Riboregulation in Agrobacterium tumefaciens
Final Report Abstract
Agrobacterium tumefaciens is a model organism of significant ecological and economic importance. The bacterium is known for inducing crown gall tumors in numerous agriculturally relevant plants and serves as a molecular tool for generating genetically engineered plants. RNA-sequencing approaches have identified hundreds of small RNAs (sRNAs) in this plant pathogen. We selected several candidates for detailed characterization. AbcR1 emerged as the founding member of a conserved sRNA family that coordinates the expression of multiple ABC transporters. In ongoing work, we are investigating another sRNA family characterized by exposed CCUCC sequences (“cuckoo” motifs), which are involved in regulating ABC transporter gene expression. Our analysis of PmaR has shown that this sRNA is a positive regulator for several peptidoglycan biosynthesis genes, motility genes as well as ampC coding for a beta-lactamase. While exploring the role of sRNAs, we found that many of them are regulated by the LysR- type regulator (LTTR) LsrB. RNA-sequencing of the lrsB mutant revealed that approximately 100 sRNA genes are regulated, raising the question of whether the previously described extensive LsrB regulon is directly controlled by this LTTR or instead regulated by LsrB- controlled sRNAs. Our results support the latter scenario. One particularly interesting gene directly and negatively regulated by LsrB is ampD, which codes for an anhydroamidase involved in peptidoglycan recycling. Overproduction of AmpD in the lsrB mutant leads to ampicillin sensitivity because intracellular muropeptides, which serve as activators for beta-lactamase induction, are depleted by AmpD. In summary, LsrB plays a dual role in the regulation of the long-observed natural ampicillin resistance by negatively regulating the expression of ampD, while positively regulating expression of the sRNA PmaR. Finally, we investigated the structure and sensory mechanism of LsrB. By mass spectrometry-based detection of disulfide bond formation, AlphaFold-assisted structural modelling, and a preliminary cryo-EM structure, we discovered that this LTTR is a redox switch, sensing hydrogen peroxide through reversible disulfide bridge formation. Comparative RNA-sequencing of lsrB and/or oxyR mutants showed that both redox sensors control partially overlapping yet distinct regulons. In addition to sensing the cellular redox status, LsrB acts as a metabolite-binding protein, sensing the plant-derived phenolic compound acetosyringone. Microscale thermophoresis along with site-directed mutagenesis confirmed the involvement of bioinformatically predicted ligand-binding residues. In summary, our research revealed LsrB as a pivotal regulator in A. tumefaciens, controlling the expression of numerous sRNAs, ampicillin resistance, nutrient acquisition and virulence in this ubiquitous plant pathogen.
Publications
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A Small Regulatory RNA Controls Cell Wall Biosynthesis and Antibiotic Resistance. mBio, 9(6).
Borgmann, Jessica; Schäkermann, Sina; Bandow, Julia Elisabeth & Narberhaus, Franz
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The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic EngineerAgrobacterium tumefaciens. Journal of Bacteriology, 201(11).
Möller, Philip; Busch, Philip; Sauerbrei, Beate; Kraus, Alexander; Förstner, Konrad U.; Wen, Tuan-Nan; Overlöper, Aaron; Lai, Erh-Min & Narberhaus, Franz
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A LysR‐type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens. Molecular Microbiology, 116(1), 126-139.
Eisfeld, Jessica; Kraus, Alexander; Ronge, Christian; Jagst, Michelle; Brandenburg, Vivian B. & Narberhaus, Franz
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The LysR‐type transcription factor LsrB regulates beta‐lactam resistance in Agrobacterium tumefaciens. Molecular Microbiology, 121(1), 26-39.
Schmidt, Janka J.; Remme, Donata C. L. E.; Eisfeld, Jessica; Brandenburg, Vivian B.; Bille, Hannah & Narberhaus, Franz
