2020, Vol. 47扩展功能
文章信息
- 荆瑞勇, 王丽艳, 孙强, 刘俊杰, 刘居东, 金剑, 刘晓冰, 王光华
- JING Rui-Yong, WANG Li-Yan, SUN Qiang, LIU Jun-Jie, LIU Ju-Dong, JIN Jian, LIU Xiao-Bing, WANG Guang-Hua
- 东北旱田土壤中Anabaena伴生细菌的分离与鉴定
- Isolation and identification of bacteria associated with Anabaena from upland soils in northeast China
- 微生物学通报, 2020, 47(1): 130-139
- Microbiology China, 2020, 47(1): 130-139
- DOI: 10.13344/j.microbiol.china.190209
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文章历史
- 收稿日期: 2019-03-16
- 接受日期: 2019-07-12
- 网络首发日期: 2019-09-09
2. 黑龙江八一农垦大学生命科学技术学院 黑龙江 大庆 163319
2. College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China
蓝细菌作为生态食物链中的初级生产者和被捕食者在自然环境中广泛存在[1]。蓝细菌在自然环境中总与一些细菌伴生，这些伴生细菌与蓝细菌关系密切，引起了学术界的关注[2-5]。目前研究发现，伴生细菌可提高或降低蓝细菌的生长[6-7]、改变蓝细菌代谢分泌物(包括蓝细菌毒素)的质量[2, 4]、矿化蓝细菌代谢分泌物(包括毒素的降解或产生)[3, 8-9]、抵御重金属污染和被捕食[10]、促进蓝细菌的聚集[11]、增强蓝细菌的光合活性[5, 11]、提高蓝细菌生长所需的CO2浓度[12]、提供蓝细菌生长所需的生长因子[13]，为蓝细菌的生存提供良好的微环境。蓝细菌对伴生细菌生长也具有重要作用，其分泌有机物为伴生细菌提供具高浓度有机营养的微环境[14-15]，促进能降解蓝细菌毒素的伴生细菌生长[16]，改变滞留在不同深度水环境中具有气囊蓝细菌的伴生细菌群落[17]。伴生细菌对蓝细菌既有有利方面，如保护蓝细菌免受噬菌体侵染[18]，也有些伴生细菌具有杀藻作用[19]，对控制具有潜在毒性的蓝细菌暴发具有重要作用[2]。
伴生细菌数量和多样性与蓝细菌丰度也存在一定关系。在蓝细菌对数生长期，其伴生菌的生长较慢，多样性丰富；而在蓝细菌的稳定生长期时，其伴生菌的生长快，多样性降低[5]。环境因子(如温度和营养[2]、溶氧和pH值[4])也影响着蓝细菌及其伴生菌的群落相互关系。蓝细菌伴生菌中除了细菌外，蓝细菌的伴生菌也包括真菌和古菌[20]。Dziallas等[4]采用PCR-DGGE方法调查了3株蓝细菌的伴生菌，测序结果发现游离细菌类群主要分布在β-变形菌纲(Betaproteobacteria)，而蓝细菌的伴生菌总有一些分布在γ-变形菌纲(Gammaproteobacteria)；游离细菌和蓝细菌的伴生细菌在拟杆菌门(Bacteriodetes)和α-变形菌纲(Alphaproteobacteria)均有分布。芬兰Joutikas湖泊水华中主要蓝细菌以鱼腥藻(Anabaena)和束丝囊(Aphanizomenon)为主，最丰富的类群异养细菌群落是疣微菌门(Verrucomicrobia)细菌，而生物量最大细菌主要分布在δ-变形菌纲(Deltaproteobacteria)和噬细胞菌属-黄杆菌属-拟杆菌属类群(CFB类群，Cytophaga-Flavobacterium-Bacteroides)中[21]。溶解蓝细菌细胞的伴生细菌如噬纤维细菌(Cytophagaceae)[22]在水华后期广泛存在。以上研究主要针对水体中蓝细菌伴生细菌的研究。
蓝细菌分布广泛[23]，土壤中伴生细菌与蓝细菌的关系是否类似于海鞘与蓝细菌的共生关系[24]，还是类似于溶藻细菌与藻类的捕杀关系[25]，相关研究鲜有报道。为了探索同一株蓝细菌在不同旱田土壤中的伴生菌系统发育地位，本文拟以模式菌株鱼腥藻(Anabaena sp. PCC7120)为诱饵，在室内培养条件下置于不同土壤中混合培养Anabaena sp. PCC7120，培养一段时间后再分离纯化Anabaena sp. PCC7120，难以除去的细菌被视为鱼腥藻的伴生细菌，通过伴生细菌的富集培养，提取其DNA，经PCR扩增、克隆测序及PCR-DGGE分子物学手段，明确在不同旱地土壤中Anabaena sp. PCC7120伴生细菌的种类，为研究土壤中蓝细菌与其伴生细菌间相关系提供数据支持。
1 材料与方法 1.1 材料蓝细菌菌株Anabaena sp. PCC7120模式菌株保藏在本实验室；供试土样来自东北旱田黑土，其基本信息见表 1。
| 样品 Sample |
采样地点 Sampling site |
