Irla Marta, Neshat Armin, Brautaset Trygve, Rückert Christian, Kalinowski Jörn, Wendisch Volker F
Genetics of Prokaryotes, Faculty of Biology & Center for Biotechnology, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany.
Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätstr. 27, 33615, Bielefeld, Germany.
BMC Genomics. 2015 Feb 14;16(1):73. doi: 10.1186/s12864-015-1239-4.
Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends.
Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.
The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.
甲醇芽孢杆菌MGA3是一种嗜热兼性核糖单磷酸（RuMP）循环甲基营养菌。鉴于其能够高产氨基酸，该微生物作为生物技术应用的一个有前景的候选者，其相关性是显而易见的。甲醇芽孢杆菌MGA3基因组由一个3337035个核苷酸（nt）的环状染色体、19174 nt的质粒pBM19和68999 nt的质粒pBM69组成。在染色体上注释了3218个蛋白质编码区，在pBM19上注释了22个，在pBM69上注释了82个。在本研究中，采用RNA测序方法全面研究甲醇芽孢杆菌MGA3的转录组，以改进基因组注释、鉴定新转录本、分析参与基因表达的保守序列基序并揭示操纵子结构。为此，应用了两种不同的cDNA文库制备方法：一种用于表征整个转录组，另一种用于富集初级转录本的5'端。
对初级转录组数据的分析能够检测到2167个推定的转录起始位点（TSS），这些位点被分类为位于已知蛋白质编码基因上游区域（5'-UTR）的1642个TSS以及新的反义、基因内或基因间转录本的525个TSS。首先，根据初级转录组数据纠正了14个错误注释的翻译起始位点（TLS）。对已鉴定的5'-UTR的进一步研究导致对其长度分布进行了详细表征，并检测到75个迄今未知的顺式调节RNA元件。此外，利用确切的TSS位置定义了甲醇芽孢杆菌MGA3中翻译起始位点、核糖体结合位点和启动子的保守序列基序。基于整个转录组数据集，确定了新转录本、操纵子结构和mRNA丰度。对操纵子结构的分析表明，几乎一半的基因是单顺反子转录的（940个），而1164个基因被组织成381个操纵子。几个与甲基营养相关的基因有高度丰富的转录本。
本研究中获得的对甲醇芽孢杆菌MGA3转录图谱的广泛见解，为进一步的比较定量转录组分析以及可能也为目前对该生物体非常有限的分子生物学工具开发提供了有价值的基础。
