Development of a systematic feedback isolation approach for targeted strains from mixed culture systems
Development of a systematic feedback isolation approach for targeted strains from mixed culture systems
复制标题
DOI：
10.1016/j.jbiosc.2016.07.019
复制
发表时间：
2017-01-01
影响因子：
2.8
通讯作者：
Sakai, Kenji
中科院分区：
文献类型：
--
作者：
Poudel, Pramod;Tashiro, Yukihiro;Sakai, Kenji
混合培养系统(MCS)如堆肥、活性污泥系统中细菌的功能很难阐明，因为该系统中有许多未分离的细菌。在这里，我们开发了一种系统的反馈分离策略，用于从MCS中分离和快速筛选多个靶标菌株。利用变性梯度凝胶电泳法对L-乳酸生产菌种进行了变性梯度凝胶电泳法检测，得到6株主要菌株(华氏柯里新杆菌、热阴沟芽孢杆菌、嗜热淀粉芽孢杆菌、史密斯芽孢杆菌、腐生芽孢杆菌和凝结芽孢杆菌)。根据文献中的适宜培养条件(如培养基数、pH值、温度)，进行反馈分离，形成136个菌落。对直接菌落基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)进行了优化，作为第二次筛选，从与6个最接近的模式菌株的相似谱型中筛选出20个候选菌落。这一步骤可以在物种水平上区分细菌，距离相似性得分>；=0.55，对应于16S rRNA基因序列相似性>；=98.2%，表明这是一种有效的技术，可以最大限度地减少菌株与目标类型菌株的距离。16S rRNA基因序列分析表明，已成功分离到2株目标菌株和1株与目标菌株相关的菌株，与DGGE条带序列具有较高的相似性(99.5%~100%)，其余候选菌株与3株与目标物种密切相关的菌株有亲缘关系。本研究提出了一种新的系统反馈分离方法，可用于从MCS中分离目标菌株以供进一步研究。(C)2016年，日本生物技术学会。版权所有。
Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Cory-nebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for L-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores >= 0.55 corresponding to 16S rRNA gene sequence similarity >= 98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.