经纬度 Longitude and latitude |
作物 Crop |
pH值 pH value |
总碳 Total carbon (g/kg) |
总N Total nitrogen (g/kg) |
含水量 Water content (%) |
| 1 | 双城，黑龙江Shuangcheng, Heilongjiang | 45°23′N, 126°22′E | Corn | 6.53 | 17.02 | 1.68 | 23 |
| 2 | 扶余，吉林Fuyu, Jilin | 45°06′N, 126°11′E | Corn | 5.78 | 19.97 | 2.03 | 18 |
| 3 | 德惠，吉林Dehui, Jilin | 44°12′N, 125°33′E | Corn | 4.79 | 17.45 | 1.44 | 22 |
| 4 | 公主岭，吉林Gongzhuling, Jilin | 43°26′N, 124°43′E | Corn | 5.50 | 14.40 | 1.12 | 23 |
| 5 | 昌图，辽宁Changtu, Liaoning | 43°05′N, 124°20′E | Corn | 5.46 | 14.58 | 1.02 | 21 |
| 6 | 哈尔滨，黑龙江Harbin, Heilongjiang | 45°41′N, 126°38′E | Soybean | 6.57 | 26.36 | 1.69 | 23 |
| 7 | 呼兰，黑龙江Hulan, Heilongjiang | 46°06′N, 127°02′E | Corn | 5.18 | 19.76 | 1.42 | 22 |
| 8 | 绥棱，黑龙江Suiling, Heilongjiang | 47°13′N, 127°07′E | Corn | 5.19 | 27.07 | 1.90 | 30 |
| 9 | 拜泉，黑龙江Baiquan, Heilongjiang | 47°35′N, 126°07′E | Corn | 4.98 | 23.41 | 1.95 | 26 |
| 10 | 克东，黑龙江Kedong, Heilongjiang | 48°09′N, 126°13′E | Soybean | 5.41 | 32.03 | 2.45 | 24 |
| 11 | 北安，黑龙江Beian, Heilongjiang | 48°09′N, 126°43′E | Soybean | 6.10 | 53.53 | 4.25 | 40 |
| 12 | 五大连池，黑龙江Wudalianchi, Heilongjiang | 48°52′N, 126°08′E | Soybean | 5.39 | 36.76 | 3.06 | 35 |
| 13 | 嫩江1，黑龙江Nenjiang1, Heilongjiang | 49°08′N, 125°37′E | Corn | 5.53 | 31.71 | 2.50 | 28 |
| 14 | 嫩江2，黑龙江Nenjiang 2, Heilongjiang | 49°26′N, 125°26′E | Corn | 5.17 | 37.23 | 2.96 | 33 |
| 注：样品列的数字代表着土壤代号，下同. Note: The numbers below the sample column indicate different soil. The same as below. |
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BG-11培养基(g/L)：NaNO3 1.500，K2HPO4·3H2O 0.040，MgSO4·7H2O 0.075，CaCl2·2H2O 0.036，柠檬酸铁铵0.006，EDTA 0.001，Na2CO3 0.001，微量元素溶液1 mL。微量元素溶液(g/L)：H3BO3 2.860，MnCl2·4H2O 1.810，NaMoO4·5H2O 0.390，CuSO5·5H2O 0.079，ZnSO4·7H2O 0.222，CoCl2·6H2O 0.050。
1.2 试验设计将用BG-11液体培养基培养10 d的Anabaena sp. PCC7120悬液5 mL分别接种于装有1 g不同地点土样和5 mL无菌水的试管中，充分混匀置于光照培养箱25 ℃培养。光周期L:D=14:10，培养2周。当Anabaena sp. PCC7120菌液变为深绿色，用接种环挑取一环菌液于选择性BG-11固体培养基(含放线菌酮100 mg/L)平板上划线分离Anabaena sp. PCC7120。固体平板置于光照培养箱培养10 d时，挑取单个蓝细菌菌落进一步纯化。纯化4次后，将平板上Anabaena sp. PCC7120及其伴生细菌用10 mL无菌水冲洗，收集于无菌试管混匀，取1 mL菌液，在无菌条件下接入装有5 mL已灭菌的液体牛肉膏蛋白胨培养基的试管中，同时设无菌水为对照，置于30 ℃、160 r/min的摇床，在黑暗条件下培养2 d，对照组试管仍为清澈，处理组变混浊。取1 mL浑浊的菌悬液在15 000 r/min离心机中离心10 min，沉淀部分提取菌体DNA。以菌体DNA为模板，采用27F/1492R细菌通用引物进行PCR扩增，PCR产物目的条带经胶回收纯化后克隆，以阳性克隆的菌液为模板，GC-357F/517R引物进行PCR扩增，其PCR产物经DGGE电泳，将不同位置条带的阳性克隆菌液送测序公司测序，系统进化分析初步确定伴生细菌的归属。
