Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure.

Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator located upstream of the Trp biosynthesis gene trpE(G). The sRNA rnTrpL contains a small ORF, trpL, encoding the 14-aa leader peptide peTrpL. If Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which subsequently acts to downregulate the trpDC operon, while peTrpL is known to have a Trp-independent role in posttranscriptional regulation of antibiotic resistance mechanisms. Here, we show that tetracycline (Tc) causes rnTrpL accumulation independently of Trp availability. In the presence of Tc, rnTrpL and peTrpL act collectively to destabilize rplUrpmA mRNA encoding ribosomal proteins L21 and L27. The three molecules, rnTrpL, peTrpL, and rplUrpmA mRNA, form an antibiotic-dependent ribonucleoprotein complex (ARNP). In vitro reconstitution of this ARNP in the presence of competing trpD and rplU transcripts revealed that peTrpL and Tc cause a shift of rnTrpL specificity towards rplU, suggesting that sRNA target prioritization may be readjusted in response to changing environmental conditions.

The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes.

Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbor small upstream open reading frames (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in S. meliloti, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. Here, we provide evidence for a tryptophan-independent role of peTrpL in trans We found that peTrpL increases the resistance toward tetracycline, erythromycin, chloramphenicol, and the flavonoid genistein, which are substrates of the major multidrug efflux pump SmeAB. Coimmunoprecipitation with a FLAG-peTrpL suggested smeR mRNA, which encodes the transcription repressor of smeABR, as a peptide target. Indeed, upon antibiotic exposure, smeR mRNA was destabilized and smeA stabilized in a peTrpL-dependent manner, showing that peTrpL acts in the differential regulation of smeABR Furthermore, smeR mRNA was coimmunoprecipitated with peTrpL in antibiotic-dependent ribonucleoprotein (ARNP) complexes, which, in addition, contained an antibiotic-induced antisense RNA complementary to smeRIn vitro ARNP reconstitution revealed that the above-mentioned antibiotics and genistein directly support complex formation. A specific region of the antisense RNA was identified as a seed region for ARNP assembly in vitro Altogether, our data show that peTrpL is involved in a mechanism for direct utilization of antimicrobial compounds in posttranscriptional regulation of multiresistance genes. Importantly, this role of peTrpL in resistance is conserved in other AlphaproteobacteriaIMPORTANCE Leader peptides encoded by transcription attenuators are widespread small proteins that are considered nonfunctional in trans We found that the leader peptide peTrpL of the soil-dwelling plant symbiont Sinorhizobium meliloti is required for differential, posttranscriptional regulation of a multidrug resistance operon upon antibiotic exposure. Multiresistance achieved by efflux of different antimicrobial compounds ensures survival and competitiveness in nature and is important from both evolutionary and medical points of view. We show that the leader peptide forms antibiotic- and flavonoid-dependent ribonucleoprotein complexes (ARNPs) for destabilization of smeR mRNA encoding the transcription repressor of the major multidrug resistance operon. The seed region for ARNP assembly was localized in an antisense RNA, whose transcription is induced by antimicrobial compounds. The discovery of ARNP complexes as new players in multiresistance regulation opens new perspectives in understanding bacterial physiology and evolution and potentially provides new targets for antibacterial control.

Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans.

Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5'-UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti.

The ribonucleases (RNases) E and J play major roles in E. coli and Bacillus subtilis, respectively, and co-exist in Sinorhizobium meliloti. We analysed S. meliloti 2011 mutants with mini-Tn5 insertions in the corresponding genes rne and rnj and found many overlapping effects. We observed similar changes in mRNA levels, including lower mRNA levels of the motility and chemotaxis related genes flaA, flgB and cheR and higher levels of ndvA (important for glucan export). The acyl-homoserine lactone (AHL) levels were also higher during exponential growth in both RNase mutants, despite no increase in the expression of the sinI AHL synthase gene. Furthermore, several RNAs from both mutants migrated aberrantly in denaturing gels at 300 V but not under stronger denaturing conditions at 1300 V. The similarities between the two mutants could be explained by increased levels of the key methyl donor S-adenosylmethionine (SAM), since this may result in faster AHL synthesis leading to higher AHL accumulation as well as in uncontrolled methylation of macromolecules including RNA, which may strengthen RNA secondary structures. Indeed, we found that in both mutants the N6-methyladenosine content was increased almost threefold and the SAM level was increased at least sevenfold. Complementation by induced ectopic expression of the respective RNase restored the AHL and SAM levels in each of the mutants. In summary, our data show that both RNase E and RNase J are needed for SAM homeostasis in S. meliloti.