Flio Is Not Required For Motility in Salmonella If Its Cytoplasmic Domain and Flip Mutant Suppressors Are Expressed

The propeller-like flagella found in bacteria, and used for motility, possess a specialized secretion apparatus, which is imbedded in the cell membrane for their formation. Its components are highly conserved not just amongst flagellar systems, but also to the Type III secretion apparatus used by some bacteria in conjunction with virulence-associated needle complexes. The flagellar secretion system of Salmonella typhimurium consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, for the virulence-associated needle complexes of S. typhimurium and the flagellum of the bacterium Aquifex aeolicus a homolog of FliO is apparently absent. In this study we showed that deleting the fliO gene from the chromosome of a motile strain of Salmonellaresulted in a drastic decrease of motility. However, incubation of the fliO mutant strain in motility agar, gave rise to mutants containing suppressors that help to restore partial motility. One class of the suppressor mutation was found in the fliP gene. Using truncation and site-directed mutagenesis analysis of the FliO protein, it was shown that expression of FliO cytoplasmic domain in cells with the fliO gene deleted can also partially restore the motility. When the FliO cytoplasmic domain was expressed in the FliP suppressor mutant strains an additive effect was observed, and near wild-type levels of motility were regained. The FliO cytoplasmic domain was purified and studied using circular dichroism spectroscopy. Based on secondary structure prediction it should contain beta-structure and alpha-helices, however, we showed that this domain is disordered and its structure is a mixture of beta-sheet and random coil. We assume that the FliO cytoplasmic domain becomes structured while interacting with its binding partners.