Riboregulation in Agrobacterium tumefaciens

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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.