ABSTRACT

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their −35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

摘要

Gac-rsm 通路是一个全球监管网络，负责管理 γ-变形菌的生活方式和代谢变化。在之前的一项研究中，我们发现了 CsrA 蛋白在模型植物病原体丁香假单胞菌pv 中促进生长和抑制运动、藻酸盐产生和毒力的作用。番茄 (Pto) DC3000。在这里，我们专注于rsm调节 sRNA 的表达和调节，因为 Pto DC3000 异常有七个变体（rsmX1-5、rsmY和rsmZ）。所呈现的结果为复杂 Gac-rsm 通路的功能及其成分之间的相互作用提供了进一步的见解。总的来说，rsm表达在高细胞密度下达到最高水平，不受表面检测的影响，并且需要 GacA 才能完全表达。rsm表达水平和 GacA 依赖性分别由在其 -35/-10 启动子区域和 GacA 结合盒中发现的序列决定。rsmX5是 Pto DC3000中唯一一个高表达不需要 GacA 的 rsm，构成了 gacA 突变体中总rsm池的主要成分。rsmY和rsmZ的缺失对 Pto DC3000 运动性和毒力表型影响较小，表明rsmX1-5可以在功能上替代它们。另一方面，gacA突变体中的rsmY或rsmZ过表达并没有恢复其表型。此外，作为这项工作的一部分，已经公开了一个负反馈调节环，其中 CsrA3 蛋白通过以 GacA 依赖性方式增加几种rsm RNA的水平来促进其自身的滴定。