The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity.
Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.
Li
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Department of Microbiology and Immunology, Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506-9177, USA.
Corum
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Morgan
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Rosey
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Stanton
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TB
Charon
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NW
D000483:Alleles; D015152:Blotting, Northern / Q000379:methods; D017401:Brachyspira hyodysenteriae / Q000235:genetics / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D005407:Flagella / Q000502:physiology* / Q000648:ultrastructure; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D016296:Mutagenesis; D019897:Periplasm / Q000502:physiology / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D013144:Spirochaetales / Q000235:genetics / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure
D011993:Recombinant Fusion Proteins; D005408:Flagellin; C067138:flaA protein, bacteria; C416532:flaB flagellin