title: Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium.
abstract: Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: K
authors.initials: K
authors.lastname: Muramoto
authors.forename: S
authors.initials: S
authors.lastname: Makishima
authors.forename: S I
authors.initials: SI
authors.lastname: Aizawa
authors.forename: R M
authors.initials: RM
authors.lastname: Macnab
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018388:Codon, Terminator / Q000235:genetics; D005407:Flagella / Q000378:metabolism* / Q000648:ultrastructure; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012329:RNA, Bacterial / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D015398:Signal Transduction; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; C099486:FliK protein, Bacteria; D012329:RNA, Bacterial; D012364:RNA, Transfer, Trp