酱香型白酒发酵中地衣芽孢杆菌前噬菌体的鉴别

Identification of prophages in Bacillus licheniformis in sauce-flavor baijiu fermentation

摘要:

【背景】噬菌体是微生物群落的重要组成部分，但传统白酒发酵中噬菌体的分类和存在尚不清楚。【目的】通过检测公共数据库和酱香型白酒发酵中地衣芽孢杆菌(Bacillus licheniformis)基因组中的前噬菌体整合区域，探究传统酱香型白酒发酵中关键功能菌株的前噬菌体分类和侵染情况。【方法】使用未培养(细菌全基因组分析)和可培养(菌株筛选和特异性PCR反应)技术对不同环境来源和来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体的分类和存在进行解析。【结果】细菌全基因组分析显示，30株来自不同环境的地衣芽孢杆菌基因组中共注释到165个前噬菌体，其中63.6%(105/165)为完整前噬菌体序列。97.1%感染地衣芽孢杆菌的噬菌体属于长尾噬菌体科(Siphoviridae)，2.9%属于肌尾噬菌体科(Myoviridae)，53.0%完整前噬菌体的基因功能未知。在来自酱香型白酒发酵的B. licheniformis MT-B06中检测到7个前噬菌体整合序列，其中57.1%(4/7)为完整前噬菌体序列，来自酱香型白酒发酵的地衣芽孢杆菌存在多种不同前噬菌体的共感染。来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体存在来自细菌基因组上相邻CotD孢子外壳蛋白(CotD family spore coat protein)基因的水平基因转移。在26株来自酱香型白酒发酵的地衣芽孢杆菌中，69.2%(18/26)存在噬菌体编码主要衣壳蛋白的基因，100.0%(26/26)存在噬菌体编码CotD孢子外壳蛋白的基因。【结论】来自不同环境的地衣芽孢杆菌和酱香型白酒发酵的地衣芽孢杆菌中存在高水平的前噬菌体整合，来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体中广泛存在来源于宿主的CotD孢子外壳蛋白基因的水平基因转移。本研究为首次对传统发酵白酒中噬菌体的分类和存在进行探究，有助于对发酵微生物群落中噬菌体-细菌相互作用加深理解。

Abstract:

[Background] Phages are an important part of microbial community, but the classification and presence of them in traditional baijiu fermentation are still unclear. [Objective] By exploring prophage regions in the genomes of Bacillus licheniformis in public databases and in sauce-flavor baijiu fermentation, we intend to clarify the classification and infection of prophages in key functional strains in traditional sauce-flavor baijiu fermentation. [Methods] With culture-independent (bacterial whole genome analysis) and culture-dependent (strain screening and specific PCR) techniques, the classification and presence of prophages in B. licheniformis from different natural habitats and sauce-flavor baijiu fermentation were analyzed. [Results] The whole genome analysis of bacteria detected 165 prophages in genomes of 30 B. licheniformis strains from different habitats, of which 63.6% (105/165) had intact sequences. A total of 97.1% of the prophages in the B. licheniformis belonged to Siphoviridae, and 2.9% Myoviridae. The functions of 53.0% genes of the intact prophages were unknown. Seven integrated prophage regions were detected in sauce-flavor baijiu-derived B. licheniformis MT-B06, of which 57.1% (4/7) were intact prophage regions. Co-infection of multiple prophages was detected in B. licheniformis from sauce-flavor baijiu fermentation. There was horizontal gene transfer of CotD family spore coat protein gene on the bacterial genome to the prophage. In the 26 sauce-flavor baijiu-derived B. licheniformis strains, 69.2% (18/26) were detected positive of prophage-encoded major capsid protein gene, and 100.0% (26/26) positive of prophage-encoded CotD family spore coat protein gene. [Conclusion] High level of prophage integration was found in B. licheniformis from different natural habitats and sauce-flavor baijiu fermentation. Horizontal gene transfer of CotD family spore coat protein gene from host to the prophage of B. licheniformis in sauce-flavor baijiu fermentation was widely observed. This study is the first time to explore the classification and presence of bacteriophages in traditional baijiu fermentation, which helps to deepen the understanding of bacteriophage-host interaction in the fermenting microbial community.