Riboregulation in Agrobacterium tumefaciens

根癌农杆菌的核糖调节

• 批准号: 289473898
• 负责人: Professor Dr. Franz Narberhaus
• 金额: --
• 依托单位: Lehrstuhl für Biologie der Mikroorganismen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/289473898?language=en
• 关键词: Riboregulation; Agrobacterium; tumefaciens

Bacterial small RNAs (sRNAs) control numerous cellular processes,in most cases by complementary base-pairing with target mRNAs.High-throughput sequencing approaches revealed hundreds ofsRNAs in the plant pathogen Agrobacterium tumefaciens. Due to theirdifferential expression, which suggested a regulatory role undercertain conditions, we selected three candidates for detailedcharacterization. AbcR1 turned out to be the founding member of aconserved family of sRNAs in Rhizobiales. It coordinates theexpression of multiple ABC transporters. In the previous fundingperiod, we discovered that another sRNA called L5 regulates anoverlapping set of ABC transporters presumably by exposedsequences in a cloverleaf-like structure. Another ABC transporter iscontrolled by the sRNA PmaR (Peptidoglycan biosynthesis, motilityand ampicillin resistance Regulator). These results suggest that anetwork comprised of at least three sRNAs coordinates nutrientacquisition in Agrobacterium. Among these sRNAs, PmaR isparticularly interesting as it is a positive regulator of several cell wallbiosynthesis genes and ampC coding for a beta-lactamase. Thisfinding explains the long-known natural resistance of Agrobacteriumtowards ampicillin. The proposed project follows two directions: First,the regulation by these three sRNAs and second, their own differentialregulation. In the first project, we want to dissect separate andoverlapping targets of the three sRNAs by RNA-Seq analyses of thecorresponding mutant strains. Validation of selected target will includequantitative real-time PCR and biochemical RNA-RNA interactionstudies. The underlying regulatory principles will be examined by RNAstability measurements and toeprinting experiments (primer extensioninhibition assays). A characteristic feature of sRNAs is their differentialabundance in response to fluctuating environmental conditions.Remarkably, we found that the expression of AbcR1, L5 and PmaR isregulated by the same transcription factor, the LysR-type regulatorLsrB. We postulate that many of the documented LsrB target genesare not directly regulated by the transcription factor but via the actionof sRNAs. To identify DNA-binding regions of the regulator, we plan touse CHAP-Seq (CHromatin-Affinity Purification and Sequencing).Selected binding sites will be characterized by EMSAs(Electrophoretic Mobility Shift Assays). Overall, we expect to gaininsights into a fascinating regulatory network comprised of protein andRNA components that promotes survival of a ubiquitous plantpathogen in an ever-changing environment.

细菌的小RNA（small RNAs，sRNAs）通过与靶mRNA的互补碱基配对控制着细胞的许多生理过程，高通量测序技术在植物病原体根癌农杆菌中发现了数百个sRNAs。由于它们的差异表达，这表明在某些条件下的调节作用，我们选择了三个候选人进行详细的表征。AbcR 1是根瘤菌目sRNA家族的创始成员。它协调多种ABC转运蛋白的表达。在之前的研究中，我们发现另一种叫做L5的sRNA调节一组重叠的ABC转运蛋白，可能是通过暴露在三叶草样结构中的序列来实现的。另一种ABC转运蛋白由sRNA PmaR（肽聚糖生物合成、运动和氨苄青霉素抗性调节因子）控制。这些结果表明，至少由三种sRNA组成的网络协调农杆菌的营养获取。在这些sRNA中，PmaR是特别令人感兴趣的，因为它是几个细胞壁生物合成基因和编码β-内酰胺酶的ampC的正调控因子。这一发现解释了农杆菌对氨苄青霉素的天然耐药性。该项目的研究方向有两个：第一，这三种sRNA的调控;第二，它们自身的差异调控。在第一个项目中，我们希望通过对相应突变株的RNA-Seq分析来剖析这三种sRNA的独立和重叠靶点。所选靶点的验证将包括定量实时PCR和生化RNA-RNA相互作用研究。将通过RNA稳定性测量和足印实验（引物延伸抑制试验）检查潜在的调控原则。sRNA的一个特征是它们对波动的环境条件的反应具有不同的丰度。值得注意的是，我们发现AbcR 1、L5和PmaR的表达受相同的转录因子LysR型调节因子LsrB的调节。我们推测，许多文献记载的LsrB靶基因不是直接受转录因子的调控，而是通过sRNA的作用。为了确定该调节子的DNA结合区域，我们计划使用CHAP-Seq（CHromatin-Affinity Purification and Sequencing），选择的结合位点将通过EMSA（Electrophoretic Mobility Shift assays）进行表征。总的来说，我们期望深入了解一个迷人的调控网络，该网络由蛋白质和RNA组成，促进无处不在的植物病原体在不断变化的环境中的生存。