Sockett H, Yamaguchi S, Kihara M, Irikura V M, Macnab R M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
J Bacteriol. 1992 Feb;174(3):793-806. doi: 10.1128/jb.174.3.793-806.1992.
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.
鼠伤寒沙门氏菌趋化蛋白CheY和CheZ中的缺陷可被鞭毛开关中的突变所抑制，使得假回复突变体在半固体平板上的群体游动能力明显优于其亲本。cheY抑制子导致顺时针开关偏向，而cheZ抑制子导致逆时针偏向。在三个已知的开关基因中，fliM产生了此类抑制突变的大多数例子。我们研究了导致抑制作用的FliM变化，以及被补偿的CheY或CheZ的变化。十个独立分离的亲本cheY突变代表九个不同的突变，一个是氨基酸重复，其余是错义突变。几个发生改变的氨基酸位于CheY三维结构的一个面上（A.M.斯托克、J.M.莫托宁、J.B.斯托克和C.E.舒特，《自然》（伦敦）337:745 - 749，1989；K.沃尔兹和P.松村，《生物化学杂志》266:15511 - 15519，1991）；这个面可能构成开关的结合位点。所有10个cheZ突变都是不同的，其中几个导致提前终止。FliM中的cheY和cheZ抑制子成簇出现，一般不重叠。一些cheZ抑制子和一个cheY抑制子涉及FliM N端附近的变化，但cheY和cheZ抑制子都不涉及C端附近的变化。最强的cheY抑制子包括从精氨酸到中性氨基酸的变化或从缬氨酸到谷氨酸的变化，这表明静电相互作用可能在开关过程中起重要作用。一个给定的cheY或cheZ突变可以被许多不同的fliM突变所抑制；相反，一个给定的fliM突变经常作为不止一个cheY或cheZ突变的抑制子出现。数据表明，抑制作用中的一个重要因素是平衡由CheY或CheZ改变引入的开关偏向变化与由FliM改变引入的适当相反变化。对于具有严重亲本突变的菌株，如cheZ缺失突变，开关偏向的调整基本上是抑制作用的唯一因素，因为吸引剂L - 天冬氨酸在没有趋化刺激的情况下最多只会使群体游动速率略有进一步提高。我们讨论了一个开关模型，其中逆时针和顺时针状态存在不同的相互作用，抑制作用通过损害其中一个状态从而相对增强另一个状态而发生。FliM也可能发生氨基酸变化，导致麻痹（Mot -）表型；这些变化局限于蛋白质中极少数的残基。
