2株小双孢菌新种的全基因组测序分析与抗菌活性测定

Whole genome sequencing and antibacterial activity determination of two novel species of Microbispora

摘要:

【背景】抗生素的滥用导致了耐药病原菌的不断增加和扩散，成为威胁人们健康的一个重要因素，因此挖掘新型抗生素显得尤其重要。新的物种很有可能会产生新的生物活性物质，特别是那些常规分离方法很少分离到的稀有放线菌。【目的】对2株小双孢菌潜在新种进行全基因组测序分析与抗菌活性测定，从多层次分子水平鉴定其新种分类地位，并预测次级代谢基因簇，为发现产生新颖活性物质的药用放线菌资源奠定基础。【方法】通过16S rRNA基因序列分析初步确定菌株H10836和H11081的种属分类；采用Illumina平台对2个菌株进行全基因组测序和注释，基于此构建全基因组系统发育树、计算ANI (average nucleotide identity)和dDDH (digital DNA-DNA Hybridization)值，最终确定菌株的新种分类地位；基于全基因组注释结果进行COG (Clusters of Orthologous Genes)聚类、KEGG (Koyto Encyclopedia of Genes and Genomes)代谢通路和antiSMASH次级代谢基因簇预测分析；采用琼脂打孔法进行抗菌活性测定。【结果】菌株H10836和H11081与小双孢菌属8个种两两之间的ANI值范围分别为85.3%?92.1%和85.2%?92.1%，dDDH值范围分别为33.0%?44.5%和31.3%?44.5%。ANI和dDDH值均低于物种判断的临界值，而且2株菌在全基因组系统发育树上与小双孢菌属聚成一簇，但独立成一亚支，从分子水平上综合确定菌株H10836和H11081是小双孢菌属的2个潜在新种。antiSMASH分析发现该2株菌基因组中有多种次级代谢产物合成基因簇，而且与已知抗生素合成基因簇相似度较低。抗菌活性测定显示该2株菌代谢产物均有抗耐甲氧西林金黄色葡萄球菌活性。【结论】菌株H10836和H11081是具有抗菌活性的新菌种，因此值得挖掘其新颖活性天然产物，试验结果将为菌株的进一步研究和应用提供参考。

Abstract:

[Background] The abuse of antibiotics leads to the increase and spread of drug-resistant pathogens, which has become an important factor threatening people’s health. Therefore, it is particularly important to explore new antibiotics. New species are likely to produce new bioactive substances, especially rare actinomycetes which are rarely isolated by conventional methods. [Objective] To confirm classification of two novel species of Microbispora and predict the secondary metabolic gene clusters by various molecular biological technologies, which will lay a foundation for the discovery of the medicinal actinobacteria producing novel active substances. [Methods] Taxonomic positions of isolates were preliminarily determined by the 16S rRNA gene sequence analysis. The whole genomes were sequenced and annotated by Illumina genome analyzer. On this basis, a whole-genome-based phylogenomic tree was constructed, and the ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) values were calculated, so as to determine the taxonomic status of novel species. Based on gene annotation, COG (clusters of orthologous genes), KEGG (Kyoto Encyclopedia of Genes and Genomes), and secondary metabolic gene cluster prediction on antiSMASH were analyzed. Antimicrobial activity was tested by cylinder plate method. [Results] The ANI and dDDH values between strain H10836 and 8 species of the genus Microbispora were 85.3%?92.1% and 33.0%?44.5%, respectively. Meanwhile, the ANI and dDDH values between strain H11081 and 8 species of the genus Microbispora were 85.2%?92.1% and 31.3%?44.5%, respectively. Therefore, all the ANI and dDDH values are well below the cut-off point recommended for delineating species. In addition, a whole-genome-based phylogenomic tree showed that strains H10836 and H11081 formed an independent monophyletic branch within the genus Microbispora. Therefore, these results of phylogenetic analysis support the conclusion that strains H10836 and H11081 could be considered to represent two potential novel species of the genus Microbispora. Furthermore, there were many kinds of biosynthetic gene clusters in the genomes of both strains H10836 and H11081 by antiSMASH analysis, and the similarities with known antibiotic synthetic gene clusters were low. The metabolites of two strains exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). [Conclusion] Strains H10836 and H11081 are two novel species with antibacterial activity, so it is worth mining their novel active natural products. The results of this experiment provide reference for further research and application of the strains.