Tsang Jennifer, Hirano Takanori, Hoover Timothy R, McMurry Jonathan L
Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, USA.
J Bacteriol. 2015 Jun;197(11):1886-92. doi: 10.1128/JB.02610-14. Epub 2015 Mar 23.
Flagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. In Helicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes, flaB and flgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). The KD (equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon = 2.9 × 10(4) M(-1)s(-1)) and slow-off (koff = 6.2 × 10(-4) s(-1)) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANT sequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANT binding did not stimulate FlgS autophosphorylation in vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.
The high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.
鞭毛生物合成是一个复杂的过程，涉及多个检查点，以协调鞭毛基因的转录与鞭毛的组装。在幽门螺杆菌中，鞭毛生物合成中期所需基因的转录由RpoN以及由组氨酸激酶FlgS和反应调节因子FlgR组成的双组分系统控制。响应未知信号时，FlgS自身磷酸化并将磷酸基团转移至FlgR，启动来自RpoN依赖性启动子的转录。在本研究中，对输出装置蛋白FlhA作为潜在信号蛋白进行了检测。缺失其N端胞质序列显著降低了两个RpoN依赖性基因flaB和flgE的表达。光学生物传感显示FlgS与由FlhA的1至25位残基组成的肽（FlhANT）之间存在高亲和力相互作用。平衡解离常数（KD）为21 nM，其特征在于结合快（kon = 2.9×10⁴ M⁻¹s⁻¹）和解离慢（koff = 6.2×10⁻⁴ s⁻¹）的动力学。FlgS不与由FlhANT序列较小片段组成的肽结合。对与纯化的FlgS片段结合的分析表明，该蛋白含有激酶结构域的C端部分与FlhANT结合。FlhANT结合在体外不刺激FlgS自身磷酸化，表明FlhA促进FlgS与刺激自身磷酸化所需的其他结构之间的相互作用。
本研究中表征的FlgS与FlhA的高亲和力结合表明FlhA在鞭毛组装中具有额外作用。除了其对III型分泌的必要性外，FlhA的N端胞质序列通过与FlgS的C端激酶结构域相互作用，对于RpoN依赖性基因表达是必需的。
