Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA.
BMC Microbiol. 2011 May 2;11:89. doi: 10.1186/1471-2180-11-89.
The temporal and spatial expression of late flagellar genes in Caulobacter crescentus is activated by the transcription factor FlbD and its partner trans-acting factor FliX. The physical interaction of these two proteins represents an alternative mechanism for regulating the activity of σ54 transcription factors. This study is to characterize the interaction of the two proteins and the consequences of the interaction on their regulatory activity.
FliX and FlbD form stable complexes, which can stand the interference of 2.65 M NaCl. The stability of FliX and FlbD was affected by the co-existence of each other. Five FliX mutants (R71A, L85K, Δ117-118, T130L, and L136K) were created by site-directed mutagenesis in conserved regions of the protein. All mutants were successfully expressed in both wild-type and ΔfliX Caulobacter strains. All but FliXL85K could rescue the motility and cell division defects of a ΔfliX mutant strain. The ability of FliX to regulate the transcription of class II and class III/IV flagellar promoters was fully diminished due to the L85K mutation. Co-immunoprecipitation experiment revealed that FliXL85K was unable to physically interact with FlbD.
FliX interacts with FlbD and thereby directly regulates the activity of FlbD in response to flagellar assembly. Mutations in highly conserved regions of FliX could severely affect the recognition between FliX and FlbD and hence interrupt the normal progression of flagellar synthesis and other developmental events in Caulobacter.
在新月柄杆菌中，晚期鞭毛基因的时空表达受转录因子 FlbD 和其伴侣反式作用因子 FliX 激活。这两种蛋白质的物理相互作用代表了调节σ54 转录因子活性的另一种机制。本研究旨在表征这两种蛋白质的相互作用及其对调节活性的影响。
FliX 和 FlbD 形成稳定的复合物，可以耐受 2.65 M NaCl 的干扰。FliX 和 FlbD 的稳定性受彼此共存的影响。通过定点突变在蛋白质的保守区域创建了 5 个 FliX 突变体（R71A、L85K、Δ117-118、T130L 和 L136K）。所有突变体都能在野生型和ΔfliX 新月柄杆菌菌株中成功表达。除 FliXL85K 外，所有突变体均能挽救ΔfliX 突变株的运动和细胞分裂缺陷。由于 L85K 突变，FliX 调节 II 类和 III/IV 类鞭毛启动子转录的能力完全丧失。共免疫沉淀实验表明，FliXL85K 无法与 FlbD 进行物理相互作用。
FliX 与 FlbD 相互作用，从而直接调节 FlbD 的活性，以响应鞭毛组装。FliX 高度保守区域的突变可能严重影响 FliX 和 FlbD 之间的识别，从而中断新月柄杆菌中鞭毛合成和其他发育事件的正常进程。
