印度洋深海沉积物可培养放线菌多样性及抗革兰阴性菌活性物质研究
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摘要
目的从印度洋深海沉积物中分离可培养海洋放线菌,对获得的一株海洋微生物进行抗革兰阴性菌活性物质研究。方法设计11种分离培养基进行海洋放线菌分离,通过16S r RNA基因序列比对分析确定菌株分类;利用多种色谱方法,对菌株IMB16-059产生的活性物质进行分离纯化,通过波谱学和化学方法鉴定其结构。结果根据菌落表型共分离得68株微生物,经16S r RNA分析表明,其中34株属于放线菌门,覆盖5科5属;其余属于厚壁菌门和变形菌门。25株发酵产物显示出抗革兰阴性菌活性。从赤杆菌IMB16-059中分离鉴定了6个化合物,其结构分别确定为L,L-环(脯氨酸-丙氨酸)(1),L,L-环(脯氨酸-酪氨酸(2)),D,L-环(脯氨酸-酪氨酸)(3),邻氨基苯甲酸(4),胸腺嘧啶(5),1H-喹啉-4-酮(6)。化合物1和4显示出抗铜绿假单胞菌活性,在样品浓度为20μg/片时,抑菌圈直径分别为13和20mm。结论印度洋深海沉积物蕴含丰富的微生物类群,其发酵产物显示出较好的抗革兰阴性菌活性,可为新抗生素发现提供重要的菌种资源。
Objective To investigate the diversity of culturable actinomycetes from the deep-sea sediment collected from the Indian Ocean and identify the antibacterial metabolites from one of the isolated marine strains.Methods The marine actinomycete strains were isolated by using 11 different media.The 16 S rRNA sequence analysis was used to characterize the diversity of culturable actinomycetes.The antibacterial metabolites were isolated and purified by using a combination of chromatographic techniques.Their structures were elucidated using spectroscopic analysesand chemical methods.Results A total of 68 colonies with phenotypical bacteria were obtained and classified by the 16 S r RNA sequence analysis.Among the identified bacteria,34 strains were classified into five genera of five families while other strains were homologous to the phyla Firmicutes and Proteobacteria.The culture extracts of 25 strains displayed antibacterial activities against Gram-negative bacteria.Six compounds were identified from the fermentation broth of Erythrobacter sp.IMB16-059.Their structures were determined as cyclo(LPro-l-Ala)(1),cyclo(L-Pro-L-Tyr)(2),cyclo(D-Pro-L-Tyr)(3),anthranilic acid(4),thymine(5),quinolin-4(1 H)-one(6).Compounds 1 and 4 showed antibacterial activity against Pseudomonas aeruginosa with inhibition zones of 1 and 20 mm at 20μg/disc,respectively.Conclusion The results indicated that the deep marine sediments from the India Ocean possess diverse and abundant actinomycetesa and antibacterial activities,which could provide potential sources for the discovery of new antibiotics.
Objective To investigate the diversity of culturable actinomycetes from the deep-sea sediment collected from the Indian Ocean and identify the antibacterial metabolites from one of the isolated marine strains.Methods The marine actinomycete strains were isolated by using 11 different media.The 16 S rRNA sequence analysis was used to characterize the diversity of culturable actinomycetes.The antibacterial metabolites were isolated and purified by using a combination of chromatographic techniques.Their structures were elucidated using spectroscopic analysesand chemical methods.Results A total of 68 colonies with phenotypical bacteria were obtained and classified by the 16 S r RNA sequence analysis.Among the identified bacteria,34 strains were classified into five genera of five families while other strains were homologous to the phyla Firmicutes and Proteobacteria.The culture extracts of 25 strains displayed antibacterial activities against Gram-negative bacteria.Six compounds were identified from the fermentation broth of Erythrobacter sp.IMB16-059.Their structures were determined as cyclo(LPro-l-Ala)(1),cyclo(L-Pro-L-Tyr)(2),cyclo(D-Pro-L-Tyr)(3),anthranilic acid(4),thymine(5),quinolin-4(1 H)-one(6).Compounds 1 and 4 showed antibacterial activity against Pseudomonas aeruginosa with inhibition zones of 1 and 20 mm at 20μg/disc,respectively.Conclusion The results indicated that the deep marine sediments from the India Ocean possess diverse and abundant actinomycetesa and antibacterial activities,which could provide potential sources for the discovery of new antibiotics.
引文
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[34]Cho J Y,Bae S H,Kim H K,et al.New quinolinone alkaloids from chestnut(Castanea crenata Sieb)Honey[J].J Agric Food Chem,2015,63(13):3587-3592.
[35]Schievano E,Morelato E,Facchin C,et al.Characterization of markers of botanical origin and other compounds extracted from unifloral honeys[J].J Agric Food Chem,2013,61(8):1747-1755.
[36]陈菲菲,王勇,王以光,等.海洋微生物来源的天然产物开发研究进展[J].应用与环境生物学报,2011,17(2):287-294.
