一株枯草芽孢杆菌原噬菌体Bsu-yong1的基因组分析

Genomic analysis of a Bacillus subtilis prophage vB_Bsu-yong1

摘要:

【目的】枯草芽孢杆菌（Bacillus subtilis）是在自然界中广泛存在的革兰氏阳性菌，其抗逆性极强，能抑制大多数有害菌的繁殖，是常用的产酶菌，用其生产的蛋白酶、淀粉酶占全球工业酶产量的50%。原噬菌体（prophage）整合在宿主基因组中，可为宿主提供基因和生物学功能，非常具有研究价值。以往，有关B. subtilis原噬菌体的报道主要集中于缺陷型原噬菌体（defective prophage），本研究对一株非缺陷型活性原噬菌体（active prophage）的基因组进行解析，以扩充对非缺陷型原噬菌体的认知。【方法】使用丝裂霉素C从枯草芽孢杆菌中诱导一株噬菌体，命名为Bacillus phage Bsu-yong1（简称Bsu-yong1）。对Bsu-yong1进行负染、透射电镜（transmission electron microscopy，TEM）观察，用Illumina MiSeq测定其基因组序列、综合运用生物信息学工具对其进行基因功能注释和系统进化分析。【结果】Bsu-yong1与PBSX类缺陷型原噬菌体在形态上相似，但Bsu-yong1具有完整的噬菌体基因组，这与缺陷型原噬菌体不同，后者在包装过程中不能正确包裹自身的基因组，而是随机包裹一段宿主染色体。Bsu-yong1基因组全长为43 590 bp，G+C含量为41%，含有62个开放阅读框（open reading frame，ORF），呈模块化分布。Bsu-yong1拥有基因编码T7SS效应器LXG多态性毒素（T7SS effector LXG polymorphic toxin）、ImmA/IrrE蛋白和SMI1/KNR4蛋白。前二者为细菌毒素（toxin），后者为抗毒素（antitoxin），toxin-antitoxin是细菌免疫系统重要成员，参与菌间竞争与环境适应。此前，尚未有编码LXG polymorphic toxin的基因在噬菌体中被发现和报道。在基于全基因组比对构建的蛋白谱进化树（proteomic tree）中，Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y聚集形成一个独立的进化支（clade），基因组比对显示它们基因组的复制与调控模块具有高度保守性，它们共享29个核心基因（core gene），均具有PBSX样形态特征。Bsu-yong1与其他噬菌体的进化距离较远。将Bsu-yong1与所有噬菌体进行比对，得到的成对序列比较（pairwise sequence comparison，PASC）最大值为46.72%，小于属边界值（70%）。【结论】vB_Bsu-yong1在有尾纲中代表一个新的未知的属；建议构建一个新的科（family），该科由Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y组成。vB_Bsu-yong携带免疫相关基因，它可能有利于宿主在菌间竞争中获胜和适应环境。本研究丰富了噬菌体基因数据库，拓展了对芽孢杆菌活性原噬菌体的认知。

Abstract:

[Objective] Bacillus subtilis, a Gram-positive bacterium ubiquitous in the nature, has strong stress tolerance and environmental adaptability and can inhibit the propagation of a variety of harmful bacteria. It is a common and important enzyme-producing bacterium, with the yield of proteases and amylases accounting for 50% of the industrial enzyme yield. Prophages integrated in host genomes are capable of providing additional genes and endow hosts with biological properties, demonstrating great research significance. The available studies about B. subtilis prophages focus on the defective ones. In this study, we analyzed the genome of an active prophage to expand the knowledge about non-defective prophages. [Methods] We induced a phage fromB. subtilis by using mitomycin C and named the phage as Bacillus phage Bsu-yong1. We employed transmission electron microscopy (TEM) to observe the negatively stained Bsu-yong1, Illumina MiSeq to sequence the genome of Bsu-yong1, and bioinformatics tools to annotate the genome. Furthermore, a proteomic tree of Bsu-yong1 was built based on genome-wide sequence alignment. [Results] Bsu-yong1 showed the morphology similar to that of PBSX-like defective prophages of B. subtilis. Unlike defective prophages which package DNA segments from random portions of the host genome, Bsu-yong1 was capable of packaging its own genome. The full-length genome of Bsu-yong1 was 43 590 bp with the G+C content of 41%. A total of 62 open reading frames (ORFs) were predicted with modular arrangement in the Bsu-yong1 genome. Bsu-yong1 harbored the ORFs encoding T7SS effector LXG polymorphic toxin, IMMA/IrrE and SMI1/KNR4. The former two were bacterial toxins and the later was an antitoxin. Toxins and antitoxins are members of the bacterial immune system, participating in bacterial competition and environmental adaptation. The gene encoding LXG polymorphic toxin in bacteriophages had never been reported before. A proteomic tree was built based on the whole genome alignment, revealing a long evolution distance between Bsu-yong1 and other phages. In the proteomic tree, Bsu-yong1 clustered with phages sv105, rho14, and vB_BteM-A9Y to form a monophyletic clade. These four phages all had PBSX-like morphology and shared 29 core genes. Pairwise sequence comparison (PASC) illustrated that the highest nucleotide sequence similarity between Bsu-yong1 and known phages was only 46.72%, which was much lower than the threshold (70%) to define a genus.[Conclusion] Bsu-yong1 represented a new unknown genus. We suggest the establishment of a novel family composed of Bsu-yong1, sv105, rho14, and vB_BteM-A9Y, which share 29 core genes. Bsu-yong1 carries immunity-related genes, which may benefit the host in bacterial competition and environmental adaptation. The findings enrich the bacteriophage gene database and enrich the knowledge of active Bacillus prophages.