Trampari Eleftheria, Stevenson Clare E M, Little Richard H, Wilhelm Thomas, Lawson David M, Malone Jacob G
From the Molecular Microbiology Department and.
the Biological Chemistry Department, John Innes Centre, Norwich NR4 7UH, United Kingdom.
J Biol Chem. 2015 Oct 2;290(40):24470-83. doi: 10.1074/jbc.M115.661439. Epub 2015 Aug 11.
The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
广泛存在的第二信使分子环二鸟苷酸(cdG)可调节多种细菌从运动性和致病性的生活方式向固着性、生物膜形成性的生活方式转变。已知许多致病性和共生性细菌与宿主的相互作用受cdG信号传导控制。尽管环二核苷酸代谢的生物化学已得到充分理解,但关于cdG结合后诱导细菌反应的下游信号通路仍有许多有待发现。作为我们正在进行的关于cdG信号在植物相关假单胞菌属物种中作用研究的一部分,我们在模式生物荧光假单胞菌SBW25中对cdG结合蛋白进行了亲和捕获筛选。鞭毛输出AAA + ATP酶FliI是此次筛选的结果,随后显示它能特异性结合cdG分子,解离常数在低微摩尔范围内。FliI与cdG之间的相互作用似乎非常广泛。除了来自不同细菌物种的FliI同源物外,还观察到III型分泌系统同源物HrcN和VI型ATP酶ClpB2有高亲和力结合。体外实验表明,添加cdG可抑制FliI和HrcN的ATP酶活性。最后,结合位点特异性诱变、质谱分析和计算机模拟分析,预测cdG在两个FliI亚基之间界面处的高度保守残基口袋中与FliI结合。我们的结果表明,cdG通过对输出ATP酶蛋白的直接变构控制,在控制多种重要细菌输出途径的功能方面发挥着新的、基本的作用。