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Field emission scanning electron microscope (FESEM) image of a flagellum-negative mutant of Vibrio cholerae O1 classical biotype strain O395 expressing toxin-coregulated pili (TCP). The use of FESEM allows for a three-dimensional view of the characteristic bundles that are formed by TCP. It can be seen that the fibers in the bundle are physically entwined to form a rope-like or “cable” structure. This property might be the basis for the mechanism by which TCP mediate bacterium-bacterium interactions within a microcolony. The image is visualized at a magnification of ×40,000 and was provided by Louisa Howard of the Dartmouth College Electron Microscope Facility.  
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cholera with tcp grey scale
Abstract:
Identification of a TcpC-TcpQ Outer Membrane Complex Involved in the Biogenesis of the Toxin-Coregulated Pilus of Vibrio cholerae
Niranjan Bose and Ronald K. Taylor*

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire

Journal of Bacteriology, April 2005, p. 2225-2232, Vol. 187, No. 7

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°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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1632. Fax: (603) 650-1318. E-mail: Ronald.K.Taylor@Dartmouth.Edu.