Evidence indicating that the cholera toxin structural genes of Vibrio cholerae RJ1 and 3083-2 are between met and trp

Abstract

An enterotoxin A subunit-specific radioactive probe was used to correlate expression of vct-1 or vct-2 with the presence of specific restriction fragments in the DNAs of several wild-type and recombinant Vibrio cholerae strains. The data are consistent with the conclusion that vct is a cholera toxin structural gene. We previously reported that the gene vct specifies the antigenic structure of cholera toxin (CT) and is located between met and trp on the Vibrio cholerae genetic map (14). The vet-I

Transcription of cholera toxin operon in wild-type and mutant strains of Vibrio cholerae

“…In contrast, all classical biotype strains showed hybridization of probe 1 to two PstI or XbaI fragments. Except for strains RJ57 and RJ57(RP4), these results are in agreement with published reports and confirm that the CT operon is present at two locations in classical strains and at one location on the chromosome of most El Tor strains (12,14,16,19,24,25). The third PstI and XbaI fragments observed in our experiments with DNA from strains RJ57 and RJ57(RP4), which are derived from El Tor strain RV79 (10), presumably hybridized with the flanking sequences of probe 1 and not with CT operon-specific sequences (16).…”

Mapping of a gene that regulates hemolysin production in Vibrio cholerae

“…Wild-type El Tor strain RJ1 and recombinants with tox-1000 are designated Tox-because these strains do not produce quantities of CT that can be detected in the Elek test or the radial passive immune hemolysis assay for CT, whereas wild-type El Tor strain 3083-2 and recombinants with tox-2000 are toxinogenic in these tests. Genetic mapping experiments indicated that tox-1000 is separated from the CT structural genes of strain RJ1 by at least one gene that is not related to CT synthesis (trp), implying that tox-1000 transregulates the CT structural genes (14)(15)(16). Miller and Mekalanos (12) and Sporecke et al (17) subsequently provided evidence supporting a similar conclusion for the CT regulatory and structural genes of strain 569B.…”

“…Two observations suggest that it is most likely that the 12-and 2.4-kb nuclease restriction fragments were derived from pSJ13, the plasmid that was used to mobilize the donor chromosome. First, we previously reported that 32P-nick-translated pBR322 DNA hybridizes with a 2.4-kb PstI nuclease restriction fragment in RJ1(pSJ13) whole cellular DNA but not in RJ1 whole cellular DNA (14). We suggested that the P-lactamase gene of pBR322 hybridized to the 3-lactamase gene of pSJ13.…”

“…Since RJ1 and 3083-2 are heterologous strains, it was possible that the Tox-recombinants were the result of a deletion or other genetic rearrangement caused by recombination between the two genomes. Indeed, we described a Tox+ recombinant derived from strains RJ1 and 3083-2 in which some CT structural gene sequences were evidently deleted (14). Accordingly, we examined the restriction nuclease digestion patterns of several Toxrecombinants.…”

“…Plasmid pCVD002 (10), which contains cloned CT Asubunit and B-subunit genes, was 32P nick translated and used in Southern hybridization analysis of PstI-cleaved whole cellular DNA from strain RJ1, strain MB1602, and several Toxrecombinants as previously described (14), except that the hybridization was performed under more stringent conditions as follows. The prehybridization (4 h at 42°C) and hybridization (overnight at 42°C) incubations were performed in 50% formamide-0.05 M Tris-5 x Denhardt solution-100 ,ug of calf thymus DNA per ml-6x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate).…”