Delaware Biotechnology Institute, Molecular Biotechnology Laboratory, University of Delaware, Newark, Delaware, USA.
mBio. 2011 Jan 25;2(1):e00340-10. doi: 10.1128/mBio.00340-10.
The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny.
梭菌属包括主要的人类病原体和对纤维素降解、碳循环和生物技术很重要的物种。小 RNA（sRNA）作为所有生物中关键的调节分子正在出现，但在梭菌中尚未进行研究。由于缺乏一种系统的方法来鉴定 sRNA 候选物，因此对梭菌 sRNA 的研究受到阻碍，这使得梭菌 sRNA 研究成为一种碰运气的过程。因此，我们希望开发一种鉴定梭菌属中潜在 sRNA 的方法，以开拓梭菌 sRNA 研究领域。我们使用比较基因组学分析结合 rho 非依赖性终止子和启动子的预测，预测了 21 株梭菌基因组中的 sRNA：丙酮丁醇梭菌、拜氏梭菌、肉毒梭菌（8 株）、纤维梭菌、艰难梭菌、克氏梭菌（2 株）、诺维梭菌、产气荚膜梭菌（3 株）、植物发酵梭菌、破伤风梭菌和产热梭菌。尽管预测的 sRNA 中有三分之一以上具有 Shine-Dalgarno（SD）序列，但只有六分之一在 SD 序列下游具有起始密码子；因此，大多数预测的 sRNA 是非编码 RNA。我们采用定量反转录 PCR（Q-RT-PCR）和 Northern 分析来检测随机选择的一组 sRNA 在丙酮丁醇梭菌和几种肉毒梭菌菌株中的存在情况，从而证实了所测试的大量 sRNA。我们鉴定了一个保守的新型 sRNA，该 sRNA与编码 ABC 转运体基因的下游基因一起对抗生素克林霉素产生响应。预测的 sRNA 数量与物种的生理功能相关（病原体的数量高，纤维素降解的数量低，溶剂形成的数量中等），但与基于 16S rRNA 的系统发育无关。
