细菌核糖体依赖性衰减子是广泛的转录后调节因子。它们带有编码前导肽的小上游开放阅读框(uORF),还没有已知的反式功能。在植物共生生物中,色氨酸生物合成基因trpE(G)在uORFtrpL之前,并根据色氨酸的可用性通过转录衰减进行调节。然而,trpLE(G)转录独立于S.meliloti中的色氨酸水平而启动,从而确保主要不依赖色氨酸的前导肽peTrpL的产生。这里,我们提供了petrpL在反式中不依赖色氨酸的作用的证据。我们发现petrpL增加了对四环素的抗性,红霉素,氯霉素,和类黄酮染料木黄酮,它们是主要的多药外排泵SmeAB的底物。与FLAG-petrpL的免疫共沉淀提示涂片mRNA,它编码meABR的转录阻遏物,作为肽靶标。的确,接触抗生素后,smeRmRNA不稳定,smeA以petrpL依赖性方式稳定,表明petrpL在smeABR的差异调节中起作用。在抗生素依赖性核糖核蛋白(ARNP)复合物中,将smeRmRNA与petrpL共免疫沉淀,which,此外,含有抗生素诱导的反义RNA,该反义RNA与smeR互补。体外ARNP重建表明,上述抗生素和金雀异黄素直接支持复合物的形成。反义RNA的特定区域被鉴定为体外ARNP组装的种子区域。我们的数据表明,peTrpL参与了抗微生物化合物在多重耐药基因的转录后调控中的直接利用机制。重要的是,peTrpL在抗性中的这种作用在其他AlphaproteobacteriaIMPORTANCE由转录衰减子编码的前导肽是广泛的小蛋白,被认为是反式无功能的我们发现,居住在土壤中的植物共生体的前导肽peTrpL是差异所必需的,抗生素暴露后多药耐药操纵子的转录后调控。通过不同抗微生物化合物的外排实现的多重抗性确保了自然界中的存活和竞争力,并且从进化和医学角度来看都是重要的。我们表明,前导肽形成抗生素和类黄酮依赖性核糖核蛋白复合物(ARNP),用于使编码主要多药抗性操纵子转录阻遏物的smeRmRNA不稳定。ARNP组装的种子区位于反义RNA中,其转录由抗微生物化合物诱导。ARNP复合物作为多抗调节新参与者的发现为理解细菌生理学和进化开辟了新的视角,并可能为抗菌控制提供新的靶标。
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.