[37]林灵,谭亿,陈菲菲,等.大连渤海老虎滩海域沉积物可培养放线菌的多样性[J].微生物学报,2011,51(2):262-269.
[38]Yonezawa K,Yamada K,Kouno I.New diketopiperazine derivatives isolated from sea urchin-derived Bacillus sp.[J].Chem Pharm Bull,2011,59(1):106-108.
[39]Rhee K H.Cyclic dipeptides exhibit synergistic,broad spectrum antimicrobial effects and have anti-mutagenic properties[J].Int J Antimicrob Agents,2004,24(5):423-427.
[40]Graz M,Hunt A,Jamie H,et al.Antimicrobial activity of selected cyclic dipeptides[J].Pharmazie,1999,54(10):772-775.
[41]Weber T,Blin K,Duddela S,et al.Anti SMASH 3.0—a comprehensive resource for the genomemining of biosynthetic gene clusters[J].Nucleic Acids Res,2015,43(W1):W237-243.
[42]Arnison P G,Bibb M J,Bierbaum G,et al.Ribosomally synthesized and post-translationally modified peptide natural products:Overview and recommendations for a universal nomenclature[J].Nat Prod Rep,2013,30(1):108-160.
[43]Maksimov M O,Pan S J,James Link A.Lasso peptides:Structure,function,biosynthesis,and engineering[J].Nat Prod Rep,2012,29(9):996-1006.
[44]Hegemann J D,Zimmermann M,Xie X,et al.Lasso peptides:An intriguing class of bacterial natural products[J].Acc Chem Res,2015,48(7):1909-1919.
[2]张静,喻玮,赵丽娜,等.我国细菌耐药现状与特征[J].中华临床感染病杂志,2016,9(2):118-128.
[3]Butler M S,Blaskovich M A T,Cooper M A.Antibiotics in the clinical pipeline at the end of 2015[J].J Antibiot,2017,70(1):3-24.
[4]Theuretzbacher U.Accelerating resistance,inadequate antibacterial drug pipelines and international responses[J].Int J Antimicrob Agents,2012,39(4):295-299.
[5]Berdy J.Thoughts and facts about antibiotics:Where we are now and where we are heading[J].J Antibiot,2012,65(8):385-395.
[6]Subramani R,Aalbersberg W.Marine actinomycetes:an ongoing source of novel bioactive metabolites[J].Microbiol Res,2012,167(10):571-580.
[7]Kamjam M,Sivalingam P,Deng Z,et al.Deep sea actinomycetes and their secondary metabolites[J].Front Microbiol,2017,8:760.
[8]Skropeta D,Wei L.Recent advances in deep-sea natural products[J].Nat Prod Rep,2014,31(8):999-1025.
[9]Wang Q,Zhang Y,Wang M,et al.Neo-actinomycins A and B,natural actinomycins bearing the 5H-oxazolo[4,5-b]phenoxazine chromophore,from the marine-derived Streptomyces sp.IMB094[J].Sci Rep,2017,7(1):3591.
[10]Gan M,Liu B,Tan Y,et al.Saccharothrixones A–D,tetracenomycin-type polyketides from the marine-derived actinomycete Saccharothrix sp.10-10[J].J Nat Prod,2015,78(9):2260-2265.
[11]Wu C,Tan Y,Gan M,et al.Identification of elaiophylin derivatives from the marine-derived actinomycete Streptomyces sp.7-145 using PCR-based screening[J].J Nat Prod,2013,76(11):2153-2157.
[12]吴月红,许学伟.赤杆菌科微生物分类研究进展[J].微生物学通报,2016,43(5):1082-1094.
[13]Feng X M,Mo Y X,Han L,et al.Qipengyuania sediminis gen.nov.,sp.nov.,a novel member of the family Erythrobacteraceae,isolated from subterrestrial sediment[J].Int J Syst Evol Microbiol,2015,65(10):3658-3665.
[14]Xu X W,Wu Y H,Wang C S,et al.Croceicoccus marinus gen.nov.,sp.nov.,a yellow-pigmented bacterium from deep-sea sediment,and emended description of the family Erythrobacteraceae[J].Int J Syst Evol Microbiol,2009,59(9):2247-2253.
[15]Takaichi S,Shimada K,Ishidsu J I.Carotenoids from the aerobic photosynthetic bacterium,Erythrobacter longus:beta-Carotene and its hydroxyl derivatives[J].Arch Microbiol,1990,153(2):118-122.
[16]Hu Y,Legako A G,Espindola A P D M,et al.Erythrolic a c i d s A-E,meroterpenoids from a marine-derived Erythrobacter sp.[J].J Org Chem,2012,77(7):3401-3407.
[17]Hu Y,Mac Millan J B.Erythrazoles A-B,Cytotoxic benzothiazoles from a marine-derived Erythrobacter sp[J].Org Lett,2011,13(24):6580-6583.
[18]Jensen P R,Gontang E,Mafnas C,et al.Culturable marine actinomycete diversity from tropical Pacific Ocean sediments[J].Environ Microbiol,2005,7(7):1039-1048.
