Structural Flexibility Of The Cytoplasmic Domain Of Flagellar Type Iii Secretion Protein Flhb Is Important For The Function Of The Protein

Bacterial flagellum is a large complex organelle made up of more than 30 different proteins. Most of the flagellar proteins are localized outside of the cell and are exported across the cytoplasmic membrane by the flagellar type III secretion system. The protein export is highly regulated. Membrane protein FlhB plays a key role in this regulation. The protein consists of two domains: a hydrophobic N-terminal part (FlhBTM), which is predicted to have four transmembrane helices, and a C-terminal cytoplasmic domain (FlhBC). Homologues of FlhB were found in all bacterial type III secretion systems. Sequences of these proteins are highly conserved suggesting that their function is also similar. In this study we have compared properties of FlhB from two organisms: Salmonella typhimurium and Aquifex aeolicus. Salmonella and Aquifex FlhB share 32% sequence identity. However, these proteins are evolutionarily very distant. Comparison of the two proteins may provide us with additional information about functionally important regions of FlhB. We have substituted flhB gene in Salmonella by flhB of A. aeolicus or by chimera gene encoding hybrid FlhB composed of the FlhBTM of S. typhimurium and FlhBC of A. aeolicus. Then we analyzed motility of the mutants on soft agar plates. Although all mutants showed some motility, they were substantially less motile than wild-type cells. We have found several spontaneous mutations in C-terminal part of FlhB that resulted in enhanced motility. To understand the effect of the mutations we have solved FlhBC structures from both organisms: Salmonella and Aquifex. We have also determined secondary structure and stability of the mutated FlhBC. Based on our findings we suggest that conformational flexibility is important for FlhB function.