1.3 主要试剂和仪器所有生化试剂、QIAquick胶回收试剂盒均购自大连宝生物工程有限公司。16S rRNA基因扩增的引物和基因测序由深圳华大基因股份有限公司完成。PCR仪、凝胶成像系统和DGGE电泳仪，Bio-Rad公司；元素分析仪，艾力蒙塔公司。
1.4 菌液DNA的摄取取1 mL蓝细菌伴生菌悬液置于2 mL离心管，15 000 r/min离心10 min，用无菌水洗涤菌沉淀3次，沉淀中加入1 mL TE buffer和终浓度为20 μg/mL的RNase A，离心管涡旋混匀后置于37 ℃温育2 h，每30 min涡旋1次；在无菌条件下移入装有不同粒径玻璃珠(1 mm׃0.5 mm׃0.1 mm=1׃1׃1)的无菌小管中，Bead-beating 15 s；离心管中再加入30 μL 10% SDS、30 μL 10 mg/mL蛋白酶K、20 μL 50 mg/mL溶菌酶，继续在37 ℃温育2 h；离心管中再加入100 μL 5 moL/L NaCl、80 μL 2% CTAB溶液，混匀后65 ℃温育10 min；最后分别采用720 μL PCI (酚׃氯仿׃异戊醇=25׃24׃1，体积比)和720 μL CIA (氯仿/异戊醇=24׃1，体积比)在15 000 r/min离心5 min条件下脱蛋白；采用440 μL异丙醇冰浴30 min析出DNA，离心收集DNA沉淀，用70%乙醇涡旋洗涤DNA，离心去除乙醇；沉淀的DNA用50 μL TE buffer溶解，4 ℃保存备用。
1.5 伴生菌PCR扩增细菌的16S rRNA基因采用引物27F (5′-AGA GTTTGATCCTGGCTCAG-3′)和1492R (5′-GGTTA CCTTGTTACGACTT-3′)进行PCR扩增。50 μL的PCR反应体系中包含上、下游引物(50 pmol/L)各0.5 μL，DNA模板1−2 μL，dNTPs (2.5 mmol/L) 5 μL，rTaq Buffer 5 μL，rTaq酶合酶(5 U/μL) 0.5 μL，加入无菌水至终体积。同时设无模板的阴性对照。PCR反应条件：94 ℃ 5 min；94 ℃ 1 min，55 ℃ 1 min，72 ℃ 1 min，35个循环；72 ℃ 10 min。
1.6 伴生细菌阳性克隆的DGGE检测对蓝细菌及其伴生菌16S rRNA基因PCR产物，从2%琼脂糖凝胶上切取约1 500 bp大小的DNA片段，将QIAquick胶回收试剂盒纯化后的DNA片段连接至pMD18-T载体，转入大肠杆菌DH5α感受态细胞中。每个处理的转化平板挑取50个白色单菌落，再用RV-M/M13-47引物进行PCR扩增来检测阳性克隆。PCR条件同伴生菌PCR扩增，循环数降至25。将检测获得的阳性克隆菌液采用GC-357F (5′-CGCCCGCCGCGCCCCGCG CCCGGCCCGCCGCCCCCGCCCCCCTACGGGAGGCAGCAG-3′)和517R (5′-ATTACCGCGGCTGC TGG-3′)引物进行PCR扩增。15 μL的PCR反应体系包含上、下游引物(50 pmol/L)各0.15 μL，0.3 μL菌液作为模板，dNTPs (2.5 mmol/L) 1.5 μL，rTaq Buffer 1.5 μL，rTaq酶(5 U/μL) 0.15 μL，补足ddH2O至终体积。设无模板的阴性对照。6 μL PCR产物用于DGGE凝胶电泳检测。DGGE变性梯度为30%−70%。DGGE凝胶图谱中不同位置相对应的阳性克隆菌液培养过夜后送至测序公司测序。
1.7 不同样品伴生细菌的DGGE检测以不同样品中蓝细菌及其伴生细菌16S rRNA基因PCR产物为模板，GC-357F和517R为引物进行PCR扩增，PCR反应体系(15 μL)：上、下游引物(50 pmol/L)各0.15 μL，模板0.3 μL，dNTPs (2.5 mmol/L) 1.5 μL，rTaq Buffer 1.5 μL，rTaq酶(5 U/μL) 0.15 μL，补ddH2O至终体积。设无模板为阴性对照。6 μL PCR产物用于DGGE凝胶电泳检测。DGGE变性梯度为30%−70%。
1.8 伴生细菌的系统发育分析在NCBI网站(http://www.ncbi.nlm.nih.gov/)对蓝细菌伴生细菌16S rRNA基因最近的亲缘菌进行BLAST比对。将其核苷酸序列用ClustalX 1.81进行同源性分析。采用MEGA 4.0软件构建Neighbor-Joining进化树。本文获得Anabaena伴生细菌16S rRNA基因GenBank登录号为MK684309−MK684345。
