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海洋放线菌是海洋微生物的重要组成部分,也是海洋微生物新天然产物挖掘的重要资源之一[1]。海洋放线菌产生多种新天然产物,结构类型包括聚酮类、生物碱类、脂肪酸类、肽类、萜烯类等,并具有多种生物活性,包括抗菌、抗寄生虫、抗疟、抗炎、抗氧化、抗肿瘤以及免疫调节剂等[2]。因此,海洋放线菌一直受到国内外海洋天然产物研究者的青睐。海洋放线菌广泛栖息于各种海洋生态环境,包括海水、海底沉积物、海洋动物(如海绵、珊瑚)、海洋植物(如海藻、海草)表面及内部组织等。其中,海绵是海洋放线菌的良好宿主,海洋放线菌与海绵形成了附生、共生或者内生的相互关系[3]。有研究表明,海绵共附生放线菌不仅种类丰富多样,而且产生结构新颖的次级代谢产物[1]。这些次级代谢产物在海绵的化学防御中起到了重要的作用,同时还具有抗菌、抗肿瘤和抗寄生虫等生物活性,具有潜在的药用开发价值[4]。因此,近年来海绵共附生放线菌已成为海洋微生物天然产物研究的热点。海南岛地处热带,四面环海,海绵种类繁多,为研究海绵共附生放线菌提供了丰富的材料。鲍时翔等[5-6]从三亚和琼海海域采集的海绵中分别分离到具抗肿瘤活性和抗流感病毒活性的共附生链霉菌。HUANG Y等[7]从南海采集的海绵样品中分离到多个种属的共附生放线菌,27.5%的菌株具有抗菌活性,91%的菌株含有聚酮类或非核糖体肽合成酶基因。本实验室从三亚海域采集的海绵中分离到放线菌新种Streptomyces spongiicola HNM0071,并从中发现了抗肿瘤先导化合物十字孢碱和棘霉素[4,8]。研究显示海南的海绵共附生放线菌资源具有种类丰富新颖、药用价值高的特点,值得广泛深入的研究。基于此,笔者从海南文昌海域采集多种海绵作为样品,分离其共附生放线菌、并分析其菌株多样性和抗菌活性,旨在为此类海洋共附生放线菌资源的前期开发提供资源和科学依据。
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笔者采用 4 种分离培养基,对采集于海南文昌海域的 4种海绵的共附生放线菌进行分离纯化,共获得50株纯化的放线菌。为确定分离菌株的分类地位,依据菌株菌落形态差异将分离菌株初步分组,挑选出各组中的代表性特征菌株进行下一步分析,共选择22株菌株,并扩增其16S rRNA序列测序分析。结果(图1)表明,这50株菌株隶属于6个科(Streptomycineae、Micromonosporineae、Pseudonocardiaceae、Nocardiaceae、Corynebacterineae和Promicromonosporaceae),7个属(链霉菌属Streptomyces、小单孢菌属Micromonospora、栖白蚁菌属Isoptericola、Krasilnikoviella属、分枝杆菌属Mycobacterium、红球菌属Rhodococcus、糖多孢菌属Saccharopolyspora)。其中链霉菌属Streptomyces 为优势属,共分离得到19株菌,约占38.00%;其次为小单孢菌属Micromonospora,共分离得到18株菌,约占36.00%;Krasilnikoviella属分离得到7株菌,约占14.00%;红球菌属Rhodococcus和栖白蚁菌属Isoptericola都分别分离得到了2株菌,约占总数的8.00%;分枝杆菌属Mycobacterium和糖多孢菌属Saccharopolyspora各分离到了1株菌,约占总数的4.00%。此外,红球菌属菌株HNM0569、链霉菌属菌株HNM0574、小单孢菌属菌株HNM0616以及分枝杆菌属菌株HNM0591与亲缘关系最近的有效发表种的 16S r RNA 基因序列的相似性都≤98.7%(表1)。根据使用基因组数据进行原核生物分类的最低标准[10],这4个菌株可能是潜在的新物种。
表 1 代表性菌株16S r RNA基因序列分析
Table 1. 16S r RNA gene sequence analysis of representative strains
编号Numbering GeneBank ID 样品来源
Sample source最相似典型菌株(登录号)
The most similar typical strain (login number)相似度/%
SimilarityHNM0594 MN446727 WC−4 Isoptericolacucumis AP-38 T(KU201961) 99.14 HNM0579 MN446724 WC−20 Isoptericolananjingensis H17 T(HQ222356) 99.93 HNM0562 MN446736 WC−33 Krasilnikoviellaflava DSM 21481 T(jgi.1053042) 99.79 HNM0577 MN446737 WC−33 Krasilnikoviellamuralis T6220-5-2bT(LC148843) 99.58 HNM0617 MN446738 WC−33 Micromonosporaaurantiaca ATCC 27029T(CP002162) 99.58 HNM0608 MN446730 WC−20 Micromonosporachalcea DSM 43026 T(X92594) 99.06 HNM0616 MN446734 WC−33 