Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus

Abstract

Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related Vibrio mimicus. The two species share many genes coding for proteins, such as ctxAB, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu, tRNALys, and tRNAVal, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla, whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla. Little variation, i.e., 0 to 0.4%, was found among V. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and V. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of V. cholerae strains from V. mimicus. The PCR system developed was subsequently evaluated by using representatives of V. cholerae from environmental and clinical sources, and of other taxa, including V. mimicus. This study provides the first molecular tool for identifying the species V. cholerae.

FIG. 1

Electrophoresis with a 1.5% agarose gel of PCR-amplified 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Lanes: M, molecular weight marker (100-bp ladder); 1, V. cholerae O1 classical RC2; 2, V. cholerae O1 El Tor RC25; 3, V. cholerae O139 RC4; 4, V. cholerae O22 RC45; 5, V. cholerae O31 RC48; 6, V. cholerae non-O1/non-O139 RC42; 7, V. cholerae non-O1/non-O139 RC44; 8, V. mimicus RC5; and 9, V. mimicus RC55.

FIG. 2

Schematic representation of 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. An alignment of ISR sequences, corresponding to positions 224 to 276 of V. cholerae RC2 ISR-2, is presented in detail, and identities are indicated by a solid box. The positions of PCR primers, namely, prVC-F and prVCM-R, are indicated by arrows. For details, see the text.

FIG. 3

Phylogenies of V. cholerae and V. mimicus strains based on ISR-1 (a), ISR-2 (b), and ISR-3 (c) sequences. A region coding for tRNA was excluded from analysis for ISR-3. Anticodons of tDNAs in ISR-3, other than tDNA^Glu, are indicated in parentheses. The unrooted evolutionary trees were inferred by using the neighbor-joining method. The scale bar represents 0.01-nucleotide substitutes per position. Abbreviations: VC, V. cholerae; VM, V. mimicus.

FIG. 4

Identification of V. cholerae by using PCR based on the 16S-23S rRNA ISR. Lanes: M, molecular weight marker (100-bp ladder): 1, V. cholerae O1 classical RC2; 2 to 5, V. cholerae O1 El Tor clinical isolates; 6 to 9, V. cholerae O139 clinical isolates; 10, V. mimicus RC5; 11 to 15, V. mimicus isolates; 16 to 18, V. cholerae non-O1/non-O139 isolates; 19, V. aestuarianus ATCC 35048^T; 20, V. alginolyticus ATCC 17749^T; 21, V. campbellii ATCC 25920^T; 22, V. carchariae ATCC 35084^T; 23, V. diazotrophicus ATCC 33466^T; 24, V. fischeri ATCC 7744^T; 25, V. fluvialis ATCC 33809^T; 26, V. furnissii ATCC 35016^T; 27, V. hollisae ATCC 33564^T; 28, V. natriegens ATCC 14048^T; 29, V. salmonicida ATCC 43839^T; and 30, V. vulnificus ATCC 27562^T.