2 结果与分析 2.1 供试土样中Anabaena sp. PCC7120伴生菌16S rRNA基因的获得供试土壤中Anabaena sp. PCC7120的伴生细菌是指在光照条件下培养Anabaena sp. PCC7120和土样的混合液，待Anabaena sp. PCC7120菌株生长后，通过平板连续分离纯化不能去除的细菌，采用NA培养基获得的细菌均被认为是伴生细菌。获得的细菌菌液通过提取其DNA，对16S rRNA基因进行PCR扩增(图 1A)，将每个土壤样品的PCR产物约1 500 bp条带胶纯化回收，进行TA克隆，每个处理中阳性克隆检测时在1 600 bp位置有条带被认为是连接成功(图 1B)，再将每个阳性克隆以GC-357F/517R进行巢式PCR扩增(图 1C)，扩增获得250 bp大小的DNA片段，将每个阳性克隆均进行DGGE检测，若图 1D中DGGE条带均具有一条相同条带(箭头所示)，被认为是大肠杆菌感受态细胞的条带，在相同处理的DGGE图谱中，选取条带位置不同的阳性克隆，将其菌液送至测序公司测序。
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| 图 1 不同土样中Anabaena sp. PCC7120伴生菌的分子生物学鉴定 Figure 1 Molecular identification of different associated bacteria of Anabaena sp. PCC7120 from different soil samples 注：A−D分别为PCR扩增、阳性克隆检测、阳性克隆巢式PCR扩增和DGGE检测.图中数字1−10分别代表土样1−10，下同. Note: A−D indicate PCR amplification, positive clone detection, nest PCR amplification of positive clone and DGGE detection, respectively. The number 1 to 10 indicates the soil sample 1 to 10, respectively, the same as below. |
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以每个样品采用27F/1492R引物PCR扩增获得产物为模板，再以GC-357F/517R进行巢式PCR，获得的PCR产物直接进行DGGE检测，从图 2可见，土样1–14中Anabaena sp. PCC7120伴生细菌种类较多，土样7中伴生细菌种类最多，呈现出8条带，其他处理处理均有3−7条带，表明不同土壤中Anabaena sp. PCC7120伴生细菌种类存在较大的差异。
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| 图 2 不同土壤中Anabaena sp. PCC7120伴生细菌DGGE图谱 Figure 2 DGGE profile of Anabaena sp. PCC7120 associated bacteria from different soils |
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克隆测序获得37条Anabaena伴生细菌的16S rRNA基因序列，经BLAST比对(表 2)，36条伴生细菌与已知细菌相似性均为98%−99%，只有克隆11-1与已知细菌Flavobacterium xinjiangense相似性为94%。相似性高的36条序列主要分布在贪噬菌属(Variovorax)和黄杆菌属(Flavobacterium)，各占7条，Sphingopyxis占6条，红球菌属(Rhodococcus)和新鞘脂菌属(Novosphingobium)各3条，无色菌属(Achromobacter)、假单胞菌属(Pseudomonas)、类芽孢菌属(Paenibacillus)、Xenophilus和Dyadobacter各有2条，假黄色单胞菌属(Pseudoxanthomonas)占1条。相似性在97%以下一株伴生细菌与Flavobacterium属亲缘关系最近。
| 克隆名 Clones |
最近亲缘细菌 Closest relative bacteria |
GenBank登录号 GenBank accession No. |
相似性 Similarity (%) |
对齐 Alignment |
来源 Sources |
| 1-1 | Sphingopyxis chilensis S37 | NR_024631 | 99 | 1 401/1 411 | [26] |
| 1-2 | Variovorax paradoxus EPS | NR_074646 | 99 | 1 454/1 457 | Lucas et al., 2013 unpublished |
