1株利用乳酸的猪源丁酸梭菌的分离鉴定及基因组序列分析

【目的】 研究1株利用乳酸的猪源丁酸梭菌的基因组信息。【方法】 采用厌氧培养法, 通过乳酸分解培养基(LADM)初筛、梭菌增殖培养基(RCM)复筛, 从饲喂乳酸菌发酵饲料的健康仔猪肠道内容物中分离利用乳酸快、丁酸转化率高的菌株, 对所筛选的菌株进行形态鉴定、16S rDNA序列分析、乳酸转化特性试验、基因组重测序和框架图测序, 并对其编码蛋白进行功能注释。【结果】 分离得到1株分离菌LY33, 基于形态和16S rDNA序列分析鉴定为丁酸梭菌。菌株LY33对乳酸具有较好的利用能力, 在生长第6天可将66 mmol的乳酸基本转化完全, 生成17 mmol左右的丁酸。LY33菌株的重测序表明, 其编码基因整体变异较小, 与参考基因组相比, LY33菌株全基因组杂合比率为0.022‰, 单核苷酸多态性(SNP)位点变异总数21 590个(占整个基因组0.4666%), 插入缺失(InDel)突变总和594个(占0.0128%), 不存在拷贝数变异(CNV), 结构变异(SV)注释的变异总数103个(占0.0003%)。突变基因主要为与菌株生长、能量代谢调节、维生素合成(特别是生物素)有关的基因, 分布于2条染色体的多条序列上。LY33菌株框架图测序及其编码基因蛋白的功能注释表明, 其基因组序列长度4 627 127 bp, GC含量28.60%, 含有4 171个基因, 编码区总长度占比84.12%, 参与了糖代谢(carbohydrate metabolism)、膜转运(membrane transport)和氨基酸代谢(amino acid metabolism)等多种代谢途径转化过程, 基因组中抗大环内酯类药物、抗杆菌肽、VanI糖肽抗性基因个数较多。【结论】 本研究从饲喂4%乳酸菌发酵饲料的健康仔猪肠道内容物中分离到1株利用乳酸快、丁酸转化率高的LY33菌株, 经鉴定为丁酸梭菌, 其具有良好的应用前景, 可作为一种新的微生物资源用于微生态制剂产品。

Isolation,Identification and Genomic Sequence Analysis of a Strain of Clostridium butyricum from Pigs Utilizing Lactic Acid

【Objective】 The study was aimed to research the genomic information of a pig-isolate Clostridium butyricum utilizing lactic acid.【Method】 Using anaerobic culture method, strains with fast lactic acid utilization and high butyric acid conversion rate were isolated from the intestinal contents of healthy piglets fed lactic acid bacteria fermented feed through primary screening of lactic acid decomposition medium (LADM) and re-screening of Clostridium proliferating medium (RCM).The screened strains were identified by morphology, 16S rDNA sequence analysis, lactic acid transformation characteristic test, genome re-sequencing and frame map sequencing, and the functional annotation of their coding proteins was carried out.【Result】 A LY33 strain was isolated.The strain LY33 was identified as Clostridium butyricum based on its morphological and 16S rDNA sequence analysis.The strain LY33 had good ability to utilize lactic acid.On the 6th day of growth, 66 mmol lactic acid could be basically transformed into about 17 mmol butyric acid.The re-sequencing of LY33 strain showed that the overall variation of its coding gene was small.Compared with the reference genome, the whole genome heterozygosity ratio of LY33 strain was 0.022‰, the total number of single nucleotide polymorphism (SNP) site variants was 21 590 (0.4666% of the whole genome), the total number of insertion/deletion (Indel) mutations was 594 (0.0128%), the variation of copy number variation (CNV) was 0 (0), and the total number of structure variation (SV) annotated variants was 103 (0.0003%).The mutant genes in the genome of LY33 strain were mainly genes related to strain growth, energy metabolism regulation and vitamin synthesis (especially biotin), which were distributed on multiple sequences of two chromosomes.The frame diagram sequencing of LY33 strain and the functional annotation of its encoded gene protein showed that its genome sequence length was 4 627 127 bp, GC content was 28.60%, contained 4 171 genes, and the total length of coding region accounted for 84.12%, which was involved in the metabolic pathway transformation of various substances such as carbohydrate metabolism, membrane transport and amino acid metabolism.There were many genes resistant to macrolides, bacitracin and VanI glycopeptide in the genome of LY33 strain.【Conclusion】 In this study, a LY33 strain with fast lactic acid utilization and high butyric acid conversion rate was isolated from the intestinal contents of healthy piglets fed with 4% lactic acid bacteria fermented feed, which was identified as Clostridium butyricum.It had a good application prospect and could be used as a new microbial resource for probiotics products.