一株肝素降解菌的分离鉴定及基因组测序分析

Isolation, identification and whole genome analysis of a heparin-degrading bacterium

以白头叶猴粪便为材料分离到一株不形成芽孢、可产黄色色素的细菌,革兰氏染色为阴性,接触酶阳性,经形态学观察和分子生物学鉴定为Sphingobacterium daejeonense NS6-1。NS6-1不能水解淀粉,但能降解肝素,肝素酶酶活为46.60 U/mL,当添加100 mmol/L的Ca2+为辅助剂时肝素酶活性可达107.29 U/mL。通过K-B纸片琼脂扩散法检测菌株对几种常规药物的敏感性,发现NS6-1对卡那霉素和庆大霉素耐药,对利福平、四环素、多黏菌素B、氧氟沙星、头孢哌酮和万古霉素敏感。基于Illumina Hiseq 4000平台对NS6-1进行全基因组序列测定,结果显示其基因组大小为4 381 042 bp, GC含量为37.21%,预测到的蛋白质编码基因数为3 852个。功能基因注释结果显示,基因组中含1个编码肝素酶Ⅱ/Ⅲ基因,1个氨基糖苷类抗生素耐药功能基因以及1个与芳香烃化合物降解相关的邻苯二酚1,2-双加氧酶基因。利用antiSMASH预测到6个次级代谢产物生物合成基因簇,其中,2个为类胡萝卜素合成基因簇,1个为高分子表面活性剂emulsan合成基因簇。综上,NS6-1具有肝素类物质降解能力以及潜在的石油降解能力和类胡萝卜素合成能力。以上结果为该菌的后续研究及实际应用奠定了理论基础。

A bacterium species was isolated from fecal specimens of white-headed langur.This bacterium is non-spore-forming,yellow pigment producing,gram-negative and catalase-positive.It was identified as Sphingobacterium daejeonense NS6-1 based on 16S rRNA gene sequence.NS6-1 can’t hydrolyze starch but is a heparin-degrading bacteria.The heparinase activity of NS6-1 was 46.60 U/mL,and can be increased to 107.29 U/mL in the present of 100 mmol/L Ca 2+.Routine drug susceptibility test by K-B disk agar diffusion method showed that NS6-1 was resistant to kanamycin and gentamicin,while sensitive to rifampicin,tetracycline,polymyxin B,ofloxacin,cefoperazone and vancomycin.The genome was 4381042 bp in length with 37.21%overall GC content,including 3852 protein coding sequences.Functional annotation revealed a heparinaseⅡ/Ⅲ-like protein in CAZy,a aminoglycoside resistance gene in CARD,a gene encoding catechol 1,2-dioxygenas which can catalyze hydrocarbon-related catabolic in oil.Six gene clusters related to secondary metabolite biosynthesis were predicted by using antiSMASH,of which two were identified as carotenoid synthesis gene clusters and one emulsan synthesis gene clusters.In summary,NS6-1 is a heparin-degrading bacterium,with potential for degradation aromatic hydrocarbons in petroleum and carotenoid synthesis.The present results would lay a theoretical foundation for the further application of the isolated strain.