Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. Clostridium beijerinckii NCIMB 8052

Background: Clostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood. Results: In this study, we conducted a single-nucleotide resolution analysis of the C. beijerinckii NCIMB 8052 transcriptome using high-throughput RNA-Seq technology. We identified the transcription start sites and operon structure throughout the genome. We confirmed the structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; we also defined important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others. We discovered 20 previously non-annotated regions with significant transcriptional activities and 15 genes whose translation start codons were likely mis-annotated. As a consequence, the accuracy of existing genome annotation was significantly enhanced. Furthermore, we identified 78 putative silent genes and 177 putative housekeeping genes based on normalized transcription measurement with the sequence data. We also observed that more than 30% of pseudogenes had significant transcriptional activities during the fermentation process. Strong correlations exist between the expression values derived from RNA-Seq analysis and microarray data or qRT-PCR results. Conclusions: Transcriptome structural profiling in this research provided important supplemental information on the accuracy of genome annotation, and revealed additional gene functions and regulation in C. beijerinckii.

研究背景：拜氏梭菌（Clostridium beijerinckii）是一类重要的产溶剂微生物。近期，NCIMB 8052株拜氏梭菌的基因组已完成测序。尽管转录组（transcriptome）结构对于解析基因组的功能与调控架构至关重要，但该菌株转录组的物理结构（如操纵子（operon）连接关系与转录边界）仍未得到充分阐明。
研究结果：本研究利用高通量RNA测序（RNA-Seq）技术，对NCIMB 8052株拜氏梭菌的转录组开展了单核苷酸分辨率水平的分析。我们鉴定出了全基因组范围内的转录起始位点（transcription start site）与操纵子结构，验证了拜氏梭菌8052中参与酸与溶剂合成代谢通路的重要基因操纵子结构，包括pta-ack、ptb-buk、hbd-etfA-etfB-crt（bcs）以及ald-ctfA-ctfB-adc（sol）操纵子；同时明确了与趋化/运动、转录调控、应激响应及脂肪酸生物合成等相关的重要操纵子。本研究共发现20个此前未被注释的具有显著转录活性的区域，以及15个翻译起始密码子可能存在注释错误的基因，由此显著提升了现有基因组注释的准确性。此外，基于测序数据的标准化转录水平检测，我们鉴定出78个潜在沉默基因与177个潜在持家基因（housekeeping gene）；同时观察到超过30%的假基因（pseudogene）在发酵过程中呈现出显著的转录活性。RNA-Seq分析得到的表达量数据与基因芯片（microarray）及实时定量PCR（qRT-PCR）结果之间存在极强的相关性。
研究结论：本研究开展的转录组结构解析工作，为提升基因组注释准确性提供了重要的补充信息，并揭示了拜氏梭菌中更多的基因功能与调控机制。