An Avirulent Vibrio cholerae StrainA Producer of the Cholera
Toxin B Subunit: Obtainment and Molecular Genetic Analysis

N. I. Smirnova^a, I. M. Krepostnova^a, L. F. Livanova^a,
S. P. Zadnova^a, S. A. Eremin^a, and T. S. Il’ina^b

^a Microbe Russian Antiplague Research Institute, Saratov, Russia

^b Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences,
Moscow, Russia

Received November 7, 2006

Abstract—The conjugative recombinant plasmid plEM3 (Km^R Tc^R) was constructed in order to introduce a
cloned ctxB gene encoding the cholera toxin (CT) B subunit into Vibrio cholerae cells. The plasmid was
obtained by cointegration of two plasmids: a conjugative plasmid, pIEMl (Km^R), carrying mini-kan transposon
and IS1 element, and pCT27 (Tc^R) plasmid, a derivative of the BR322 carrying the cloned ctxB gene. The
avirulent Vibrio cholerae strain ftTetr biovar devoid, as follows from PCR analysis, of key structural and regu-
latory genes allowing for pathogenesis and carrying a mutation in one of the O1-antigen genes was chosen as
a pIEM3 plasmid carrier strain. Uncoupling of the cointegrated construction occurs in 5% of Vibrio cholerae
cells, retaining only the multicopy pCT27 plasmid. This leads to the appearance of Tc^RKm^S clones producing
the secreted B subunit of CT at a high level (10 to 14 g/ml). One of these clones (KM93) was selected as a pro-
ducer strain of this protein. Patterns of inheritance and expression of a cloned ctxAB gene in KM93 cells were
studied by molecular-genetic and biochemical assays. It was found that expression of a cloned ctxB gene did
not depend on the presence of key regulatory genes of toxRt tcpP, tcpH, toxT in the chromosome, suggesting
the existence of some other mechanisms that might control transcriptional activity of the cholera toxin B subunit
gene. Effective production of the B subunit of the CT was also obtained upon rearing the constructed producer
strain under the conditions of industrial process.

DOI:10.3103/S0891416807040027

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