| 2-1 | Rhodococcus qingshengii djl-6 | NR_043535 | 99 | 1 438/1 440 | [27] |
| 2-2 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 419/1 436 | [28] |
| 2-3 | Rhodococcus qingshengii djl-6 | NR_043535 | 99 | 1 437/1 440 | [27] |
| 3-1 | Rhodococcus erythropolis PR4 | NR_074622 | 99 | 1 438/1 440 | [29] |
| 4-1 | Sphingopyxis chilensis S37 | NR_024631 | 99 | 1 398/1 411 | [26] |
| 4-2 | Sphingopyxis chilensis S37 | NR_024631 | 99 | 1 397/1 411 | [26] |
| 5-1 | Achromobacter xylosoxidans A8 | NR_074754 | 99 | 1 440/1 453 | [30] |
| 6-1 | Dyadobacter fermentans DSM 18053 | NR_074368 | 99 | 1 408/1 429 | [31] |
| 6-2 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 420/1 436 | [28] |
| 7-1 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 420/1 436 | [28] |
| 7-2 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 421/1 436 | [28] |
| 7-3 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 427/1 436 | [28] |
| 8-1 | Pseudomonas aeruginosa PAO1 | NR_074828 | 99 | 1 453/1 459 | [32] |
| 8-2 | Pseudomonas aeruginosa PAO1 | NR_074828 | 99 | 1 452/1 458 | [32] |
| 9-1 | Paenibacillus agaridevorans DSM 1355 | NR_025490 | 98 | 1 444/1 481 | [33] |
| 9-2 | Variovorax paradoxus EPS | NR_074646 | 99 | 1 456/1 457 | Lucas et al., 2013 unpulished |
| 9-3 | Sphingopyxis ginsengisoli Gsoil 250 | NR_041366 | 98 | 1 389/1 411 | [34] |
| 9-4 | Sphingopyxis chilensis S37 | NR_024631 | 99 | 1 402/1 411 | [26] |
| 10-1 | Dyadobacter fermentans DSM 18053 | NR_074368 | 98 | 1 405/1 429 | [31] |
| 10-2 | Variovorax paradoxus EPS | NR_074646 | 99 | 1 456/1 457 | Lucas et al., 2013 unpulished |
| 10-3 | Variovorax paradoxus EPS | NR_074646 | 99 | 1 455/1 457 | Lucas et al., 2013 unpulished |
| 10-4 | Sphingopyxis chilensis S37 | NR_024631 | 99 | 1 405/1 411 | [26] |
| 11-1 | Flavobacterium xinjiangense AS 1.2749 | NR_025201 | 94 | 1 347/1 438 | [35] |
| 11-2 | Novosphingobium resinovorum NCIMB 8767 | NR_044045 | 99 | 1 372/1 373 | [36] |
| 11-3 | Xenophilusa-ovorans KF46F | NR_025114 | 98 | 1 426/1 455 | [37] |
| 11-4 | Pseudoxanthomonas mexicana AMX 26B | NR_025105 | 99 | 1 447/1 467 | [38] |
| 11-5 | Xenophilusa-ovorans KF46F | NR_025114 | 98 | 1 426/1 455 | [37] |