[19]夏涵,府伟灵,陈鸣,等.快速提取细菌DNA方法的研究[J].现代预防医学,2005,32(5):571-573.
[20]Stach J E M,Maldonado L A,Masson D G,et al.Statistical approaches for estimating actinobacterial Diversity in marine sediments[J].Appl Environ Microbiol,2003,69(10):6189-6200.
[21]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:Open-source,platform-independent,communitysupported software for describing and comparing microbial communities[J].Appl Environ Microbiol,2009,75(23):7537-7541.
[22]Tamura K,Stecher G,Peterson D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30(12):2725-2729.
[23]Saitou N,Nei M.The neighbor-joining method:A new method for reconstructing phylogenetic trees[J].Mol Biol Evol,1987,4(4):406-425.
[24]Fujii K,Ikai Y,Oka H,et al.A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide:Combination of Marfey's method with mass spectrometry and its practical application[J].Anal Chem,1997,69(24):5146-5151.
[25]Fujii K,Ikai Y,Mayumi T,et al.A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide:Elucidation of limitations of Marfey's method and of its separation mechanism[J].Anal Chem,1997,69(16):3346-3352.
[26]Kreitlow S,Mundt S,Lindequist U.Cyanobacteria—a potential source of new biologically active substances[J].J Biotechnol,1999,70(1-3):61-63.
[27]National Committee for Clinical Laboratory Standards(CLSI/NCCLS)Approved Standard M7-A9.Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically[S].Wayne,PA,USA:Clinical and Laboratory Standards Institute,2012.
[28]Huang R,Yan T,Peng Y,et al.Diketopiperazines from the marine sponge Axinella sp[J].Chem Nat Comp,2014,50(1):191-193.
[29]Li T,Wang G C,Huang X J.Whitmanoside A,a new a-pyrone glycoside from the leech Whitmania pigra[J].Heterocycles,2013,87(7):1537-1543.
[30]Nalli Y,Gupta S,Khajuria V,et al.TNF-αand IL-6inhibitory effects of cyclic dipeptides isolated from marine bacteria Streptomyces sp[J].Med Chem Res,2017,26(1):93-100.
[31]Campbell J,Lin Q,Geske G D,et al.New and unexpected insights into the modulation of Lux R-Type quorum sensing by cyclic dipeptides[J].ACS Chem Biol,2009,4(12):1051-1059.
[32]Zhao H,Fu H,Qiao R.Copper-catalyzed direct amination of ortho-functionalized haloarenes with sodium azide as the amino source[J].J Org Chem,2010,75(10):3311-3316.
[33]Sun J F,Wu Y,Yang B,et al.Chemical constituents of marine sponge Halichondria sp.from South China Sea[J].Chem Nat Comp,2015,51(5):1-3.
[34]Cho J Y,Bae S H,Kim H K,et al.New quinolinone alkaloids from chestnut(Castanea crenata Sieb)Honey[J].J Agric Food Chem,2015,63(13):3587-3592.
[35]Schievano E,Morelato E,Facchin C,et al.Characterization of markers of botanical origin and other compounds extracted from unifloral honeys[J].J Agric Food Chem,2013,61(8):1747-1755.
[36]陈菲菲,王勇,王以光,等.海洋微生物来源的天然产物开发研究进展[J].应用与环境生物学报,2011,17(2):287-294.
[37]林灵,谭亿,陈菲菲,等.大连渤海老虎滩海域沉积物可培养放线菌的多样性[J].微生物学报,2011,51(2):262-269.
[38]Yonezawa K,Yamada K,Kouno I.New diketopiperazine derivatives isolated from sea urchin-derived Bacillus sp.[J].Chem Pharm Bull,2011,59(1):106-108.
[39]Rhee K H.Cyclic dipeptides exhibit synergistic,broad spectrum antimicrobial effects and have anti-mutagenic properties[J].Int J Antimicrob Agents,2004,24(5):423-427.
[40]Graz M,Hunt A,Jamie H,et al.Antimicrobial activity of selected cyclic dipeptides[J].Pharmazie,1999,54(10):772-775.
[41]Weber T,Blin K,Duddela S,et al.Anti SMASH 3.0—a comprehensive resource for the genomemining of biosynthetic gene clusters[J].Nucleic Acids Res,2015,43(W1):W237-243.
[42]Arnison P G,Bibb M J,Bierbaum G,et al.Ribosomally synthesized and post-translationally modified peptide natural products:Overview and recommendations for a universal nomenclature[J].Nat Prod Rep,2013,30(1):108-160.
[43]Maksimov M O,Pan S J,James Link A.Lasso peptides:Structure,function,biosynthesis,and engineering[J].Nat Prod Rep,2012,29(9):996-1006.
[44]Hegemann J D,Zimmermann M,Xie X,et al.Lasso peptides:An intriguing class of bacterial natural products[J].Acc Chem Res,2015,48(7):1909-1919.