An in vivo reporter assay for sRNA-directed gene control in Gram-positive bacteria: identifying a novel sRNA target in Staphylococcus aureus

Bacterial small regulatory RNAs (sRNAs) play a major role in the regulation of various cellular functions. Most sRNAs interact with mRNA targets via an antisense mechanism, modifying their translation and/or degradation. Despite considerable progresses in discovering sRNAs in Gram-positive bacteria, their functions, for the most part, are unknown. This is mainly due to difficulties in identifying their targets. To aid in the identification of sRNA targets in Gram-positive bacteria, we set up an in vivo method for fast analysis of sRNA-mediated post-transcriptional control at the 5' regions of target mRNAs. The technology is based on the co-expression of an sRNA and a 5' sequence of an mRNA target fused to a green fluorescent protein (GFP) reporter. The system was challenged on Staphylococcus aureus, an opportunistic Gram-positive pathogen. We analyzed several established sRNA-mRNA interactions, and in addition, we identified the ecb mRNA as a novel target for SprX2 sRNA. Using our in vivo system in combination with in vitro experiments, we demonstrated that SprX2 uses an antisense mechanism to prevent ecb mRNA translation initiation. Furthermore, we used our reporter assay to validate sRNA regulations in other Gram-positive organisms, Bacillus subtilis and Listeria monocyto-genes. Overall, our method is broadly applicable to challenge the predicted sRNA-mRNA interactions in Gram-positive bacteria.