Karlinsey J E, Tsui H C, Winkler M E, Hughes K T
Department of Microbiology, University of Washington, Seattle, Washington 98195, USA.
J Bacteriol. 1998 Oct;180(20):5384-97. doi: 10.1128/JB.180.20.5384-5397.1998.
The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium. The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion. The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings. FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J. E. Karlinsey et al., J. Bacteriol. 179:2389-2400, 1997). In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains. The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected. This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter. Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings. Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell. Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains. With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains. No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains. We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.
钩基体（HBB）是鼠伤寒沙门氏菌鞭毛组装途径中的关键中间结构。在完整的HBB结构缺失时，FlgM蛋白会抑制鞭毛特异性转录因子sigma28，并且在HBB完成后会分泌到细胞外。flk基因在HBB完成前的一个组装步骤（即P环和L环组装时）编码FlgM活性的正调控因子。通过在flk基因中引入无效突变，在P环和L环组装突变体（flgA、flgH和flgI）中，FlgM对sigma28依赖性3类鞭毛基因转录的抑制作用得到缓解（J. E. Karlinsey等人，《细菌学杂志》179:2389 - 2400，1997）。在P环和L环突变菌株中，flk中的隐性突变导致细胞内FlgM水平降低至野生型（Fla +）菌株中的水平。与同基因flk +菌株相比，flk基因中的突变导致细胞内FlgM水平降低的同时，flgMN操纵子的转录增加了10倍，而flgAMN操纵子的转录不受影响。通过核糖核酸酶T2保护试验直接测量flgAMN和flgMN mRNA转录本以及将lac操纵子与flgAMN或flgMN启动子融合时都是如此。Flk的缺失不允许FlgM通过缺乏P环和L环的基体结构进行分泌。细胞内的FlgM对蛋白水解稳定，周转主要发生在分泌到细胞外之后。在P环或L环突变菌株中，Flk的缺失都不会导致FlgM周转增加。对于与flgM的lacZ翻译融合，flk中的无效突变导致在P环和L环突变菌株中，由3类flgMN启动子表达的flgM - lacZ mRNA翻译显著减少。在Fla +、环突变体或HBB缺失菌株中，在没有Flk的情况下，未检测到flgAMN或flgMN mRNA稳定性的降低。我们得出结论，flk基因中的突变导致细胞内FlgM水平降低仅在flgM mRNA翻译水平。
