A Vibrio cholerae relaxed (relA) mutant expresses major virulence factors, exhibits biofilm formation and motility, and colonizes the suckling mouse intestine.

We have constructed a relaxed mutant of El Tor biotype Vibrio cholerae strain C7258 by disruption of the RelA catalytic domain. The ability of the V. cholerae relaxed mutant to biosynthesize guanosine tetraphosphate and pentaphosphate was severely affected; the mutant showed a reduced growth rate in minimal medium that could be reversed by the addition of Casamino Acids, and it was thermosensitive. Contrary to published findings, the new relA mutant still produced significant cholera toxin and toxin-coregulated pilus. The V. cholerae relA mutant was motile, produced normal biofilms, and colonized the suckling mouse intestine. Our data suggest that levels of basal guanosine nucleotides pppGpp and ppGpp, rather than the availability of a stringent response, could influence expression of virulence factors, depending on strain and culture conditions. Production of hemagglutinin (HA)/protease, which requires HapR, RpoS, and the cyclic AMP receptor protein, was not strongly affected. Nevertheless, overexpression of RelA protein from an isopropyl-beta-d-thiogalactopyranoside-inducible promoter posttranscriptionally diminished production of HA/protease.