Inhaltspezifische Aktionen

Schwerpunkte der wissenschaftlichen Arbeit:

Research topics

We are investigating regulation at the level of RNA in bacteria. We are also interested in very small proteins translated from own small ORFs. Particularly, we are using rhizobia and E. coli to study the trans-acting products of the trp attenuator: the attenuator sRNA rnTrpL and the leader peptide peTrpL. Furthermore, we are analyzing SAM-II riboswitches and the small proteome of S. meliloti.

The versatile attenuator sRNA rnTrpL and the leader peptide peTrpL

In prokaryotes, transcription and translation can be coupled. Therefore, in bacteria efficiency of translation of small upstream open reading frames (uORFs) can determine whether the downstream genes are transcribed. This mechanism depends on mutually exclusive RNA secondary structures and is known as ribosome-dependent 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 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, trpL is efficiently translated (and thus the so called leader peptide is produced) and a small attenuator RNA (which harbors trpL) is generated [1, 2].

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 RNA attenuator (sRNA) rnTrpL, which are generated upon transcription attenuation of the trp operon trpE(G), are functional in trans. We also found that the trp attenuator responds not only to tryptophan availability, but also to translation inhibition. Moreover, we demonstrated the also in E. coli the rnTrpL sRNA is functional in trans [3-7].

 

The conserved attenuator sRNA rnTrpL: a central riboregulator, which responds to nutrient availability.

In contrast to E. coli, S. meliloti harbors three trp operons and only one of them, trpE(G), is regulated by transcription attenuation. Since trpE(G) is not regulated by a transcriptional repressor and is constitutively transcribed during growth. When enough Trp is available, trpE(G) transcription is abolished and the sRNA rnTrpL is generated. This sRNA base-pairs with trpD in the polycistronic trpDC  mRNA and destabilizes the transcript [3]. Additionally, it downregulates the quorum sensing autoinducer synthase gene sinI [8]. Bioinformatic predictions suggested that rnTrpL base-pairs with and determines the level of mRNAs encoding several transcription regulators, suggesting that it is a central riboregulator in S. meliloti [8].

In E. coli at low Trp concentration, transcription of the trpEDCBA operon is derepressed and its expression is regulated by attenuation. Under conditions of Trp sufficiency, the attenuator sRNA rnTrpL is produced [1]. We have shown that rnTrpL directly downregulates expression of dnaA, which encodes the master regulator of initiation of chromosome replication. Thus, the sRNA rnTrpL links the availability of Trp, which is the most costly to synthesize amino acid, to the initiation of chromosome replication. Bioinformatic prediction suggested that also in E. coli, rnTrpL is a part of an extended network [6].

 

 

Trp-related roles of the attenuator sRNA rnTrpL in S. meliloti (A) and E. coli (B). From: Melior H and Evguenieva-Hackenberg E (2021) RNA-Regulation und Antibiotikaresistenz: Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien. Biospektrum, 02.21:127-130, DOI: 10.1007/s12268-021-1545-0


Role of the trp attenuator and its trans-acting products in adaptation to antibiotic exposure.

The attenuator sRNA rnTrpL is produced not only under conditions of tryptophan shortage, but also upon translation inhibition. Upon exposure to subinhibitory tetracycline (Tc) amount, transcription of trpE(G) is attenuated even under conditions of Trp insufficiency [5]. We found that the specificity of the sRNA rnTrpL is reprogrammed in the the presence of Tc. Together with the leader peptide peTrpL (14) and Tc, it forms an antibiotic-dependent ribonucleoprotein complex (ARNP) which destabilizes rplUrpmA mRNA encoding ribosomal proteins L21 and L27. Similar ARNPs are also formed upon exposure to several other translation-inhibiting antibiotics [5].

Furthermore, peTrpL forms a second ARNP type dedicated to the differential posttranscriptional regulation of the multiresistance operon smeABR. The smeR-ARNP is supported by substrates of the major multidrug resistance efflux pump SmeAB and comprises an antisense RNA, which is induced upon exposure to specific, but structurally different antimicrobial compounds [4].

The novel, ARNP-based mechanism for mRNA destabilization is a major research topic in the lab.

