FliO Regulation of FliP in the Formation of the Salmonella enterica Flagellum

Abstract

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the Δ fliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO _43–125 or FliO _1–95 was able to rescue motility of the Δ fliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO _43–125, should contain beta-structure and alpha-helices. FliO _43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.

Author Summary

The propeller-like flagella, which some bacteria use to swim, possess a specialized secretion apparatus, which is imbedded in the cell membrane for their formation. The components are highly conserved among flagella systems and also to the Type III secretion apparatus used by some bacteria in conjunction with virulence-associated needle complexes. The ubiquity of these secretion apparatuses and their function as intricate nanomachines has made them fascinating for biologists. The most studied flagellar system is that of Salmonella enterica, which consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. Among these proteins, FliO shows a sporadic distribution in bacteria, and its function is unknown, suggesting it might have a specialized role to play where it is present. In this study, we show that FliO has an important role in maintaining stability of FliP, which is a highly conserved member of the secretion apparatus. We have characterized the important regions of FliO through mutagenesis. We have shown that it is possible to bypass the effect of not producing the FliO protein, by encoding mutations within FliP or by overexpressing the cytoplasmic domain of FliO only.