TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae

Abstract

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Figure 1

(A) Genomic map of the region in the tcp gene cluster affected by two independent transposon insertions (arrowheads). The BclI sites show the extent of the deletion in the ΔtcpP strain. (B) Comparison of the tcpP and tcpH genes to psaE and psaF from Yersinia pestis (20) and toxR and toxS from V. cholerae (6, 7). Arrows indicate the direction and sizes of the ORFs. Sequences encoding hydrophobic regions representing potential transmembrane domains are shown in black. (C) Proposed membrane topology of ToxR, ToxS, TcpP, and TcpH. Carboxyl (C) and amino (N) termini of the proteins are indicated.

Figure 2

Amino acid sequence alignment of the amino-terminal region of the V. cholerae TcpP protein and the proteins PsaE from Y. pestis (20), HilA from Salmonella typhimurium (21), and ToxR from V. cholerae (6). Identical amino acids are shown in black boxes, whereas gray boxes indicate similar amino acid residues.

Figure 3

β-Galactosidase activities of V. cholerae strains carrying a toxT∷lacZ reporter construct in the chromosome with plasmids carrying the tcpP or toxR gene under arabinose-inducible promoters. Arabinose was added to a final concentration of 0.04%.

Figure 4

β-Galactosidase activities of V. cholerae strains carrying a toxT∷lacZ reporter construct in the chromosome with plasmids carrying the tcpP and/or tcpH genes under arabinose-inducible promoters. Arabinose was added to a final concentration of 0.04%.