 

 

Antibiotic-related roles of peTrpL and rnTrpL in rhizobia. A) posttranscriptional regulation of the smeABR operon by peTrpL and an antisense RNA. B) Two types of peTrpL-containing ARNP complexes, one of them involved in reprograming of the sRNA rnTrpL. From: Melior H and Evguenieva-Hackenberg E (2021) RNA-Regulation und Antibiotikaresistenz: Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien. Biospektrum, 02.21:127-130, DOI: 10.1007/s12268-021-1545-0

 

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

 

SAM homeostasis in S. meliloti

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) [9]. To analyze the mechanisms of SAM homeostasis, we are manipulating the level of the SAM synthetase (encoded by metK) in the cell, and are analyzing the regulation of metZ and metA genes, which are preceded by SAM II riboswitches.

 

The small proteome in rhizobia

Recently, we mapped genome-wide transcription start sites and predicted promoters in the reannotated genome of the soybean symbiont Bradyrhizobium japonicum (Bradyrhizobium diazoefficiens). This analysis led to the discovery and annotation of many small ORFs [10-12]. Currently, in the frame of SPP 2002, we are analyzing the small proteome (proteins encoded by own ORFs that are < 70 codons) in the alfalfa symbiont S. meliloti. For this, our cooperation partners identified small proteins by mass spectrometry (AG Becher, Greifswald) and translated small ORFs by Ribo-Seq analysis (AG Sharma, Würzburg, and AG Backofen, Freiburg). Currently we are validating translation of tagged small proteins using western blot analysis. One of the small proteins, found by mass spectrometry to accumulate strongly upon exposure to tetracycline, is the leader peptide peTrpL, which plays a role in multidrug resistance and regulation of ribosomal genes [4, 5].

 

References:

  1. Yanofsky C (1981) Attenuation in the control of expression of bacterial operons. Nature, 289:751-758.
  2. Bae YM and Crawford IP (1990) The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition. J Bacteriol, 172, 3318-3327.
  3. Melior H, Li S, Madhugiri R, Stötzel M, Azarderakhsh S, Barth-Weber S, Baumgardt K, Ziebuhr J and Evguenieva-Hackenberg E (2019) Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans. Nucleic Acids Res., 47, 6396–6410.
  4. Melior H, Maaß S, Li S, Förstner KU, Azarderakhsh S, Varadarajan AR, Stötzel M, Elhossary M, Barth-Weber S, Ahrens CH, Becher D and Evguenieva-Hackenberg E (2020) The leader peptide petrpl forms antibiotic-containing ribonucleoprotein complexes for posttranscriptional regulation of multiresistance genes. mBio, 11, 1–22.
  5. Melior H, Li S, Stötzel M, Maaß S, Schütz R, Azarderakhsh S, Shevkoplias A, Barth-Weber S, Baumgardt K, Ziebuhr J, Förstner KU, Chervontseva Z, Becher D and Evguenieva-Hackenberg E (2021) Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure. Nucleic Acids Res., 49, 2894–2915.
  6. Li S, Edelmann D, Berghoff BA, Georg J and Evguenieva-Hackenberg E (2021) Bioinformatic prediction reveals posttranscriptional regulation of the chromosomal replication initiator gene dnaA by the attenuator sRNA rnTrpL in Escherichia coli. RNA Biol., 18, 1324–1338.
  7. Melior H and Evguenieva-Hackenberg E (2021) RNA-Regulation und Antibiotikaresistenz: Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien. Biospektrum, 02.21:127-130.
  8. Baumgardt K, Šmídová K, Rahn H, Lochnit G, Robledo M and 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.
  9. Baumgardt K, Melior H, Madhugiri R, Thalmann S, Schikora A, McIntosh M, Becker A and Evguenieva-Hackenberg E (2017) RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti. Microbiology. 163(4):570-583.
  10. Čuklina J, Hahn J, Imakaev M, Omasits U, Förstner KU, Ljubimov N, Goebel M, Pessi G, Fischer HM, Ahrens CH, Gelfand MS and 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.
  11. Hahn J, Thalmann S, Migur A, von Boeselager RF, Kubatova N, Kubareva E, Schwalbe H and Evguenieva-Hackenberg E (2017) Conserved small mRNA with an unique, extended Shine-Dalgarno sequence. RNA Biol. 14(10):1353-1363.
  12. Hahn J, Tsoy OV, Thalmann S, Čuklina J, Gelfand MS and Evguenieva-Hackenberg E (2016) Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110. PLoS One 11:e0165429.