| 11-6 | Novosphingobium resinovorum NCIMB 8767 | NR_044045 | 99 | 1 372/1 373 | [36] |
| 11-7 | Novosphingobium resinovorum NCIMB 8767 | NR_044045 | 99 | 1 372/1 373 | [36] |
| 12-1 | Variovorax boronicumulans BAM-48 | NR_041588 | 99 | 1 453/1 455 | [39] |
| 12-2 | Variovorax paradoxus EPS | NR_074646 | 99 | 1 454/1 457 | Lucas et al. 2013 unpublished |
| 12-3 | Flavobacterium johnsoniae UW101 | NR_074455 | 99 | 1 421/1 436 | [28] |
| 13-1 | Variovorax paradoxus S110 | NR_074654 | 99 | 1 448/1 457 | Lucas et al., 2013 unpublished |
| 13-2 | Paenibacillus validus JCM 9077 | NR_040892 | 99 | 1 454/1 458 | [40] |
| 14-1 | Achromobacter xylosoxidans A8 | NR_074754 | 99 | 1 449/1 453 | [30] |
| 注：表中克隆名前面的数字为土样编号，后面的数字为克隆序号. Note: Numbers in the front of clones indicate soil serial number, the following numbers indicate clone serial number in the table. |
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将获得的伴生细菌16S rRNA基因序列与相近同源细菌序列构建系统树，由图 3可知，这37株伴生细菌分布在4个门中，变形菌门(Protebacteria) (22条)占总数的59.5%；拟杆菌门(Bacteroidetes) (9条)占总数的24.3%；厚壁菌门(Firmicutes) (3条)和放线菌门(Actinobacteria) (3条)各占总数的8.1%。在Protebacteria中，α-Protebacteria占9条，β-Protebacteria占10条，γ-Protebacteria占3条。从进化树上可知，相似性较低的克隆11-1形成独立的分支，初步确定该克隆相对应的菌株为新的菌株，其余36条伴生菌的克隆序列均与相似性最高的菌株处于同一进化分支。
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| 图 3 基于16S rRNA基因的不同土样中Anabaena伴生菌系统进化分析 Figure 3 Phylogenetic tree of Anabaena associated bacteria from different soils based on 16S rRNA gene 注：进化树中分支点上数字代表Bootstrap值，Bootstrap值< 50未显示，括号中的序号代表相应菌株在NCBI上的核酸序列登录号；标尺代表每个核苷酸替代物残基的数量. Note: The numbers of branching points in phylogenetic tree indicate bootstrap value, bootstrap values < 50 are not shown; Serial number in bracket indicates nucleic acid sequence landing number of corresponding strain in NCBI website; The scale bar represents the number of nucleotide substitutions per residue. |
