POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
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<strong>BLAST</strong> X Poster #11<br />
THE COMPLEX HOOK BASAL BODY STRUCTURE OF THE LYME DISEASE SPIROCHETE<br />
BORRELIA BURGDORFERI<br />
K. Miller 1 , R. Duda 2 , R. Hendrix 2 , M. Motaleb 1,3 , and N.W. Charon 1<br />
1<br />
West Virginia <strong>University</strong> Health Sciences Center, Microbiology and Immunology Department,<br />
Box 9177, Room 2077, Morgantown, WV 26506<br />
2<br />
<strong>University</strong> <strong>of</strong> Pittsburgh, Department <strong>of</strong> Biological Sciences, 4249 Fifth Avenue, 357A,<br />
Crawford Hall, Pittsburgh, PA 15260<br />
3<br />
East Carolina <strong>University</strong>, Department <strong>of</strong> Microbiology and Immunology, Greenville, NC 27834<br />
FlgE is the structural protein <strong>of</strong> the flagellar hook <strong>of</strong> B. burgdorferi. Previous western blot<br />
analysis using polyclonal FlgE antibody indicated that the majority <strong>of</strong> the hook migrated as a<br />
high molecular weight complex in SDS-PAGE gels instead <strong>of</strong> at the position <strong>of</strong> monomeric FlgE<br />
(46 kDa). Both the high molecular complex and a small amount <strong>of</strong> the monomer were present in<br />
wild-type and complemented cells but absent in flgE mutants. These data suggest that FlgE<br />
may be cross-linked into oligomers in a manner similar to that found in bacteriophage capsid<br />
proteins and in some bacterial pili. High molecular weight FlgE complexes have been observed<br />
in other spirochete species (Treponema phagedenis and Treponema denticola), suggesting that<br />
this property is conserved. We are interested in determining whether or not FlgE is cross-linked,<br />
and what amino acids are involved in the cross-linking.<br />
The hook-basal body complexes were purified using a refined version <strong>of</strong> methods<br />
previously reported (Sal, et al, 2008). Following purification <strong>of</strong> periplasmic flagella that contained<br />
attached hook-basal body complexes, the filament portions were depolymerized by acidic<br />
conditions or the use <strong>of</strong> urea. Hook-basal body complexes were then isolated by pelleting using<br />
an ultracentrifuge recovered in a neutralizing buffer. Electron microscopy <strong>of</strong> these preparations<br />
showed that acidic conditions resulted in more intact hook-basal body complexes than urea<br />
treatment, although the high molecular weight forms <strong>of</strong> FlgE were found using both methods. In<br />
order to determine results from each experiment, the experimental samples were run on 10%<br />
SDS-PAGE gels. We are using proteolytic finger printing and peptide mapping methods to<br />
compare the high molecular weight complexes <strong>of</strong> FlgE with the monomeric form (histidinetagged<br />
recombinant). Hook-basal body complexes and recombinant FlgE were treated with<br />
proteases or hydroxylamine at various temperatures (37, 45, and 65°C) and run in SDS-PAGE<br />
gels. Western blotting was used to assess the stability <strong>of</strong> the FlgE complex as well as to<br />
determine the size <strong>of</strong> (antigenic) peptide fragments resulting from proteolytic digestion. Digest<br />
patterns were compared between recombinant FlgE and the isolated hook-basal body<br />
complexes.<br />
Refining the hook-basal body isolation procedure resulted in improved hook-basal body<br />
preparations. The FlgE complex is stable under harsh conditions (both urea and acid treatment).<br />
Differences and similarities in hydroxylamine digest patterns between recombinant FlgE and<br />
purified hook-basal body complexes supports the hypothesis that FlgE is cross-linked.<br />
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