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Molecular analysis of VcfQ protein involved in Vibrio cholerae type IV pilus biogenesis
Tomoko Miyazato1, Claudia Toma1, Noboru Nakasone1
1Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, Uehara 207, Nishihara, Okinawa 903-0215, Japan 2Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan.
Abstract:
The nucleotide sequence of an ORF (vcfQ) within the type IV pilus gene cluster of Vibrio cholerae O34 strain NAGV14 was determined, thereby completing the sequence analysis of the structural operon. The vcfQ gene showed homology to the mshQ gene of the mannose-sensitive haemagglutinin pilus gene cluster. The vcfQ was 651 bp larger than mshQ, and the G+C content of the extra 651 bp portion (35.6 mol%) was lower than that of the overall vcfQ gene (42.5 mol%). Except for the first 270 aa residues, the deduced amino acid sequence of VcfQ showed high homology to the MshQ protein. There was immunological cross-reaction between VcfQ and MshQ by Western blotting. Cell fractionation studies showed that VcfQ is located in both the inner and the outer membranes. Mutational analysis showed that vcfQ-deficient mutant expressed detectable levels of major pilin (VcfA), but failed to assemble them into pili, indicating that VcfQ is essential for pilus assembly. Colony-blotting analyses showed that the N-terminal region of vcfQ is variable in V. cholerae strains.