Citrus Research and Education Center, Department of Microbiology and Cell Sciences, University of Florida, 700 Experiment Station Road, Lake Alfred FL 33850, U.S.A.
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan, 655011, China.
Mol Plant Microbe Interact. 2021 Nov;34(11):1236-1249. doi: 10.1094/MPMI-05-21-0113-R. Epub 2021 Nov 16.
The RNA-binding protein CsrA is a global posttranscriptional regulator and controls many physiological processes and virulence traits. Deletion of caused loss of virulence, reduced motility and production of xanthan gum and substantial increase in glycogen accumulation, as well as enhanced bacterial aggregation and cell adhesion in spp. How CsrA controls these traits is poorly understood. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis was conducted to compare the protein profile of wild-type strain subsp. and the isogenic Δ strain. A total of 2,374 proteins were identified, and 284 were considered to be differentially expressed proteins (DEP), among which 151 proteins were up-regulated and 133 were down-regulated in the Δ strain with respect to the wild-type strain. Enrichment analysis and a protein-protein interaction network analysis showed that CsrA regulates bacterial secretion systems, flagella, and xanthan gum biosynthesis. Several proteins encoded by the operon were down-regulated, whereas proteins associated with flagellum assembly and the type IV secretion system were up-regulated in the Δ strain relative to the Xcc306 strain. These results were confirmed by β-glucuronidase assay or Western blot. RNA secondary structure prediction and a gel-shift assay indicated that CsrA binds to the Shine-Dalgarno sequence of . In addition, the iTRAQ analysis identified 248 DEPs that were not previously identified in transcriptome analyses. Among them, CsrA regulates levels of eight regulatory proteins (ColR, GacA, GlpR, KdgR, MoxR, PilH, RecX, and YgiX), seven TonB-dependent receptors, four outer membrane proteins, and two ferric enterobactin receptors. Taken together, this study greatly expands understanding of the regulatory network of CsrA in subsp. .[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
RNA 结合蛋白 CsrA 是一种全局转录后调控因子，控制着许多生理过程和毒力特性。缺失 导致毒力丧失、运动性降低、黄原胶产量减少、糖原积累大量增加，以及 spp. 中的细菌聚集和细胞黏附增强。CsrA 如何控制这些特性还知之甚少。在这项研究中，我们进行了基于等重同位素标记相对和绝对定量（iTRAQ）的蛋白质组学分析，比较了野生型菌株 subsp. 及其同源缺失突变株 的蛋白质谱。共鉴定了 2374 种蛋白质，其中 284 种被认为是差异表达蛋白（DEP），在缺失突变株中，有 151 种蛋白上调，133 种蛋白下调。富集分析和蛋白质-蛋白质相互作用网络分析表明，CsrA 调节细菌分泌系统、鞭毛和黄原胶生物合成。 操纵子编码的几种蛋白下调，而与鞭毛组装和 IV 型分泌系统相关的蛋白在 Δ 菌株中上调。这些结果通过β-葡萄糖醛酸酶测定或 Western blot 得到了验证。RNA 二级结构预测和凝胶迁移实验表明，CsrA 结合到 的 Shine-Dalgarno 序列。此外，iTRAQ 分析鉴定了 248 个在转录组分析中未被识别的差异表达蛋白。其中，CsrA 调节了 8 种调控蛋白（ColR、GacA、GlpR、KdgR、MoxR、PilH、RecX 和 YgiX）、7 种 TonB 依赖受体、4 种外膜蛋白和 2 种铁 enterobactin 受体的水平。综上所述，这项研究极大地扩展了我们对 CsrA 在 subsp. 中的调控网络的理解。
