The phzA2-G2 Transcript Exhibits Direct RsmA-Mediated Activation in Pseudomonas aeruginosa M18

In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz) gene clusters, phzA1-G1 (phz1) and phzA2-G2 (phz2), each of which is responsible for phenazine-1-carboxylic acid (PCA) biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5′-untranslated region (UTR) of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3), located 21 nucleotides upstream of the Shine-Dalgarno (SD) sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes.在细菌中，RsmA/CsrA家族的RNA结合蛋白作为转录后调控因子，可调控靶标转录本的翻译起始过程。铜绿假单胞菌（Pseudomonas aeruginosa）的基因组包含两个吩嗪生物合成（phenazine biosynthetic, phz）基因簇：phzA1-G1（phz1）与phzA2-G2（phz2），二者分别负责吩嗪-1-羧酸（phenazine-1-carboxylic acid, PCA）的生物合成。本研究证实，RsmA可在转录后水平对铜绿假单胞菌M18的两个phz基因簇发挥差异化基因调控作用。经序列分析，我们在phz2转录本的5'非翻译区（5′-untranslated region, UTR）中发现了4个GGA基序，它们是潜在的RsmA结合位点。通过一系列lacZ报告基因融合（lacZ reporter fusions）实验与凝胶迁移率变动分析（gel mobility shift assays），研究结果显示：位于SD序列（Shine-Dalgarno sequence）上游21个核苷酸处的第3个GGA基序（S3），参与了RsmA直接介导的phz2表达激活过程。据此，我们提出了一种全新的调控模型：RsmA与靶标S3基序结合后，可使茎环结构不稳定，从而增强核糖体的结合能力。该模型得到了RNA结构预测、自由能计算以及核苷酸替换实验的全面验证。与之相反，此前已报道多种RsmA介导的翻译抑制机制：RsmA结合至靶标转录本的SD序列附近，从而阻断核糖体的结合。同理，RsmA可负调控phz1的表达。本研究的新发现表明，RsmA对两个phz基因簇的差异化调控，可能使铜绿假单胞菌相较于其他基因组中仅含单个phz基因簇的假单胞菌具备生存优势。