Lysogenic Conversion of Environmental Vibrio mimicusStrains by CTXΦ

ABSTRACTThe filamentous bacteriophage CTXΦ, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. choleraeby using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. choleraefrom nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicusstrains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicusstrains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXΦ and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicusstrains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmΦ, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicusstrains by CTXΦ. When a genetically marked derivative of the replicative form of the CTXΦ genome carrying a functionalctxABoperon, pMSF9.2, was introduced into nontoxigenicV. mimicusstrains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicusstrains by wild-type CTXΦ may lead to the origination of toxigenic V. mimicusstrains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXΦ and showed that at least one species ofVibrioother than V. choleraemay contribute to the propagation of the phage.