美洲大蠊肠道内生分枝杆菌的分离鉴定及其抑菌活性的初步研究
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摘要
目的对美洲大蠊肠道内生分枝杆菌进行形态学和分子生物学鉴定,初步研究其抑菌活性并筛选其次级代谢产物生物合成基因簇。方法采用平板划线法对美洲大蠊肠道内生分枝杆菌进行分离、纯化及革兰染色检查;PCR扩增其16S rDNA并测序,进行BLAST比对及系统发育树分析,对生物合成基因簇聚酮合酶(PKS I)基因、非核糖体肽合成酶(NRPS)基因与FADH2依赖型卤化酶基因进行初步筛选。采用牛津杯法初步测定分离菌对常见致病菌的抑菌活性。结果从美洲大蠊肠道分离到4株内生分枝杆菌,在高氏I号培养基上形成的菌落较小,颜色呈橘红色,革兰染色呈紫色;系统发育树分析表明,4株分枝杆菌与已知分枝杆菌相似度均在99%以上,且与植物、土壤及临床标本来源的分枝杆菌亲缘关系较近。4株美洲大蠊肠道内生分枝杆菌的生物合成基因PKS I表达均呈阳性,其中3株菌的NRPS基因表达呈阳性。抑菌试验显示,其中3株分枝杆菌均对8株常见致病菌表现出不同程度的选择性抑制作用。结论美洲大蠊肠道内生分枝杆菌具有抑菌活性,可能具有产聚酮类或多肽类等活性物质的潜力。
Objectives To determine the morphology and identify via molecular biology the endophytic Mycobacteria from Periplaneta americana, to determine its antibacterial activity, and to detect the expression of secondary metabolite biosynthesis gene clusters. Methods Endophytic Mycobacteria in the intestine were isolated and purified using a streak plate and then subjected to Gram staining. The 16S rDNA of Mycobacteria was amplified with PCR and sequenced, and sequencing results were used for BLAST comparison and construction of a phylogenetic tree using the neighbor-joining method. Expression of the polyketide synthase I(PKS I) gene, non-ribosomal peptide synthetase(NRPS) gene, and FADH2-dependent halogenase gene encoded in biosynthetic gene clusters were identified using PCR and agarose gel electrophoresis. Eight strains of common pathogenic bacteria were used as indicator bacteria, and the antibacterial activity of the endophytic Mycobacteria was preliminarily tested using the Oxford cup method. Results Four strains of endophytic Mycobacteria were isolated in the gut of P. americana. The colonies formed on Gauze's medium No. 1 were small, orange-red in color, and stained purple in Gram staining. A phylogenetic tree analysis indicated that the similarity between four strains of Mycobacteria and the known Mycobacteria was over 99%, and these four strains of Mycobacteria were high homologous to Mycobacteria from plant, soil, and clinical specimens. The four strains of endophytic Mycobacteria from the intestine of P. americana tested positive for expression of PKS I encoded in a secondary metabolite biosynthesis gene cluster, only three strains of endophytic Mycobacteria from the intestine tested positive for expression of NRPS, and no strains expressed the FADH2-dependent halogenase gene. An antibacterial activity assay indicated that three strains of Mycobacteria selectively inhibited eight strains of common pathogenic bacteria to different degrees. The strain WA20-1-3 had antibacterial activity against Staphylococcus aureus and Escherichia coli. The strain WA20-2-6 had antibacterial activity against Candida albicans, Trichophyton rubrum, Aspergillus niger, and Aspergillus fumigatus. The strain WA22-2-3 had antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae, and E. coli. Conclusion Endophytic Mycobacteria from P. americana have antibacterial activity and might have the ability to produce active substances such as polyketides or non-ribosomal peptides.
Objectives To determine the morphology and identify via molecular biology the endophytic Mycobacteria from Periplaneta americana, to determine its antibacterial activity, and to detect the expression of secondary metabolite biosynthesis gene clusters. Methods Endophytic Mycobacteria in the intestine were isolated and purified using a streak plate and then subjected to Gram staining. The 16S rDNA of Mycobacteria was amplified with PCR and sequenced, and sequencing results were used for BLAST comparison and construction of a phylogenetic tree using the neighbor-joining method. Expression of the polyketide synthase I(PKS I) gene, non-ribosomal peptide synthetase(NRPS) gene, and FADH2-dependent halogenase gene encoded in biosynthetic gene clusters were identified using PCR and agarose gel electrophoresis. Eight strains of common pathogenic bacteria were used as indicator bacteria, and the antibacterial activity of the endophytic Mycobacteria was preliminarily tested using the Oxford cup method. Results Four strains of endophytic Mycobacteria were isolated in the gut of P. americana. The colonies formed on Gauze's medium No. 1 were small, orange-red in color, and stained purple in Gram staining. A phylogenetic tree analysis indicated that the similarity between four strains of Mycobacteria and the known Mycobacteria was over 99%, and these four strains of Mycobacteria were high homologous to Mycobacteria from plant, soil, and clinical specimens. The four strains of endophytic Mycobacteria from the intestine of P. americana tested positive for expression of PKS I encoded in a secondary metabolite biosynthesis gene cluster, only three strains of endophytic Mycobacteria from the intestine tested positive for expression of NRPS, and no strains expressed the FADH2-dependent halogenase gene. An antibacterial activity assay indicated that three strains of Mycobacteria selectively inhibited eight strains of common pathogenic bacteria to different degrees. The strain WA20-1-3 had antibacterial activity against Staphylococcus aureus and Escherichia coli. The strain WA20-2-6 had antibacterial activity against Candida albicans, Trichophyton rubrum, Aspergillus niger, and Aspergillus fumigatus. The strain WA22-2-3 had antibacterial activity against Bacillus subtilis, Klebsiella pneumoniae, and E. coli. Conclusion Endophytic Mycobacteria from P. americana have antibacterial activity and might have the ability to produce active substances such as polyketides or non-ribosomal peptides.
