Analysis of an autoregulatory loop controlling ToxT, cholera toxin, and toxin-coregulated pilus production in Vibrio cholerae

Coordinate expression of many virulence genes in the human pathogen Vibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membran e proteins, are required to activate toxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose prod ucts are required for the biogenesis of cholera toxin (CTX) and the toxin-c oregulated pilus, the major subunit of which is TcpA. This work examined co ntrol of toxT transcription. We tested a model whereby activation of toxT b y ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-d ependent transcription of the tcpA promoter reads through a proposed termin ator between the tcpF and toxT genes to result in continued ToxT production . Primer extension analysis of RNA from wild-type classical strain O395 sho wed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxT(Delta pro)) result ed in the abolishment of toxT transcription, as predicted. Deletion of the tcpA promoter (tcpA(Delta pro)) had no effect on subsequent detection of th e smaller toxT primer extension product, but the larger toxT product was no t detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT, Ne ither mutant strain produced detectable TcpA, but the CTX levels of the str ains were different, The toxT(Delta pro) strain produced little detectable CTX, while the tcpA(Delta pro) strain produced CTX levels intermediate betw een those of the wild-type and toxT(Delta pro) strains. Dependence of toxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectable toxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest that toxT transcription is dependent on two differen t promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.