The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins

Abstract

Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D) are required for efficient intestinal colonization. Expression of acf genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesis of the toxin- coregulated pilus. The gene for acfB was cloned by using an acfB::TnphoA fusion junction to probe a V. cholerae O395 bacteriophage lambda library. DNA sequence analysis revealed that acfB is predicted to encode a 626-amino-acid protein related to the V. cholerae HlyB and TcpI proteins. These three Vibrio proteins have amino acid sequence similarity in a region highly conserved among bacterial methyl-accepting chemotaxis proteins. Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a membrane topology and domain organization similar to those of methyl-accepting chemotaxis proteins. Heterologous expression of acfB in Escherichia coli generates four polypeptide species with apparent molecular masses of 34, 35, 74, and 75 kDa. The 74- and 75-kDa proteins appear to represent modified forms of the full-length AcfB protein. The 34- and 35-kDa polypeptide species most likely correspond to a C-terminal 274- amino-acid polypeptide that results from internal translation initiation of acfB mRNA. Localization studies with AcfB-PhoA hybrid proteins indicate that AcfB resides in the V. cholerae inner membrane. V. cholerae acfB::TnphoA mutants display an altered motility phenotype in semisolid agar. The relationship between AcfB and Vibrio motility and the amino acid similarities between AcfB and chemotaxis signal-transducing proteins suggest that AcfB may interact with the V. cholerae chemotaxis machinery. The data presented in this report provide preliminary evidence that acfB encodes an environmental sensor/signal-transducing protein involved in V. cholerae colonization.