Identification of a TcpC-TcpQ outer membrane complex involved in the biogenesis of the toxin-coregulated pilus of Vibrio cholerae.
The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65 degrees C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.
Bose
Niranjan
N
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755, USA.
Taylor
Ronald K
RK
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology*; D036781:Fimbriae Proteins / Q000235:genetics / Q000502:physiology*; D010861:Fimbriae, Bacterial / Q000502:physiology*; D008074:Lipoproteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D014734:Vibrio cholerae / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; C077900:TcpC protein, Vibrio cholerae; C099203:TcpQ protein, Vibrio cholerae; D036781:Fimbriae Proteins