Micromonosporaechinofusca DSM 43913 T(LT607733) 98.70 HNM0609 MN446731 WC−4 Micromonosporasediminicola DSM 45794T(FLRH01000004) 99.85 HNM0615 MN446733 WC−33 Micromonosporasediminis CH3-3 T(AB889541) 99.78 HNM0619 MN446735 WC−33 Micromonosporaterminaliae TMS7 T(KX394339) 99.28 HNM0581 MN446725 WC−20 Micromonosporatrujilloniae234402T(HQ123435) 99.03 HNM0611 MN446732 WC−33 Micromonosporatulbaghiae DSM 45142 T(jgi.1058868) 99.13 HNM0591 MN446726 WC−4 Mycobacterium fragaeDSM 45731T(LQOW01000023) 92.67 HNM0569 MN446720 WC−33 Rhodococcusgannanensis M1T(KX887333) 98.05 HNM0563 MN446717 WC−33 Rhodococcusphenolicus DSM 44812 T(LRRH01000094) 98.89 HNM0595 MN446728 WC−4 Saccharopolysporaindica VRC122 T(JX411621) 99.28 HNM0578 MN446723 WC−33 Streptomyces araujoniaeASBV-1T (EU792889) 99.50 HNM0565 MN446718 WC−33 Streptomyces diastaticusNRRL B-1773 T(DQ026631) 99.28 HNM0575 MN446722 WC−33 Streptomyces nanshensis SCSIO 01066T(EU589334) 98.78 HNM0596 MN446729 WC−24 Streptomyces plicatus NBRC 13071 T(AB184291) 99.64 HNM0568 MN446719 WC−33 Streptomyces sedi YIM 65188 T(EU925562) 99.30 HNM0574 MN446721 WC−33 Streptomyces xishensis YIM M 10378 T(HQ585118) 97.44 -
各海绵样品中共附生放线菌分离结果(表2)表明,样品WC−33分离得到的菌株数量为26株,占分离种属的52%,其次为样品WC−4,共分离得到16株菌,样品WC−20上分离得到5株菌,样品WC−24上分离得到的菌株最少,只有3株。各种样品上分离的菌株不仅数量上有差别,种类上也存在一定的差异。其中,样品WC−33多样性最丰富,共分离得到7种不同属的放线菌。样品菌株WC−4虽然分离到了16株菌,但是其多样性不太丰富,仅为2个属,而样品WC−20和样品WC−24不仅得到的菌株数量少,而且种类也不丰富。另外,表2结果表明,不同海绵样品上放线菌种类的分布具有较大的差异,其中小单孢属Micromonospora在4种不同的海绵样品上均有分布,链霉菌属Streptomyces在3种不同的海绵样品上均有分布,而其他种属放线菌只在样品WC−33中分离得到,如栖白蚁菌属Isoptericola、Krasilnikoviella属、分枝杆菌属Mycobacterium、红球菌属Rhodococcus、糖多孢菌属Saccharopolyspora等均来源于WC−33号样品。
表 2 4种海绵样品上的共附生放线菌分布
Table 2. Distribution of symbiotic actinomycetes on 4 sponge samples
放线菌种属
Species of actinomycetes放线菌数量/株
Number of actinomycetes海绵 WC−20 海绵 WC−24 海绵 WC−33 海绵 WC−4 Isoptericola sp - - 2 - Krasilnikoviella sp - - 7 - Micromonospora sp 5 1 1 11 Mycobacterium sp - - 1 - Rhodococcus sp - - 2 - Saccharopolyspora sp - - 1 - Streptomyces sp - 2 12 5 分离菌株数(种类)
Strains (species)5(1) 3(2) 26(7) 16(2) -
对50株海洋放线菌的发酵提取物进行抑菌活性分析,结果(表3)表明,24株海洋放线菌的发酵产物对4种指示细菌显示出不同程度的抑菌作用,占总菌数的48%。其中16株海洋放线菌显示出抗无乳链球菌活性,14株海洋放线菌显示出抗金黄色葡萄球菌活性,11株海洋放线菌显示出抗耐甲氧西林金黄色葡萄球菌活性,仅有2株海洋放线菌显示出抗大肠杆菌活性。此外,各海洋放线菌种属之间抗细菌活性比例呈现明显差异。除了Mycobacterium和Rhodococcus属海洋放线菌未发现抗细菌的菌株之外,其他5个属的海洋放线菌均含有抗细菌活性菌株,其中链霉菌属海洋放线菌的抗细菌活性比例为52.63%,Micromonospora属为33.33%,其他3个属(Isoptericola、Krasilnikoviella、Saccharopolyspora)的分离菌株数尽管较少,但活性菌株比例却相当高。
