ORGANIZATION OF TCP, ACF, AND TOXT GENES WITHIN A TOXT-DEPENDENT OPERON

The toxin coregulated pilus (ICP) is required for Vibrio cholerae to c olonize the human intestine, The expression of the pilin gene, tcpA, i s dependent upon ToxR and upon ToxT, The toxT gene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of act ivating a tcpA::TnphoA fusion when cloned in Escherichia coil, In this study, we determined that ToxR/ToxT activation occurs at the level of tcpA transcription, ToxT expressed in E, coli could activate a top op eron fusion, while ToxR, ToxR with ToxS, or a ToxR-PhoA fusion failed to activate the top operon fusion and we could not demonstrate binding of a ToxR extract to the tcpA promoter region in DNA mobility-shift a ssays, The start site for the regulated promoter was shown by primer e xtension to lie 75bp upstream of the first codon of tcpA. An 800-base tcpA message was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin-l oop sequence between tcpA and tcpB, The more-sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstream top genes, including toxT a nd the adjacent accessory colonization factor (acf) genes, An in-frame tcpA deletion, but not a polar tcpA::TnphoA fusion, could be compleme nted for pilus surface expression by providing tcpA in trans. This evi dence suggests that the top genes, including toxT, are organized in an operon directly activated by ToxT in a ToxR-dependent manner. Most of the toxT expression under induced conditions requires transcription o f the tcpA promoter, Further investigation of how tcp::TnphoA insertio ns that are polar on toxT expression retain regulation showed that a l ow basal level of toxT expression is present in toxR and tcp::TnphoA s trains. Overall, these observations support the ToxR/ ToxT cascade of regulation for fcp,Once induced, toxT expression becomes autoregulator y via the top promoter, linking top expression to that of additional c olonization factors, exotoxin production, and genes of unknown functio n in cholera pathogenesis.