The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.
The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.
Ohnishi
K
K
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Fan
F
F
Schoenhals
G J
GJ
Kihara
M
M
Macnab
R M
RM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D005407:Flagella / Q000378:metabolism*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009876:Operon; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D001426:Bacterial Proteins; C085247:FliO protein, Bacteria; C085248:FliR protein, Bacteria; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C074890:FliP protein, Bacillus; C074891:FliQ protein, Bacteria