引文
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[22] 李航,朱丽,陈代杰.参与抗生素生物合成的FADH_2依赖型卤化酶研究进展[J].中国抗生素杂志,2010,35(1):1-6.
[23] 王海强,安向向,侯淑芬,等.39株内生放线菌次级代谢产物的合成潜能[J].河北大学学报(自然科学版),2017,37(3):254-61.
[24] Chen R,Liao L,Zhang X,et al.Cloning and analysis of a halogenase gene of Streptomyces sp.604F from the Arctic Ocean[J].Acta Microbiol Sinica,2014,54(6):703-12.
[2] 方霞,沈娟,王影姣,等.药用昆虫美洲大蠊内生放线菌的分离和鉴定[J].中国病原生物学杂志,2016,11(6):550-3,565.
[3] 孙翼飞,巩宗强,苏振成,等.一株高浓度多环芳烃降解菌的鉴定和降解特性[J].生态学杂志,2011,30(11):2503-8.
[4] 刘相岑,郝晓蔚,张瑞婕,等.降解植物甾醇产雄甾1,4-二烯-3,17-二酮工程菌株的构建及转化培养基优化[J].食品工业科技,2018,39(18):110-6.
[5] Goodfellow M,Kam pfer P,Busse HJ,et al.Begey′s manual of systematic bacteriology[M].2nd Eds.New York:Springer,2012.
[6] Zhou RQ,Nie K,Huang HC,et al.Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China[J].Vet Res Commun,2009,33(8):855-63.
[7] 梁丽琪,冯娇,蒋晓圆,等.同时产碳青霉烯酶IMP与NDM的肺炎克雷伯菌和霍氏肠杆菌的分离及耐药性研究[J].中华医院感染学杂志,2018(21):3201-6.
[8] Tamura K,Peterson D,Peterson N,et al.MEGA 5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Mol Biol Evol,2011,28(10):2731-9.
[9] Stach JE,Maldonado LA,Masson DG,et al.Statistical approa-ches for estimating actino-bacterial diversity in marine sedi-ments[J].Appl Environ Microb,2003,69:6189-200.
[10] Schloss PD,Westcott SL,Ryabin T,et al.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J].Appl Environ Microb,2009,75(23):7537.
[11] Izumikawa M,Murata M,Tachibana K,et al.Cloning of modular type I polyketide synthase genes from salinomycin producing strain of Streptomyces albus[J].Bioorg Med Chem,2003,11(16):3401-5.
[12] González I,Ayuso-Sacido A,Anderson A,et al.Actinomycetes isolated from lichens:evaluation of their diversity and detection of biosynthetic gene sequences[J].FEMS Microbiol Ecol,2005,54(3):401-15.
[13] Hornung A,Bertazzo M,Dziarnowski,et al.A genomic screening approach to the structure-guided identification of drug candidates from natural sources[J].Chembiochem,2007,8(7):757-66.
[14] 王四宝,曲爽.昆虫共生菌及其在病虫害防控中的应用前景[J].中国科学院院刊,2017,32(8):863-72.
[15] 梅承,范硕,杨红.昆虫肠道微生物分离培养策略及研究进展[J].微生物学报,2018,58(6):985-94.
[16] Barretto DA,Vootla SK.In vitro anticancer activity of Staphyloxanthin Pigment extracted from Staphylococcus gallinarum KX912244,a gut microbe of Bombyx mori[J].Indian J Microbiol,2018,58(2):146-58.
[17] Dillon RJ,Vennard CT,Buckling A,et al.Diversity of locust gut bacteria protects against pathogen invasion[J].Ecol Lett,2005,8(12):1291-8.
[18] Akbar N,Siddiqui R,Iqbal M,et al.Gut bacteria of cockroaches are a potential source of antibacterial compound(s)[J].Lett Appl Microbiol,2018,66(5):416-26.
[19] Gupta RS,Lo B,Son J.Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera[J].Front Microbiol,2018(9):67.
[20] Sun S,Wang H,Chen Y,et al.Salicylate and phthalate pathways contributed differently on phenanthrene and pyrene degradations in Mycobacterium sp.WY10[J].J Hazard Mater,2018(364):509-18.
[21] Su L,Shen Y,Xia M,et al.Overexpression of cytochrome p450 125 in Mycobacterium:a rational strategy in the promotion of phytosterol biotransformation[J].J Ind Microbiol Biot,2018,45(10):857-67.
[22] 李航,朱丽,陈代杰.参与抗生素生物合成的FADH_2依赖型卤化酶研究进展[J].中国抗生素杂志,2010,35(1):1-6.
[23] 王海强,安向向,侯淑芬,等.39株内生放线菌次级代谢产物的合成潜能[J].河北大学学报(自然科学版),2017,37(3):254-61.
[24] Chen R,Liao L,Zhang X,et al.Cloning and analysis of a halogenase gene of Streptomyces sp.604F from the Arctic Ocean[J].Acta Microbiol Sinica,2014,54(6):703-12.