title: Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium.
abstract: Defects in the chemotaxis proteins CheY and CheZ of Salmonella typhimurium can be suppressed by mutations in the flagellar switch, such that swarming of a pseudorevertant on semisolid plates is significantly better than that of its parent. cheY suppressors contribute to a clockwise switch bias, and cheZ suppressors contribute to a counterclockwise bias. Among the three known switch genes, fliM contributes most examples of such suppressor mutations. We have investigated the changes in FliM that are responsible for suppression, as well as the changes in CheY or CheZ that are being compensated for. Ten independently isolated parental cheY mutations represented nine distinct mutations, one an amino acid duplication and the rest missense mutations. Several of the altered amino acids lie on one face of the three-dimensional structure of CheY (A. M. Stock, J. M. Mottonen, J. B. Stock, and C. E. Schutt, Nature (London) 337:745-749, 1989; K. Volz and P. Matsumura, J. Biol. Chem. 266:15511-15519, 1991); this face may constitute the binding site for the switch. All 10 cheZ mutations were distinct, with several of them resulting in premature termination. cheY and cheZ suppressors in FliM occurred in clusters, which in general did not overlap. A few cheZ suppressors and one cheY suppressor involved changes near the N terminus of FliM, but neither cheY nor cheZ suppressors involved changes near the C terminus. Among the strongest cheY suppressors were changes from Arg to a neutral amino acid or from Val to Glu, suggesting that electrostatic interactions may play an important role in switching. A given cheY or cheZ mutation could be suppressed by many different fliM mutations; conversely, a given fliM mutation was often encountered as a suppressor of more than one cheY or cheZ mutation. The data suggest that an important factor in suppression is a balancing of the shift in switch bias introduced by alteration of CheY or CheZ with an appropriate opposing shift introduced by alteration of FliM. For strains with a severe parental mutation, such as the cheZ null mutations, adjustment of switch bias is essentially the only factor in suppression, since the attractant L-aspartate caused at most a slight further enhancement of the swarming rate over that occurring in the absence of a chemotactic stimulus. We discuss a model for switching in which there are distinct interactions for the counterclockwise and clockwise states, with suppression occurring by impairment of one of the states and hence by relative enhancement of the other state. FliM can also undergo amino acid changes that result in a paralyzed (Mot-) phenotype; these changes were confined to a very few residues in the protein.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
authors.forename: H
authors.initials: H
authors.lastname: Sockett
authors.forename: S
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: M
authors.initials: M
authors.lastname: Kihara
authors.forename: V M
authors.initials: VM
authors.lastname: Irikura
authors.forename: R M
authors.initials: RM
authors.lastname: Macnab
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D002633:Chemotaxis / Q000235:genetics*; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D005407:Flagella / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000235:genetics*; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C066821:FliM protein, Bacteria