THE VIBRIO-CHOLERAE ACFB COLONIZATION DETERMINANT ENCODES AN INNER MEMBRANE-PROTEIN THAT IS RELATED TO A FAMILY OF SIGNAL-TRANSDUCING PROTEINS

Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D ) are required for efficient intestinal colonization. Expression of ac f genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesi s of the toxin-coregulated pilus. The gene for acfB was cloned by usin g an acfB::TnphoA fusion junction to probe a V. cholerae O395 bacterio phage lambda library. DNA sequence analysis revealed that acfB is pred icted to encode a 626-amino-acid protein related to the V. cholerae Hl yB and TcpI proteins. These three Vibrio proteins have amino acid sequ ence similarity in a region highly conserved among bacterial methyl-ac cepting chemotaxis proteins. Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a memb rane topology and domain organization similar to those of methyl-accep ting chemotaxis proteins. Heterologous expression of acfB in Escherich ia coli generates four polypeptide species with apparent molecular mas ses of 34, 35, 74, and 75 kDa. The 74- and 75-kDa proteins appear to r epresent modified forms of the full-length AcfB protein. The 34- and 3 5-kDa polypeptide species most likely correspond to a C-terminal 274-a mino-acid polypeptide that results from internal translation initiatio n of acfB mRNA, Localization studies with AcfB-PhoA hybrid proteins in dicate that AcfB resides in the V. cholerae inner membrane. V. cholera e acfB::TnphoA mutants display an altered motility phenotype in semiso lid agar. The relationship between AcfB and Vibrio motility and the am ino acid similarities between AcfB and chemotaxis signal-transducing p roteins suggest that AcfB may interact with the V. cholerae chemotaxis machinery. The data presented in this report provide preliminary evid ence that acfB encodes an environmental sensor/signal-transducing prot ein involved in V. cholerae colonization.