表 3 海洋放线菌抗细菌和抗真菌活性分布
Table 3. Distribution of antibacterial activity of marine actinomycetes
分类
Classification总菌数/株
Total bacterial count抗细菌菌数/株
Antibacterial strains抗真菌菌数/株
Antifungal strains活性菌数(比例)/株
Active strainC1 C2 C3 C4 Z1 Z2 Z3 Z4 Z5 Z6 细菌 真菌 Isoptericola 2 1 1 1 - 0 0 0 0 1 0 2(100%) 1(50%) Krasilnikoviella 7 0 2 3 4 0 0 0 0 0 0 5(71.43%) 0 Micromonospora 18 0 3 3 5 1 0 0 0 1 1 6(33.33%) 1(5.56%) Mycobacterium 1 0 0 0 0 0 0 0 0 0 0 0 0 Rhodococcus 2 0 0 0 0 0 0 0 0 0 0 0 0 Saccharopolyspora 1 0 0 0 1 1 0 1 1 0 0 1(100%) 1(100%) Streptomyces 19 1 8 4 6 4 3 4 4 8 6 10(52.63%) 9(47.37%) 总计(比例) 50 2(4%) 14(28%) 11(22%) 16(32%) 6(12%) 3(6%) 5(10%) 5(10%) 10(20%) 7(14%) 24(48%) 12(24%) 注:C1:大肠杆菌,C2:金黄色葡萄球菌,C3:耐甲氧西林金黄色葡萄球菌,C4:无乳链球菌。Z1.香蕉枯萎病菌;Z2.芒果炭疽病菌;Z3.水稻稻瘟病菌;Z4.木薯根腐病菌;Z5.薯蓣炭疽病菌;Z6.橡胶炭疽病菌。
Note: C1: Escherichia coli; C2: Staphylococcus aureus; C3: Methicillin-resistant Staphylococcus aureus; C4: Streptococcus agalactiae. Z1. Fusarium oxysporum;Z2.Colletotrichum asianum; Z3. Magnaporthe grisea; Z4. Lasiodiplodia theobromae; Z5.Colletotrichum gloeosporioides; Z6.Colletotrichum gloeosporioides.12株海洋放线菌的发酵产物对6种指示丝状病原真菌显示出不同程度的抑菌作用,占总菌数的24%(表3)。其中20%的海洋放线菌对薯蓣炭疽病菌显示抑菌活性,14%的海洋放线菌对橡胶炭疽病菌显示抑菌活性,12%的海洋放线菌对香蕉枯萎病菌显示抑菌活性,10%的海洋放线菌对水稻稻瘟病菌和木薯根腐病菌显示抑菌活性,仅6%的海洋放线菌对芒果炭疽病菌显示抑菌活性。此外,各海洋放线菌种属之间抗真菌活性比例也呈现明显差异,其中Mycobacterium,Krasilnikoviella和Rhodococcus属海洋放线菌未发现抗真菌活性的菌株,而Isoptericola属海洋放线菌抗真菌活性比例最高,活性比例为50%,链霉菌属海洋放线菌的抗真菌活性比例次之,活性比例为47.37%;Micromonospora属海洋放线菌最少,活性比例仅为5.56%。
Isolation, Identification and Antibacterial Activity of Actinomycetes Associated with Marine Sponge
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摘要: 为了探究海南海绵共附生放线菌资源的多样性及潜在的药用价值,采用 4 种分离培养基分离海南文昌海域海绵的共附生放线菌,利用 16S rRNA序列分析对分离的放线菌进行鉴定,采用滤纸片扩散法对分离放线菌的发酵产物进行抗菌活性分析。结果表明,从4种海绵中共获得50株放线菌,所分离菌株归属于5 个亚目、6个科和 7 个属,包括链霉菌属、小单孢菌属、红球菌属、糖多孢菌、栖白蚁菌属、分枝杆菌属和Krasilnikoviella属,其中Krasilnikoviella为首次从海绵中分离得到,4株海洋放线菌为潜在的新物种。抗菌活性测试显示48%的分离菌株呈现抗细菌活性,24%的分离菌株呈现抗植物丝状病原真菌活性。本研究结果初步揭示了海南文昌海域海绵共附生放线菌的可培养物种多样性及其发酵产物的抑菌活性。