The spirochete FlaA periplasmic flagellar sheath protein impacts flagellarhelicity

Spirochete periplasmic flagella (PFs), including those from Brachyspira (Se rpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique struct ure. In most spirochete species, the periplasmic flagellar filaments consis t of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a shea th protein (FlaA). Each of these proteins is encoded by a separate gene. Us ing Brachyspira hyodysenteriae as a model system for analyzing PF Function by allelic exchange mutagenesis, we analyzed purified PFs from previously c onstructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly construct ed 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::ca t mutants were still motile, but all were less motile than the wild-type st rain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resu lted in the specific loss of the cognate gene product in the assembled puri fied PFs, Consistent with these results, Northern blot analysis indicated t hat 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 wi ld-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 s heath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a sma ller helix pitch and helix diameter compared to the wild-type strain and fl aB mutants. These results indicate that the interaction of FlaA with the Fl aB core impacts periplasmic flagellar helical morphology.