAGAGCAAGCTCC-3′ 14 J 103 rjg2 5′-CCCGTGACACTCGCCTTCCCTCCGTGATGAACC-3′ 14 M 177 gmhD1 5′-TTACTTACGATTAATCAGCGCCAT-3′ 45 M 178 gmhD2 5′-GGCGGCGCTGGCATGATTGGCAGC-3′ 45 M 179 rjg1 5′-CATGGAAGTGGTTCATCACGGAGG-3′ 45 J 414 rjg2 5′-GTGGACGCGTTCAAAGCACCGAATATCCGAGTT-3′ 45 M 310 orf21 5′-GGTGACATCAAAGGGACCACTTTTTC 22; this study M 311 orf22 5′-GGTGTATGCCACTAGTGTAGGTAAT 22; this study M 459 IS10041 5′-CCCCAGCTTTTGACGCTTATTGTGAACGT-3′ 22; this study M 460 IS10042 5′-GATCGATATCTTTCTAACTTCTGTATAAGG-3′ 22; this study M 277 ctxA1 5′-ACAGAGTGAGTACTTTGACC-3′ 22; this study M 278 ctxA2 5′-ATACCATCCATATATTTGGGAG-3′ 22; this study S 86 ctxAB1 5′-GGCTGTGGGTAGAAGTGAAACGG-3′ 22; this study S 87 ctxAB2 5′-CTAAGGATGTGGAATAAAAACATC-3′ 22; this study M 279 tcpA1 5′-AAAACCGGTCAAGAGGG-3′ (same as KAR 24) 28 M 280 tcpA2 5′-CAAAAGCTACTGTGAATGG-3′ (same as KAR 25) 28 M 281 tcpA3 5′-CAAATGCAACGCCGAATGG-3′ (same as KAR 82) 28 M 590 tcpAL1 5′-GATCGCATGCCAGAGTTCTATCTTTCGTC-3′ 22; this study M 591 tcpAL2 5′-GATCGTCGACATAGTGATAAGAGTCTTACCC-3′ 22; this study M 668 smt1 (smt-VCA0198) 5′-CCGAAATACGGTCATTACTTGGGC-3′ 22; this study M 669 smt2 5′-CACTTCATTATTCCCGTAAGCAGC-3′ 22; this study M 680 smt1.1 (nupC-VCA0179) 5′-AATAGCCAATCACGCACCAAG-3′ 22; this study M 681 smt1.2 5′-TAATCGCACTGCGGCTTTCAG-3′ 22; this study M 682 smt2.1 (hmpA-VCA0183) 5′-TGACCCACCAGAAAACCGGAC-3′ 22; this study M 683 smt2.2 5′-GCGCCTTATCCACACCAAGCG-3′ 22; this study M 684 smt3.1 (rhlE-VCA0204) 5′-CGCTCAATCGCAAATAATTCC-3′ 22; this study M 685 smt3.2 (dcuB-VCA0205) 5′-TGCTCTCTCTCCCCAAATGAC-3′ 22; this study M 686 smt4.1 (VCA0206) 5′-GTATTGTCGGATTTCATTTGC-3′ 22; this study M 687 smt4.2 (VCA0208) 5′-AGTGACGGCCTCTGGCGGAGC-3′ 22; this study M 688 smt5.1 (hlyA-VCA0219) 5′-GGGTTCCGCGACACCGGATGC-3′ 22; this study M 689 smt5.2 5′-TGTTTAATGGCTATGTTGACG-3′ 22; this study M 698 ctxrgn1.1 (VC1444) 5′-TAATCTGCTATTTCACTGAAG-3′ 22; this study M 699 ctxrgn1.2 5′-TTCCTGAGTGATCCCCAATCC-3′ 22; this study M 700 ctxrgn2.1 (VC1451-rtxA) 5′-GCGGAAAAGCTGAAAGGCACC-3′ 22; this study M 701 ctxrgn2.2 5′-ACCTTCATGGTGTGAAATCAC-3′ 22; this study M 702 ctxrgn3.1 (VC1465) 5′-CCGCTGTCTCAATAGAACCTG-3′ 22; this study M 703 ctxrgn3.2 5′-GGACATCATACAAGAGAAGAC-3′ 22; this study M 704 ctxrgn4.1 (VC1470) 5′-GAACATGAACCTTAATGCGAG-3′ 22; this study M 705 ctxrgn4.2 5′-CACGTCATTTATGAATTACGG-3′ 22; this study M 706 ctxrgn5.1 (VC1476-VC1477) 5′-GGTATCAGCATGAGACTTTTTTG-3′ 22; this study S 122 ctx core (orfU) 5′-CGTCACACCAGTTACTTTTCG-3′ 22; this study S 123 ctx core (zot) 5′-AACCCCGTTTCACTTCTAC-3′ 22; this study M 707 ctxrgn5.2 5′-CCAATAGTGATAACTACTTCG-3′ 22; this study M 452 ald2 5′-TTTTCTTGATTGTTAGGATGC-3′ 12 M 453 ald1 5′-ATTCTTCTGAGGATTGCTGAT-3′ 12 M 644 tagD1 5′-GCGGTGACACTAAAGTAGTGTTTG-3′ 22; this study M 645 tagD2 5′-GATGGTCAGATAAAAGAACGCAGG-3′ 22; this study M 664 tcpAdn1 5′-TTCGCAATTACAGTCGGTGGCTTG-3′ 22; this study M 665 tcpAdn2 5′-AGCCAACTCAGTTAAAACTTGTTC-3′ 22; this study M 448 toxT2 5′-CTTGGTGCTACATTCATGG-3′ 12 M 449 toxT1 5′-AGGAGATGGAAGTGGTGTG-3′ 12 M 646 vpi08451 5′-ATCATTCCAGATAAAGTTACGCAGA-3′ 22; this study M 647 vpi08452 5′-TCTACTTCCGGCTTCCCTGCCACG-3′ 22; this study M 407 rstR1 5′-GACGTAGCGTGCGGAGTCGCGTTG-3′ 22; this study M 408 rstR2 5′-TGAAGCATAAGGAACCGACCAAGC-3′ 22; this study M 573 rstA1 5′-ACTCGATACAAACGCTTCTC-3′ 22; this study M 574 rstA2 5′-AGAATCTGGAGGTTGAGTG-3′ 22; this study
