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Schwerpunkte der wissenschaftlichen Arbeit:

 

We are investigating ribonucleases (RNases), small regulatory RNAs and very small proteins in rhizobia, soil-dwelling bacteria capable to fix molecular nitrogen in symbiosis with plants.

A major goal of our work is to uncover the physiological roles and mechanisms of a small RNA and a small protein originating from a bacterial transcription attenuator. In prokaryotes transcription and translation are coupled. Therefore, in bacteria efficiency of translation of small upstream ORFs (uORFs) can determine whether the downstream genes are transcribed. This mechanism depends on mutually exclusive RNA secondary structures and is known as transcription attenuation. Many Gram-negative bacteria have transcription attenuators upstream of amino acid (aa) biosynthesis operons. A prime example is the trpEDCBA operon in Escherichia coli. Its 5′ mRNA leader harbors the uORF trpL, which contains two consecutive tryptophan (Trp) codons. Under conditions of Trp shortage, ribosome pausing at the Trp codons prevents the formation of a transcriptional terminator between trpL and trpE. When Trp is available, the leader peptide (small protein) peTrpL is efficiently translated and the small attenuator RNA rnTrpL (which harbors trpL) is generated (Yanofski, 1981).

Usually attenuator RNAs and leader peptides are considered non-functional. We found that, in the soil-dwelling plant symbiont Sinorhizobium meliloti and in other related bacteria, both the leader peptide peTrpL and the small RNAs (sRNAs) rnTrpL generated upon transcription attenuation of the trp operon trpE(G) are functional in trans. Furthermore, we found that the trp attenuator responds not only to tryptophan availability, but also to translation inhibition.

 

The network of the attenuator sRNA rnTrpL

(Project EV42/6-1, funded by DFG)

The attenuator sRNA rnTrpL is a central riboregulator, which responds to nutrient availability. In S. meliloti, the sRNA rnTrpL regulates the tryptophan biosynthesis genes trpDC (Melior et al., 2019) and the quorum sensing autoinducer synthase gene sinI (Baumgardt et al., 2016). Bioinformatic predictions suggested that rnTrpL base-pairs with and determines the level of mRNAs encoding several transcription regulators. Currently we are analyzing the network of this sRNA.

 

 

 

Role of the trp attenuator and its trans-acting products in adaptation to translation inhibitors. The attenuator sRNA rnTrpL is released not only under conditions of tryptophan shortage, but also upon translation inhibition. We found that the sRNA rnTrpL, the leader peptide (14 aa) peTrpL and several translation-inhibiting antibiotics form an antibiotic-dependent ribonucleoprotein complex (ARNP) which destabilizes rplUrpmA mRNA encoding ribosomal proteins L21 and L27 (https://www.biorxiv.org/content/10.1101/606483v2). The analysis of this novel mechanism for posttranscriptional gene regulation is a major research topic in the lab.

 

 

 

 

The S. meliloti trp attenuator and ARNP formation, image generated by deepdreamgenerator. https://deepdreamgenerator.com/

 

Roles of small ORFs and small proteins in rhizobia

(Project EV42/7-1 in SPP 2002, funded by DFG)

Recently we mapped genome-wide transcription start sites and predicted promoters in the reannotated genome of the soybean symbiont Bradyrhizobium japonicum (Bradyrhizobium diazoefficiens) (Čuklina et al., 2016). This analysis led to the discovery and annotation of many small ORFs (Hahn et al., 2016; Hahn et al., 2017). Currently we are analyzing the small proteome (proteins encoded by own ORFs that are < 50 codons) in rhizobia. In addition to a small protein inventory, functional analysis of selected small proteins is a major goal of this project. We found that the small protein peTrpL (14 aa leader peptide of the trpE(G) operon) is a multiresistance factor in rhizobia. Analysis of peTrpL in S. meliloti revealed that this small protein is involved in the posttranscriptional upregulation of the multidrug efflux pump SmeAB (https://www.biorxiv.org/content/10.1101/606483v1). Currently we are analyzing the molecular mechanisms of action of the new multiresistance factor peTrpL

 

 

 

Roles of ribonucleases in gene regulation and RNA modification in Sinorhizobium meliloti

(Part project in RTG 2355, funded by DFG)

Ribonucleases play pivotal roles in posttranscriptional gene regulation. In S. meliloti, RNase E and RNase J are necessary for the homeostasis of the major methyl donor in the cell, S-adenosylmethionine (SAM) (Baumgardt, Melior et al., 2017). Specific aims of this project are, first, to uncover the post-transcriptional mechanisms leading to increased SAM concentration upon depletion of either RNase E or RNase J in S. meliloti; and second, to identify ″hot spots″ of m6A methylation in mRNAs and regulatory RNAs in this organism as a first step towards the identification of the corresponding methylating enzyme and our understanding of the physiological role of this RNA modification in bacteria. Altogether, we aim to reveal new mechanisms for post-transcriptional regulation of gene expression in bacteria.

 

 

 

References:

Baumgardt K, Melior H, Madhugiri R, Thalmann S, Schikora A, McIntosh M, Becker A, Evguenieva-Hackenberg E. (2017) RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti. Microbiology. 163(4):570-583.

Baumgardt K, Šmídová K, Rahn H, Lochnit G, Robledo M, Evguenieva-Hackenberg E. (2016) The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti. RNA Biol., 13, 486-499.

Čuklina J, Hahn J, Imakaev M, Omasits U, Förstner KU, Ljubimov N, Goebel M, Pessi G, Fischer HM, Ahrens CH, Gelfand MS, Evguenieva-Hackenberg E. (2016) Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation. BMC Genomics. 2016 17:302.

Hahn J, Thalmann S, Migur A, von Boeselager RF, Kubatova N, Kubareva E, Schwalbe H, Evguenieva-Hackenberg E. (2017) Conserved small mRNA with an unique, extended Shine-Dalgarno sequence. RNA Biol. 14(10):1353-1363.

Hahn J, Tsoy OV, Thalmann S, Čuklina J, Gelfand MS, Evguenieva-Hackenberg E. (2016) Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110. PLoS One 11:e0165429.

Melior H, Li S, Madhugiri R, Stötzel M, Azarderakhsh S, Barth-Weber S, Baumgardt K, Ziebuhr J, Evguenieva-Hackenberg E (2019) Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans. Nucleic Acids Res 47, 6396-6410.

Merino E, Jensen RA, Yanofsky C. (2008) Evolution of bacterial trp operons and their regulation. Curr. Opin. Microbiol. 11, 78-86.

Yanofsky C. (1981) Attenuation in the control of expression of bacterial operons. Nature 289:751-758.