Llewellyn Midge, Dutton Rachel J, Easter Jesse, O'donnol Danielle, Gober James W
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095-1569, USA.
Mol Microbiol. 2005 Aug;57(4):1127-42. doi: 10.1111/j.1365-2958.2005.04745.x.
The expression of the flagellin proteins in Caulobacter crescentus is regulated by the progression of flagellar assembly both at the transcriptional and post-transcriptional levels. An early basal body structure is required for the transcription of flagellin genes, whereas the ensuing assembly of a hook structure is required for flagellin protein synthesis. Previous experiments have shown that the negative regulatory protein, FlbT, operates this second post-transcriptional checkpoint by associating with the 5' untranslated region (UTR) of the fljK flagellin transcript, inhibiting translation and destabilizing the mRNA. In this paper we examine the role of flaF in flagellar biogenesis. The flaF gene, which is conserved in several speices of flagellated alpha-proteobacteria, is required for motility and flagellin protein synthesis. A deletion of flbT in a DeltaflaF strain restored flagellin protein expression, but not motility, indicating that FlaF functions in filament assembly. Mutant strains with a deletion in flaF had no detectable fljK mRNA, the levels of which were restored by an additional mutation in flbT. Assay of fljK gene expression using transcription and translation reporter fusions indicated that FlaF was essential for the translation of fljK mRNA. FlaF protein levels were under cell cycle control, peaking during the period of flagellin expression and filament assembly, whereas FlbT was present throughout the cell cycle. These results suggest that FlbT and FlaF activities oppose one another in the regulation of flagellin expression in response to both the progression of flagellar assembly and the cell cycle.
新月柄杆菌中鞭毛蛋白的表达在转录和转录后水平上均受鞭毛组装进程的调控。鞭毛蛋白基因的转录需要早期的基体结构，而鞭毛蛋白的合成则需要随后的钩状结构组装。先前的实验表明，负调控蛋白FlbT通过与fljK鞭毛蛋白转录本的5'非翻译区（UTR）结合，抑制翻译并使mRNA不稳定，从而操作这第二个转录后检查点。在本文中，我们研究了flaF在鞭毛生物发生中的作用。flaF基因在几种有鞭毛的α-变形杆菌中保守，是运动性和鞭毛蛋白合成所必需的。在ΔflaF菌株中缺失flbT可恢复鞭毛蛋白的表达，但不能恢复运动性，这表明FlaF在丝状体组装中起作用。flaF缺失的突变菌株没有可检测到的fljK mRNA，其水平通过flbT中的额外突变得以恢复。使用转录和翻译报告融合体对fljK基因表达进行检测表明，FlaF对于fljK mRNA的翻译至关重要。FlaF蛋白水平受细胞周期控制，在鞭毛蛋白表达和丝状体组装期间达到峰值，而FlbT在整个细胞周期中均存在。这些结果表明，在响应鞭毛组装进程和细胞周期时，FlbT和FlaF的活性在鞭毛蛋白表达的调控中相互拮抗。
