Riboregulation in Agrobacterium tumefaciens

细菌小RNA(SRNAs)控制着许多细胞过程，在大多数情况下是通过与目标mRNAs互补碱基配对。高通量测序方法发现植物病原菌根癌农杆菌中有数百个sRNAs。由于他们的差异表达，这意味着在某些条件下的调节作用，我们选择了三个候选人的详细特征。结果表明，AbcR1是根瘤目中一个有服务功能的sRNA家族的创始成员。它协调多个ABC转运蛋白的表达。在前一期的研究中，我们发现另一种称为L5的SRNA可能通过暴露三叶草样结构中的序列来调节一组重叠的ABC转运蛋白。另一种ABC转运蛋白由sRNA PmaR(肽聚糖的生物合成、动力和氨苄青霉素抗性调节因子)控制。这些结果表明，至少由三个sRNA组成的网络协调农杆菌的营养获取。在这些sRNA中，PmaR特别有趣，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酶的正调控基因。这一发现解释了众所周知的农杆菌对氨苄西林的天然抗药性。拟议的项目遵循两个方向：第一，这三个sRNA的调节；第二，它们各自的差异调节。在第一个项目中，我们希望通过对相应的突变株进行RNA-Seq分析来剖析这三个sRNA的单独和重叠靶标。对选定目标的验证将包括定量实时聚合酶链式反应和生化RNA-RNA相互作用研究。潜在的调控原理将通过RNA稳定性测量和脚印实验(引物延伸抑制试验)进行检验。SRNAs的一个特征是它们对波动的环境条件的不同丰度。值得注意的是，我们发现AbcR1、L5和PmaR的表达受到同一转录因子-LysR型调节因子LsrB的调节。我们推测，许多已报道的LsrB靶基因不是由转录因子直接调节的，而是通过sRNA的作用而调节的。为了鉴定该调节子的DNA结合区，我们计划使用CHAP-SEQ(染色质亲和纯化和测序)。选定的结合部位将用EMSA(凝胶迁移率位移分析)进行表征。总体而言，我们希望获得对一个由蛋白质和RNA组件组成的迷人调控网络的见解，该网络促进无处不在的植物病原体在不断变化的环境中生存。

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.