Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.
Nucleic Acids Res. 2011 May;39(10):4235-48. doi: 10.1093/nar/gkr033. Epub 2011 Jan 29.
Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.
小型非编码 RNA（sRNAs）是细菌中转录后基因调控的广泛效应因子。目前，关于李斯特菌在细胞外环境中生长时表达的 sRNAs 有大量信息。我们使用从细胞外生长的细菌和李斯特菌感染的巨噬细胞中分离的小于 500nt 的分馏 RNA 获得的 cDNA 进行深度测序，以编目中继细菌生长过程中的 sRNA 谱。在这里，我们报告了发现的 150 个假定调控 RNA，其中 71 个以前没有描述过。共有 29 个调控 RNA，包括小非编码反义 RNA，专门在细胞内表达。我们通过 northern blot 验证了高表达的 sRNA，并通过构建和表征 rli31、rli33-1 和 rli50* 的同基因突变体，证明了候选细胞内表达 sRNA 的表达对于细菌在巨噬细胞中的有效生长是必需的。在评估感染的小鼠和昆虫模型中的生长时，所有三个突变体均表现出衰减。比较基因组分析显示，谱系特异性 sRNA 候选物的存在以及天然感染减毒细菌基因组中 sRNA 基因座的缺失，以及非致病性李斯特菌基因组中额外的缺失。我们的分析揭示了广泛的 sRNA 表达是细菌在细胞内生长过程中调节的一个重要特征。
