To view Journal of Bacteriology, April 2005, Vol. 187, No. 7 cover click here
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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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.