CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia.
CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia.
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DOI：
10.1099/mic.0.000283
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发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
Fineran PC
中科院分区：
文献类型：
--
作者：
Hampton HG;McNeil MB;Paterson TJ;Ney B;Williamson NR;Easingwood RA;Bostina M;Salmond GPC;Fineran PC
SdhE 是琥珀酸脱氢酶和延胡索酸还原酶（FRD）进行黄素化修饰及激活所必需的。此外，SdhE 在变形菌（α、β 和 γ 变形菌纲）和真核生物中具有保守性。尽管这个最近被鉴定出来的蛋白质家族的功能已被确定，但对于其基因的调控机制几乎一无所知。在此，研究鉴定出了转录后和翻译后调控因子 RsmA（CsrA）和 RsmC（HexY），并表明它们可抑制粘质沙雷氏菌 ATCC 39006 中 sdhEygfX 的表达。相反，鞭毛主调控因子复合物 FlhDC 可激活 sdhEygfX 的转录。为了探究调控层级，我们开发了一种新方法，即利用 I - F 型内源性成簇规律间隔短回文重复序列（CRISPR）- CRISPR 相关蛋白（Cas）基因组编辑系统，在 flhC 基因上产生染色体点突变。flhC 基因突变减轻了 RsmC 对 sdhEygfX 表达的抑制能力，而 RsmA 则以 FlhDC 依赖和非依赖两种方式抑制 sdhEygfX。rsmA 或 rsmC 基因突变，或者 FlhDC 过表达，都会导致灵菌红素、生物表面活性剂的产量增加，以及泳动和群集运动能力增强。与运动调控因子对 sdhE 的调节作用一致，我们已证明 SdhE 和 FRD 对于鞭毛依赖的最大程度泳动是必需的。综上所述，这些结果表明，代谢和运动的调控因子（RsmA、RsmC 和 FlhDC）共同控制着 sdhEygfX 操纵子的转录。
SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.
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