A method of producing a thy A<；SUP>；-<；/SUP>；strain of vibrio cholerae comprising the step of site-directed mutagenesis in the V. cholerae chromosome at the locus of the thy A gene SEQ ID NO: 1 of FIG. 1 , is described. Particularly, a Delta thy A strain of Vibrio cholerae lacking the functionality of the thy A is disclosed. This strain may comprise one or several episomal autonomously replicating DNA elements, such as plasmids, having an optionally foreign, e.g. E. coli, functional thy A gene that enables the strain to grow in the absence of thymine in the growth medium, and optionally having a structural gene encoding a homologous or heterologous protein. Further, proteins encoded by a structural thy A gene and the 5'-flanking region are described as SEQ ID NO: 4 of FIG. 4 and SEQ ID NO: 5 of FIG. 5 , respectively. Additionally, a vaccine comprising a Vibrio cholerae Delta thy A strain of the invention or a thy A<；SUP>；-<；/SUP>；strain of Vibrio cholerae produced by the method of the invention is disclosed.
