Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene

Abstract

Listen

Translate article

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 Δ motAB strain was rescued better by a p motA + B + plasmid than the non-motile phenotype of a Δ motB strain was rescued by a p motB + 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 p motB. However, introduction of an optimized RBS into p motB (to generate p motB 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 p motB, and at codon 261 or earlier in p motB o or p motAB, 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 p motB. In contrast, motility defects conferred by most motB am mutations in p motB o or p motAB 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.