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Isolation and Use of Lytic Bacteria against Nitrogen-Fixing Cyanobacteria for Protoplast Preparation

固氮蓝藻裂解菌的分离及其在原生质体制备中的应用

基本信息

批准号：
62440013
负责人：
KAI Hideaki
金额：
$ 6.02万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62440013/
关键词：
Microbial nitrogen fixation Cyanobacteria(Blue-green algae)Lytic bacteria Lytic enzyme Flexithrix sp.Arthrobacter sp.Bacillus sp.プロトプラスト Azolla Anabaena azollae ラン藻類 Flexithrix sp 分離プラーク

项目摘要

The ability to reduce the dinitrogen molecule to ammonia is restricted to prokaryotes where it is relatively scarce but spread widely through the systematic groups. Among these organisms, a prominent role is occupied by the cyanobacteria. They fix N_2 both in the free-living state and in symbiosis with a wide range of partners, and they are important contributors to the global supply of fixed nitrogen, including use in agriculture as a source of "green fertilizer."The screening and isolation of microorganisms capable of lysing cyanobacteria were performed on Dermocapra sp. and Anabaena cylindrica with the conventional double-agar overlay technique. Lysed zones were caused by dissolution of the cyanobacteria after digestion of cell walls by the lytic nicroorganisms.All of the single-colony isolations were finally retested on medium containing cyanobacteria whole cells, after the microorganisms had been examined for the presence of contamination on several different media. By this procedure, six strains of bacteria were iso;ated from paddy and upland soil samples, of which bacterium CL-101, CL-105 and CL-106 showed superior lytic activity.Morphological and physiological properties of the three strains were examined and identified as Flexithrix sp., Arthrobacter sp. and Bacillus sp., respectively.Protoplasts were obtained from Anabaena cyrindrica and Anabaena variabilis by using lytic enzyme from Bacillus CL-106. Several buffer sustems and osmotic agents were tried for effectiveness.Sucrose-Tris HCl buffer was finally chosen for the incubation period for the enzyme digestion. The MDM was added as stabilizers.

将二氮分子还原为氨的能力仅限于原核生物，在那里它相对稀缺，但在系统群中广泛传播。在这些生物中，一个突出的作用是由蓝藻占领。它们在自由生活状态和与广泛的伙伴共生中固定N_2，它们是全球固定氮供应的重要贡献者，包括在农业中作为“绿色肥料”的来源。“用常规的双琼脂覆盖技术在Dermocapra sp.和Anabaena cylindrica上筛选和分离能够裂解蓝细菌的微生物。溶解区是由溶解性微生物消化细胞壁后蓝细菌溶解造成的。在几种不同培养基上检查微生物是否存在污染后，最后在含有蓝细菌全细胞的培养基上对所有单菌落分离物进行重新测试。从水稻土和高地土壤中分离到6株细菌，其中CL-101、CL-105和CL-106具有较上级的溶菌活性，经形态学和生理学鉴定，它们均为嗜热菌属（Atomthrix sp.）节杆菌属和芽孢杆菌属，分别用芽孢杆菌CL-106的裂解酶从鱼腥藻和变异鱼腥藻中获得原生质体。对几种缓冲液和渗透剂进行了试验，最后选择蔗糖-Tris HCl缓冲液作为酶消化的孵育期。添加MDM作为稳定剂。