Abstract: To investigate microbial diversity and potential pharmaceutical value of actinomycetes associated with marine sponges from Hainan actinomycetes associated with marine sponges from Wenchang sea area in Hainan were collected, isolated and identified, and their anti-microbial activities were analyzed. Four media were used to isolate and purify the actinomycetes from the marine sponges, and the isolates were identified according to their 16S rRNA sequences. the antibacterial activities of their fermentated products were tested using standard disk diffusion. Results showed that a total of 50 strains of actinomycetes were isolated from 4 types marine sponges. The isolates belonged to 7 genera within 6 families, 5 suborders of Actinobacteria, and contained Streptomyces, micromonospora, Rhodococcus, Saccharopolyspora, Isoptericola, Mycobacterium and Krasilnikoviella, among which Krasilnikoviella was first recorded isolated from marine sponges. Four isolates were potential novel species. The antimicrobial test showed that 48% of the total isolates displayed antibacterial activity while 24% inhibited plant filamentous pathogenic fungi. The diversity and antimicrobial activity of the fermentated products of the actinomycetes associated with marine sponges collected from Wenchang sea area in Hainan were preliminarily revealed.
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Key words:
- marine sponge /
- isolation /
- actinomycetes /
- antibacterial activity
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表 1 代表性菌株16S r RNA基因序列分析
Table 1 16S r RNA gene sequence analysis of representative strains
编号Numbering GeneBank ID 样品来源
Sample source最相似典型菌株(登录号)
The most similar typical strain (login number)相似度/%
SimilarityHNM0594 MN446727 WC−4 Isoptericolacucumis AP-38 T(KU201961) 99.14 HNM0579 MN446724 WC−20 Isoptericolananjingensis H17 T(HQ222356) 99.93 HNM0562 MN446736 WC−33 Krasilnikoviellaflava DSM 21481 T(jgi.1053042) 99.79 HNM0577 MN446737 WC−33 Krasilnikoviellamuralis T6220-5-2bT(LC148843) 99.58 HNM0617 MN446738 WC−33 Micromonosporaaurantiaca ATCC 27029T(CP002162) 99.58 HNM0608 MN446730 WC−20 Micromonosporachalcea DSM 43026 T(X92594) 99.06 HNM0616 MN446734 WC−33 Micromonosporaechinofusca DSM 43913 T(LT607733) 98.70 HNM0609 MN446731 WC−4 Micromonosporasediminicola DSM 45794T(FLRH01000004) 99.85 HNM0615 MN446733 WC−33 Micromonosporasediminis CH3-3 T(AB889541) 99.78 HNM0619 MN446735 WC−33 Micromonosporaterminaliae TMS7 T(KX394339) 99.28 HNM0581 MN446725 WC−20 Micromonosporatrujilloniae234402T(HQ123435) 99.03 HNM0611 MN446732 WC−33 Micromonosporatulbaghiae