Mutational analysis of the flagellar rotor protein FliN: identification of surfaces important for flagellar assembly and switching.
FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.
Paul
Koushik
K
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Harmon
Jacob G
JG
Blair
David F
DF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D005407:Flagella / Q000502:physiology*; D005838:Genotype; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011487:Protein Conformation; D020124:Thermotoga maritima / Q000235:genetics
D001426:Bacterial Proteins; C077389:FliN protein, Bacteria