Transcriptome landscape of <i>Lactococcus lactis</i> reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species.RNA测序（RNA-seq）已彻底革新全基因组转录组分析技术，细菌中非编码调控RNA的鉴定研究亦同步蓬勃发展。本研究依托差分RNA测序（dRNA-seq）结合人工与自动化转录组挖掘手段，解析了乳酸菌模式菌株乳酸乳球菌MG1363（Lactococcus lactis MG1363）的转录组图谱。该策略实现了乳酸乳球菌的高分辨率基因组注释，并鉴定出60个顺式编码反义RNA（asRNAs）、186个反式编码推定调控RNA（sRNAs）以及134个新型小开放阅读框（small ORFs）。基于asRNAs的推定靶标，本研究提出了一种全新的分类方案。研究人员在（反义）RNA的启动子序列中鉴定出多个转录因子DNA结合基序，为解析乳球菌调控RNA与转录因子之间的相互作用提供了新的研究视角。通过差分Northern印迹杂交，研究人员实验验证了14个推定sRNAs的存在及其长度，其中包括丰度较高的6S RNA——该RNA的表达水平会随可利用碳源的变化发生差异调控。针对另一sRNA LLMGnc_147的功能分析表明，其参与碳摄取与代谢过程。乳酸乳球菌中存在13%的无先导mRNA（lmRNAs），通过基因本体（GO）类别富集分析发现，这类mRNA主要参与核苷酸代谢及DNA/RNA结合过程。此外，研究人员还发现起始密码子下游紧邻一段富含A的序列基序，这为解析lmRNAs的翻译机制提供了全新思路。综上，本研究首次通过实验手段完成了乳酸乳球菌全基因组转录组景观分析，后续针对sRNA的相关研究亦为乳酸乳球菌及其相关乳球菌物种的调控RNA研究提供了坚实的研究基础。