DSM 45142 T(jgi.1058868) 99.13 HNM0591 MN446726 WC−4 Mycobacterium fragaeDSM 45731T(LQOW01000023) 92.67 HNM0569 MN446720 WC−33 Rhodococcusgannanensis M1T(KX887333) 98.05 HNM0563 MN446717 WC−33 Rhodococcusphenolicus DSM 44812 T(LRRH01000094) 98.89 HNM0595 MN446728 WC−4 Saccharopolysporaindica VRC122 T(JX411621) 99.28 HNM0578 MN446723 WC−33 Streptomyces araujoniaeASBV-1T (EU792889) 99.50 HNM0565 MN446718 WC−33 Streptomyces diastaticusNRRL B-1773 T(DQ026631) 99.28 HNM0575 MN446722 WC−33 Streptomyces nanshensis SCSIO 01066T(EU589334) 98.78 HNM0596 MN446729 WC−24 Streptomyces plicatus NBRC 13071 T(AB184291) 99.64 HNM0568 MN446719 WC−33 Streptomyces sedi YIM 65188 T(EU925562) 99.30 HNM0574 MN446721 WC−33 Streptomyces xishensis YIM M 10378 T(HQ585118) 97.44 表 2 4种海绵样品上的共附生放线菌分布
Table 2 Distribution of symbiotic actinomycetes on 4 sponge samples
放线菌种属
Species of actinomycetes放线菌数量/株
Number of actinomycetes海绵 WC−20 海绵 WC−24 海绵 WC−33 海绵 WC−4 Isoptericola sp - - 2 - Krasilnikoviella sp - - 7 - Micromonospora sp 5 1 1 11 Mycobacterium sp - - 1 - Rhodococcus sp - - 2 - Saccharopolyspora sp - - 1 - Streptomyces sp - 2 12 5 分离菌株数(种类)
Strains (species)5(1) 3(2) 26(7) 16(2) 表 3 海洋放线菌抗细菌和抗真菌活性分布
Table 3 Distribution of antibacterial activity of marine actinomycetes
分类
Classification总菌数/株
Total bacterial count抗细菌菌数/株
Antibacterial strains抗真菌菌数/株
Antifungal strains活性菌数(比例)/株
Active strainC1 C2 C3 C4 Z1 Z2 Z3 Z4 Z5 Z6 细菌 真菌 Isoptericola 2 1 1 1 - 0 0 0 0 1 0 2(100%) 1(50%) Krasilnikoviella 7 0 2 3 4 0 0 0 0 0 0 5(71.43%) 0 Micromonospora 18 0 3 3 5 1 0 0 0 1 1 6(33.33%) 1(5.56%) Mycobacterium 1 0 0 0 0 0 0 0 0 0 0 0 0 Rhodococcus 2 0 0 0 0 0 0 0 0 0 0 0 0 Saccharopolyspora 1 0 0 0 1 1 0 1 1 0 0 1(100%) 1(100%) Streptomyces 19 1 8 4 6 4 3 4 4 8 6 10(52.63%) 9(47.37%) 总计(比例) 50 2(4%) 14(28%) 11(22%) 16(32%) 6(12%) 3(6%) 5(10%) 5(10%) 10(20%) 7(14%) 24(48%) 12(24%) 注:C1:大肠杆菌,C2:金黄色葡萄球菌,C3:耐甲氧西林金黄色葡萄球菌,C4:无乳链球菌。Z1.香蕉枯萎病菌;Z2.芒果炭疽病菌;Z3.水稻稻瘟病菌;Z4.木薯根腐病菌;Z5.薯蓣炭疽病菌;Z6.橡胶炭疽病菌。
Note: C1: Escherichia coli; C2: Staphylococcus aureus; C3: Methicillin-resistant Staphylococcus aureus; C4: Streptococcus agalactiae. Z1. Fusarium oxysporum;Z2.Colletotrichum asianum; Z3. Magnaporthe grisea; Z4. Lasiodiplodia theobromae; Z5.Colletotrichum gloeosporioides; Z6.Colletotrichum gloeosporioides. -
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