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.

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.

The essential nature of YqfG, a YbeY homologue required for 3' maturation of Bacillus subtilis 16S ribosomal RNA is suppressed by deletion of RNase R.

Ribosomal RNAs are processed from primary transcripts containing 16S, 23S and 5S rRNAs in most bacteria. Maturation generally occurs in a two-step process, consisting of a first crude separation of the major species by RNase III during transcription, followed by precise trimming of 5' and 3' extensions on each species upon accurate completion of subunit assembly. The various endo- and exoribonucleases involved in the final processing reactions are strikingly different in Escherichia coli and Bacillus subtilis, the two best studied representatives of Gram-negative and Gram-positive bacteria, respectively. Here, we show that the one exception to this rule is the protein involved in the maturation of the 3' end of 16S rRNA. Cells depleted for the essential B. subtilis YqfG protein, a homologue of E. coli YbeY, specifically accumulate 16S rRNA precursors bearing 3' extensions. Remarkably, the essential nature of YqfG can be suppressed by deleting the ribosomal RNA degrading enzyme RNase R, i.e. a ΔyqfG Δrnr mutant is viable. Our data suggest that 70S ribosomes containing 30S subunits with 3' extensions of 16S rRNA are functional to a degree, but become substrates for degradation by RNase R and are eliminated.

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.

The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti.

Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti.

RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-ß-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.