Department of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USA.
Marsico Lung Institute, Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Bacteriol. 2018 May 9;200(11). doi: 10.1128/JB.00736-17. Print 2018 Jun 1.
is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA. CsrA homologs are present in many bacteria. The opportunistic pathogen uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity and are more stringent than those for RsmA.
是一种革兰氏阴性机会致病菌，具有明显的急性和慢性毒力表型。急性毒力通常与 III 型分泌系统 (T3SS) 的表达有关，而慢性毒力的特征是生物膜的形成。许多与急性和慢性毒力相关的表型受到 RsmA 和 RsmF 的反向调节。RsmA 和 RsmF 都是 CsrA 家族 RNA 结合蛋白的成员，在转录后水平调节蛋白质合成。RsmA 的活性受两个小的非编码调节 RNA (RsmY 和 RsmZ) 控制。生物信息学分析表明，RsmY 和 RsmZ 每个都有 3 或 4 个假定的 RsmA 结合位点。每个预测的结合位点都包含一个 GGA 序列，位于茎环结构的环部分。RsmY 和 RsmZ 通过将这些蛋白质与靶 mRNA 隔离来调节 RsmA，可能还有 RsmF。在这项研究中，我们使用选择性 2'-羟基酰化分析引物延伸和突变分析 (SHAPE-MaP) 化学来确定 RsmY 和 RsmZ 的二级结构，并进行功能测定以表征每个 GGA 位点对 RsmY/RsmZ 活性的贡献。我们的数据表明，RsmA 在 RsmY 和 RsmZ 上有两个优先结合位点，而 RsmF 在 RsmY 上有一个优先结合位点，在 RsmZ 上有两个结合位点。尽管 RsmF 和 RsmA 具有共同的共识位点，但 RsmF 的结合特性比 RsmA 更严格。CsrA 同源物存在于许多细菌中。机会致病菌 利用 RsmA 和 RsmF 来反向调节与急性和慢性毒力表型相关的因素。RsmA 对 RsmY 和 RsmZ 的亲和力高于 RsmF。本研究的目的是利用 RsmY 和 RsmZ 调节性小 RNA (sRNA) 作为模型，了解 RsmA 和 RsmF 的差异结合特性。对 RsmY 和 RsmZ 上预测的 RsmA/RsmF 结合位点进行突变，揭示了控制 RsmA 和 RsmF 活性所需的位点的相似性。虽然 RsmA 的结合相对耐受结合位点突变，但 RsmF 对除两个位点之外的所有位点的突变都很敏感，进一步表明 RsmF 结合活性的要求 比 RsmA 更严格。
