title: Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene.
abstract: The 308 residue MotB protein anchors the stator complex of the Escherichia coli flagellar motor to the peptidoglycan of the cell wall. Together with MotA, it comprises the transmembrane channel that delivers protons to the motor. At the outset of the mutational analysis of MotB described here, we found that the non-motile phenotype of a DeltamotAB strain was rescued better by a pmotA(+)B(+) plasmid than the non-motile phenotype of a DeltamotB strain was rescued by a pmotB(+) plasmid. Transcription in each case was from the inducible tac promoter but relied on the native ribosome-binding site (RBS). This result confirms that translational coupling to motA is important for normal translation of the motB mRNA, since overproduction of MotA in trans did not improve complementation by pmotB. However, introduction of an optimized RBS into pmotB (to generate pmotB(o)) did. To dissect the function of the periplasmic domain of MotB, site-directed mutagenesis was used to replace Gln, Ser, and Tyr codons scattered throughout motB with amber (UAG) codons. Plasmid-borne motB(am) genes were introduced into sup(o), supE, and supF strains to see what motility defects were imposed by particular amber mutations and whether the defects could be suppressed by amber-suppressor tRNAs inserting the native or heterologous amino acids. Amber mutations at codon 268 or earlier in pmotB, and at codon 261 or earlier in pmotB(o) or pmotAB, eliminated motility. Thus, in agreement with the deletion analysis of motB by another laboratory, we conclude that the portion of MotB carboxyl-terminal to its peptidoglycan-binding motif (residues 161 to 264) is not essential. In strains containing supE or supF alleles, motility defects associated with motB(am) mutations were suppressed weakly, if at all, in pmotB. In contrast, motility defects conferred by most motB(am) mutations in pmotB(o) or pmotAB could be suppressed to a significant extent. However, the S18(am), Q100(am), Q112(am), Q124(am), Y201(am), and Y208(am) mutations were still suppressed extremely poorly. Full-length MotB was present at very low levels in suppressor strains containing the first four mutations, but Y201(am) and Y208(am) were suppressed efficiently at the translational level. We suggest that a translational pause by suppressor tRNAs reading UAG at these two positions may divert the nascent polypeptide into an alternative folding pathway that traps MotB in a non-functional conformation. We further propose that MotA and MotB form a stable pre-assembly complex in the membrane. In this complex, MotB exists in a form that cannot associate with peptidoglycan and blocks the proton-conducting channel. Opening of the channel and attachment to the cell wall may occur when the complex collides with a flagellar basal body and MotA makes specific contacts with the C ring and/or the MS ring.
authors.affiliation: Department of Biology, Texas A&M University, TX 77843-3258, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Van Way
authors.forename: E R
authors.initials: ER
authors.lastname: Hosking
authors.forename: T F
authors.initials: TF
authors.lastname: Braun
authors.forename: M D
authors.initials: MD
authors.lastname: Manson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D005407:Flagella / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D016153:Genes, Suppressor / Q000235:genetics; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; D008565:Membrane Proteins; D020409:Molecular Motor Proteins; C063010:MotA protein, Bacteria; C064779:MotB protein, Bacteria; D010446:Peptide Fragments; D010457:Peptidoglycan; D012329:RNA, Bacterial