Grünberger Felix, Reichelt Robert, Bunk Boyke, Spröer Cathrin, Overmann Jörg, Rachel Reinhard, Grohmann Dina, Hausner Winfried
Institute of Microbiology and Archaea Center, University of Regensburg, Regensburg, Germany.
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.
Front Microbiol. 2019 Jul 12;10:1603. doi: 10.3389/fmicb.2019.01603. eCollection 2019.
DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other species, the genome of DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.
DSM 3638是嗜热古菌的一种模式生物，其最佳生长温度接近100°C。该基因组大约在18年前进行了测序。然而，一些出版物表明，与其他物种不同，DSM 3638的基因组容易发生基因组重排。因此，我们使用第三代测序技术对该基因组进行了重新测序。新组装的基因组大小为1,889,914 bp，与已发表的序列具有高度的序列同一性。然而，检测到两个主要偏差：（1）由于最近描述的一次缺失，该基因组比NCBI参考基因组小18,342 bp。（2）PF0349和PF0388之间的区域发生了倒位，最有可能是由于原始序列的组装问题。此外，还鉴定出了许多微小变异，范围从单核苷酸交换、缺失或插入。新序列中插入序列（IS）元件的总数也从30个减少到了24个。使用纳米孔测序对一份保存了两年的“实验室培养物”进行重新测序，证实了即使在正常实验室条件下且未采取任何特殊处理措施的情况下，DSM 3638基因组仍具有总体稳定性。为了改进基因组注释，将更新后的DNA序列与RNA测序方法相结合。在这里，汇集了来自八种不同生长条件的RNA，以增加检测到的转录本数量。此外，采用了差异RNA-Seq方法来鉴定转录起始位点（TSS）。总共检测到2515个TSS，并将其分为834个初级（pTSS）、797个反义（aTSS）、739个内部和145个二级TSS。我们对上游区域的分析揭示了一种保守的古菌启动子结构。对pTSS和aTSS之间距离的研究揭示了大量的反义转录本，这是由同一个TATA框双向转录产生的结果。这种反义转录本产生机制可以通过转录实验进一步证实。我们假设双向转录会产生无功能的反义RNA，并且由于TATA元件的结构和TATA结合蛋白的对称结构，这种现象在古菌中普遍存在。
