Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites
Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites
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DOI：
10.1046/j.1365-2958.1998.00924.x
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发表时间：
1998-07-01
影响因子：
3.6
通讯作者：
Chatterjee, AK
中科院分区：
文献类型：
--
作者：
Liu, Y;Cui, YY;Chatterjee, AK
胡萝卜软腐欧文氏菌（Erwinia carotovora ssp.）胡萝卜软腐菌菌株71（下文称为Ecc 71）产生胞外酶，例如果胶酸裂解酶同工酶（Pels）、纤维素酶（Cel）、多聚半乳糖醛酸酶（Peh）和蛋白酶（Prt）。这些酶降解植物细胞壁成分，并在很大程度上负责诱发植物和植物产品中的软腐病，Ecc 71还产生Harpin（Ecc），过敏反应（HR）的激发子和群体感应信号，N-（3-oxo-hexanoyl）-L-高丝氨酸内酯（OHL）。OHL控制胞外酶和Harpin（Ecc）的产生。这些酶的释放以及Harpin（Ecc）的结构基因hrpN（Ecc）和OHL合成的基因ohll的表达受RsmA负调控，另一方面，rsmB（以前称为aepH）正调控胞外酶的产生。6从大肠杆菌中纯化的His-RsmA重组蛋白。如凝胶迁移率变动分析所示，大肠杆菌结合rsmB RNA。rsmB包含547 bp DNA，其从σ（70）样启动子后的单个起始位点转录。在Ecc 71中，检测到两种rsmB RNA种类：全长479个碱基的rsmB RNA和259个碱基的rsmB ′ RNA，rsmB ′ DNA与259个碱基和479个碱基的转录物杂交，3' RNase保护试验表明，259个碱基和479个碱基的RNA种类在推定的rho非依赖性终止子之后立即终止于相同的位置。rsmB-lacZ转录融合体的表达证实，由于内部启动子的激活，rsmB' RNA不产生。在Ecc 71中，由lac启动子驱动的rsmB ′表达引起Pel、Peh、Cel和Prt的过量产生，以及pel-1、peh-1、hrpN（Ecc）和ohll转录物的积累。相反，缺失rsmS' DNA序列的质粒不能引起Ecc 71中胞外酶的过量产生。rsmB'效应也发生在大肠杆菌中，因为在rsmB '存在下刺激糖原积累。rsmB'的体内和体外翻译以及突变分析已经确定rsmB' RNA不产生翻译产物。因此，我们得出结论，rsmS’RNA本身起调节剂的作用。实际上，rsmS’DNA的表达导致Ecc 71和粘质沙雷氏菌菌株SM 274中RNA结合蛋白RsmA的负面作用的中和。我们提出了一个模型，解释如何RsmA和rsmB控制胞外酶的基因表达。
The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of softrot diseases in plants and plant products, Ecc71 also produces Harpin(Ecc), the elicitor of hypersensitive reaction (HR) and the quorum-sensing signal, N-(3-oxo-hexanoyl)-L-homoserine lactone (OHL). OHL controls extracellular enzyme and Harpin(Ecc) production. The revels of these enzymes, as well as the expression of hrpN(Ecc), the structural gene for Harpin(Ecc) and ohll, the gene specifying OHL synthesis, are negatively regulated by RsmA, rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His-RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a sigma(70)-like promoter, In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA, rsmB' DNA hybridizes with the 259 base and the 479 base transcripts, A 3' RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho-independent terminator. The expression of rsmB-lacZ transcriptional fusions established that the rsmB' RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB' RNA is derived by processing of the primary rsmB RNA, In Ecc71, rsmB' expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrpN(Ecc) and ohll transcripts. By contrast, a plasmid with the rsmS' DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71, The rsmB' effect also occurs in Escherichia coli as glycogen accumulation is stimulated in the presence of rsmB'. In vivo and in vitro translation as well as mutational analysis of rsmB' have established that rsmB' RNA does not yield a translational product. Therefore, we concluded that the rsmS' RNA itself functions as the regulator, Indeed, the expression of rsmS' DNA leads to neutralization of the negative effects of the RNA-binding protein, RsmA, in Ecc71 and Serratia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.