表型特征结合基因组分析鉴定不同菌落形态Bacillus velezensis ACCC 19742

在中国农业微生物菌种中心(ACCC)菌株的定期转接保藏过程中,发现解淀粉芽孢杆菌ACCC 19742在同一培养基上出现两种不同的菌落形态,将这两个不同形态的菌株编号为19742-1和19742-2。通过形态学、生理生化及基因组分析相结合鉴定不同菌落形态ACCC 19742,并进一步确定该菌株的分类地位。首先将菌株进行分离与纯化,其次将纯化后的菌株进行16S rRNA及gyrB基因扩增及序列分析,通过MEGA 7.0软件构建系统发育树;API 20NE、BIOLOG及脂肪酸等分析菌株的生理生化特性;全基因组分析菌株的ANI和DDH值。两株菌在API 20NE中,仅葡萄糖酸盐同化反应存在差异;脂肪酸检测中主要组成相同,仅是百分含量方面略有差别;两株菌的16S rRNA基因相似性为100%,gyrB基因的相似性为99.4%;全基因组测序表明,两株菌的ANI值为99.95%,DDH值为99.62%。综合遗传学特征和表型特征,证实两者为来源于同一菌株不同的形态变异型,而并非污染所致。同时,19742-1和19742-2与Bacillus velezensis NRRL_B 41580~T的ANI及DDH值最高,分别为97%和77%,且16S rRNA和gyrB系统进化分析也表明,该菌株在分类地位上属于贝莱斯芽孢杆菌(Bacillus velezensis),而非解淀粉芽孢杆菌。这为菌株的保藏提供了一定的参考价值。

Phenotypic Characteristics Combined with Genomic Analysis to Identify Different Colony Morphology Bacillus velezensis ACCC 19742

During the period of regular transfer and preservation of bacteria in Agricultural Culture Collection of China(ACCC),it was found that Bacillus amyloliquefaciens ACCC 19742 showed two different colonies on the same medium,named these two different colonies as 19742-1 and 19742-2. The morphology,physiology,biochemistry and genome analysis were used to identify ACCC 19742,and to further determine the taxonomic status of the strain. Firstly,the strains were isolated and purified. Secondly,16S rRNA and gyrB genes were amplified and sequenced. Phylogenetic tree was constructed by MEGA 7.0 software. Physiological and biochemical characteristics of the strains were analyzed by API 20NE,BIOLOG and fatty acid. The ANI and DDH values of the strains were analyzed by whole genome analysis. Only the gluconate assimilation reaction in 2 strains was different according to API 20NE detection,the main fatty acid components were consistent,only the percentage was slightly different. Based on the phylogenetic analysis,16S rRNA gene similarity of the two strains was 100%,while the gyrB gene similarity was 99.4%. The ANI value of the two strains was 99.95% and the DDH value was 99.62%. Combining with genetic and phenotypic characteristics,it was confirmed that the two strains were derived from different morphological variants of the same strain,not caused by pollution. Meanwhile,ANI and DDH values of 19742 and Bacillus velezensis NRRL_B 41580~T were the highest(97% and 77%,respectively). Evolutionary analysis of 16S rRNA and gyrB also indicated that the strain belonged to the taxonomic status Bacillus velezensis,but not B. amyloliquefaciens. This provides a certain reference value for the preservation of the strain.