Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus
Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus
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DOI：
10.1128/jb.183.5.1655-1662.2001
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发表时间：
2001-03-01
影响因子：
3.2
通讯作者：
Macnab, RM
中科院分区：
文献类型：
--
作者：
Kihara, M;Minamino, T;Macnab, RM
沙门氏菌鞭毛基体的 MS 环是一个完整的膜结构，由 61 kDa 蛋白质 FliF 的约 26 个亚基组成。在测试的许多非鞭毛类 fliF 突变体中，其中三种在鞭毛区 II 中产生了基因间抑制子。假回复体蜂拥而至，尽管效果很差。运动功能的部分恢复被证明是由于输出功能和鞭毛组装的部分恢复。三个亲本突变体均被发现携带相同的突变，即与预测的 FliF 蛋白周质结构域中 Ala-174 和 Ser-175 缺失相对应的六碱基缺失。鉴定出的19个基因间抑制因子均位于flhA中，它们在核苷酸水平上由10个独立实例组成，在氨基酸水平上由9个独立实例组成。由于九个中的两个对应于同一氨基酸位置处的不同取代，因此FlhA蛋白中只有八个位置产生了抑制子。因此，FliF-FlhA 基因间抑制是相当罕见的事件。 FlhA 是鞭毛蛋白输出装置的一个组成部分，具有包含序列 N 端一半的完整膜结构域和细胞质 C 端结构域。所有抑制突变都位于整合膜域内。当这些突变置于野生型 fliF 背景中时，没有突变表型。在fliF突变体背景中，突变体FlhA占主导地位，产生假回复表型。野生型FlhA在假回复背景中没有发挥显着的负显性，这表明它不能与突变型FlhA有效竞争与突变型FliF的相互作用，突变型FliF在野生型和第二位点FlhA背景中均相对于野生型FliF具有部分显性。膜分级分离实验表明，fliF 突变虽然阻止输出，但足够温和，允许 MS 环本身组装，以及将细胞质 C 环组装到 MS 环上。这项研究的数据为模型提供了遗传支持，在该模型中，至少输出装置的 FlhA 组件与其所在的 MS 环发生物理相互作用。
The MS ring of the flagellar basal body of Salmonella is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliF mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in flhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF, Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the fliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.