Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (LB medium vs. iron-limited condition)

Bacterial small RNAs (sRNAs) are trans-encoded regulatory RNAs that typically bind mRNAs by short sequence complementarities and change the expression of the corresponding proteins. Some of the well characterized sRNAs serve critical steps in the regulation of important cellular processes, such as quorum sensing (Qrr), iron homeostasis (RyhB), oxidative stress (OxyS) and carbon metabolism (Spot42). However, many sRNAs remains to be identified, and the functional roles of sRNAs are known for only a small fraction. For example, of the hundreds of candidate sRNAs from members of the bacterial family Vibrionaceae the function is known for only nine. We have in this study significantly contributed to the discovery and verification of new sRNAs in a representative of Vibrioneceae, i.e., the A. salmonicida which causes severe disease in farmed Atlantic salmon and other fishes. A computational search for intergenic non-coding (nc) RNAs in the 4.6 Mb genome identified a total of 233 potential sRNAs and 19 other types of ncRNAs with homologs in Rfam. Depending on a set threshold value our microarray approach identified 50-80 sRNAs that are expressed under different growth conditions, twelve of which were verified by Northern blot analysis. In total our data identify nine previously unrecognized sRNAs. Two-condition experiment, cells grown in LB medium (control samples) vs. cells grown under iron-limited conditions (2,2`-dipyridyl) (stimulated samples). Samples collected from three different timepoints (15, 30 and 60 min). Technical replicates for each timepoint: 3 control, 3 stimulated, independently grown and harvested. One replicate per array.

细菌小RNA（small RNAs, sRNAs）是一类反式编码的调控RNA，通常通过短序列互补结合信使RNA（mRNA），进而调控对应蛋白质的表达水平。部分已得到充分表征的sRNAs在重要细胞过程的调控中发挥关键作用，例如群体感应（quorum sensing, Qrr）、铁稳态（iron homeostasis, RyhB）、氧化应激（oxidative stress, OxyS）以及碳代谢（carbon metabolism, Spot42）。然而，仍有大量sRNAs有待发现，且仅极少数sRNAs的功能角色已被阐明。例如，在弧菌科（Vibrionaceae）细菌的数百个候选sRNAs中，仅9个的功能已被探明。本研究针对弧菌科的代表物种——杀鲑气单胞菌（A. salmonicida，该菌可引发养殖大西洋鲑及其他鱼类的严重病害），在新sRNAs的发现与验证方面取得了重要进展。研究人员对该4.6 Mb的基因组开展基因间非编码RNA（intergenic non-coding RNA, ncRNA）的计算筛选，共鉴定出233个潜在sRNAs，以及19个在Rfam数据库中存在同源序列的其他类型ncRNAs。通过设定阈值，本研究的微阵列（microarray）分析方法在不同生长条件下共鉴定出50至80个表达的sRNAs，其中12个经Northern印迹（Northern blot）验证属实。本研究数据共鉴定出9个此前未被报道的sRNAs。本实验为双条件实验：设置两组对照，分别为在LB培养基中培养的细胞（对照样本）与在铁限制条件下（添加2,2'-联吡啶）培养的细胞（刺激样本）。实验分别在15、30及60分钟三个时间点收集样本。每个时间点设置3份对照样本与3份刺激样本作为技术重复，所有样本均独立培养并收获；每个微阵列对应一个技术重复样本。