Small RNAs in the genus Clostridium.
Small RNAs in the genus Clostridium.
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DOI：
10.1128/mbio.00340-10
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发表时间：
2011-01-25
期刊：
影响因子：
6.4
通讯作者：
Papoutsakis ET
中科院分区：
文献类型：
--
作者：
Chen Y;Indurthi DC;Jones SW;Papoutsakis ET
梭菌属包括主要的人类病原体和对纤维素降解、碳循环和生物技术重要的物种。小RNA（sRNA）是所有生物体中重要的调控分子，但在梭菌中尚未研究。梭菌sRNA的研究由于缺乏系统的方法来鉴定候选sRNA而受阻，因此将梭菌sRNA的研究委托给了一个偶然的过程。因此，我们希望开发一种方法来鉴定梭菌属中潜在的sRNA，以开拓梭菌sRNA研究的领域。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，我们预测了21种梭菌基因组中的sRNA：丙酮丁醇梭菌、拜氏梭菌、肉毒梭菌（8种菌株）、解纤维梭菌、艰难梭菌、克氏梭菌（2种菌株）、诺氏梭菌、产气荚膜梭菌（3种菌株）、植物发酵梭菌、破伤风梭菌和热纤梭菌。虽然超过三分之一的预测sRNA具有Shine-Dalgarno（SD）序列，但只有六分之一的sRNA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。采用定量逆转录-PCR（Q-RT-PCR）和北方分析来测试丙酮丁醇梭菌和几种肉毒梭菌菌株中随机选择的一组sRNA的存在，从而确认大部分测试的sRNA。我们发现了一个保守的，新的sRNA，与下游基因编码的ATP结合盒（ABC）转运蛋白基因，响应抗生素克林霉素。预测的sRNA的数量与物种的生理功能相关（病原体高，纤维素分解低，溶剂生成中间），但不与16 S rRNA为基础的基因。梭菌包括主要的人类病原体和对人类生理学、纤维素降解、碳循环和生物技术重要的物种。小RNA（sRNA）越来越被认为是所有生物体中的重要调控分子，但它们在梭菌中几乎未被探索。我们提供了第一个全面的列表，计算确定和实验验证的小RNA在梭菌属，旨在加快兴趣和研究的小RNA分子在一个非常重要的属。在梭菌病原体中发现的较高数量的sRNA表明该物种的生理功能或生态位与预测和保守的sRNA数量之间存在良好的相关性。我们的预测sRNA列表显示了ATP结合盒（ABC）转运蛋白基因上游或下游sRNA的强烈富集。这一点，结合一个保守的sRNA显然参与克林霉素耐药性的鉴定，提供了一个新的视角，为未来的研究可能调控抗生素耐药基因的sRNA在细菌中。
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. Clostridia include major human pathogens and species important to human physiology, cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are increasingly recognized as crucial regulatory molecules in all organisms, but they remain virtually unexplored in clostridia. We provide the first comprehensive list of computationally identified and experimentally verified small RNAs in the genus Clostridium aiming to accelerate interest in and studies of small RNA molecules in a very important genus. The higher number of sRNAs found in clostridial pathogens suggests a good correlation between the physiological function or niche of the species and the number of predicted and conserved sRNAs. Our list of predicted sRNAs displays a strong enrichment of sRNAs upstream or downstream of ATP-binding cassette (ABC) transporter genes. This, combined with the identification of a conserved sRNA apparently involved in clindamycin resistance, provides a new perspective for future studies of possible regulation of antibiotic resistance genes by sRNAs in bacteria.