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本研究以Anabaena sp. PCC7120菌株为材料，对不同旱田土壤的该蓝细菌伴生菌进行调查，共得到37条不同的Anabaena sp. PCC7120伴生细菌，根据16S rRNA基因序列相似性大于95%的菌株可鉴定到属水平[41]，也有研究认为97%以上可鉴定到属水平[42]。核苷酸序列相似性在98.7%−99%的菌株间认为是种间水平[43]。本研究获得的伴生菌中有1株不能明确其在属水平上的归属，其余36株伴生细菌可确定至属水平甚至种间水平。从门水平来看，获得的Anabaena伴生细菌主要分布在变形菌门(Proteobacteria)，其次是拟杆菌门(Bacteroidetes)，放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)也有分布，这与湖水中伴生细菌相似。Berg等[44]从湖水中分离获得的460株异养细菌中，基于16S rRNA基因序列的系统进化分析发现，这些异养细菌分布在变形菌门、拟杆菌门、放线菌门、厚壁菌门和异常球菌-栖热菌类群(Deinococcus-Thermus)中，这些异养细菌有些是潜在的病原菌，有些抑制或提高蓝细菌的生长，大部分异养细菌可促进蓝细菌的生长。而本研究从旱田土壤中获得的Anabaena sp. PCC7120伴生细菌中未发现潜在的病原菌株。
在属水平上，由构建的系统进化树可见，不同土壤中Anabaena伴生细菌的16S rRNA基因序列与鞘氨醇盒菌属(Sphingopyxis) (6条)、贪噬菌属(Variovorax) (6条)、黄杆菌属(Flavobacterium) (5条)和红球菌属(Rhodococcus) (3条)的近亲缘性菌株最多。Sphingopyxis alaskensis RB2256菌株在海洋寡营养环境中异养生长[45]，该株菌也可在土壤环境中生长；Sphingopyxis chilensis S37菌株能降解氯酚[26]；Sphingopyxis ginsengisoli Gsoil 250菌株分离自人参田[34]，该菌株分布广泛且能在贫营养环境下降解芳香类化合物。Sphingopyxis和Sphingomonas、Sphingobium、Novosphingbium是从系统发育、化学分类及表型特征方面分化出的4个属。因Novosphingbium resinovorum系统进化地位与Sphingopyxis相近，于2007年由Flavobacterium resinovorum修订成Novosphingbium resinovorum[36]，该类菌可降解异类生物质[46]。Variovorax boronicumulans是一株分离自土壤可富集硼的细菌[39]，Variovorax paradoxus属于有机营养型兼性无机化能营养型，可分泌胞外多糖[4]，与Xenophilus azovorans在分类学上隶属于丛毛单孢菌科(Comamonaduceae)[37]。噬细胞菌属/黄杆菌属(Cytophaga/Flavobacterium)和δ变形菌纲细菌(δ-Proteobacteria)与湖泊中微囊藻浓度同步变化，该类细菌具有降解微囊藻细胞分泌的大分子，可清除水体的毒素[9]。红球菌属(Rhodococcus)可降解醇类和烷类化合物[48]，也是贫营养环境中常被检测到的蓝细菌伴生细菌[1]。本研究分离鉴定的蓝细菌伴生细菌还包含假黄色单胞菌(Pseudoxanthomonas)和可降解废水中的苯环类化合物假单胞菌(Pseudomonas)[8]等。蓝细菌菌株的类群也影响着其伴生菌的类群，同一种蓝细菌的不同菌株其伴生细菌的类群更近些[49]，而本研究调查了Anabaena sp. PCC7120在不同农田中的伴生细菌，通过PCR克隆测序，获得的伴生细菌推测其具有分布广、耐受贫营养、提供微量元素、清除蓝细菌毒素等作用，具体机理有待进一步研究。
分离蓝细菌伴生菌的方法不同也可能造成伴生菌分离菌株的差异，如使用不同配方的培养基。本研究用的BG-11培养基含有NO3−作为氮源、Na2CO3为碳源，主要培养自养细菌，我们推测生长在BG-11培养基上的蓝细菌产生的分泌物可作为伴生细菌的有机碳源，伴生菌的生长也可提供蓝细菌生长所需的微量元素或消除蓝细菌生长产生的有毒物质。Berg等[44]在分离蓝细菌伴生菌时采用了5种培养基(Z8、R2A、CYA、TOX及BA)，每种培养基针对的角度不同，可获得更多蓝细菌伴生菌的信息。比如，培养基配方中磷含量影响到伴生细菌的种类[11]。此外，本文采用PCR克隆测序的方法获得蓝细菌伴生菌的16S rRNA基因全长序列，获得的一些序列是嵌合体或由于PCR扩增引起错配的非细菌源序列，这些序列已被剔除。采用PCR-DGGE图谱主要有两种用途：(1)利用GC-357F/517R引物PCR扩增16S rRNA基因全长序列的阳性克隆子，PCR产物经DGGE图谱分析，同一样品多个阳性克隆在DGGE图谱中条带位置相同被认为是相同的克隆子，反之对应的阳性克隆子被认为是不同的克隆子，将不同的阳性克隆子对应的菌液进行16S rRNA基因测序；(2)每个土样DNA先采用27F/1492R引物PCR扩增，以其PCR产物为模板，再用GC-357F/517R引物进行巢式PCR扩增，获得的PCR产物用于DGGE图谱分析，可以直观地看到不同土样Anabaena伴生细菌对应的条带数，同时也为阳性克隆子的测序数量提供参考。文中采用相同蓝细菌菌株在不同土壤中获得的伴生细菌，其种属分布与土壤理化性质没有直接关系，有待下一步深入探讨。
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