来源于Micrococcus luteus的复苏促进因子异源表达及其对活性污泥中休眠细菌的促生长作用  

Heterologous expression of resuscitation promoting factor from Micrococcus luteus and promotes growth dormant bacteria from activated sludge

作　　者：张宇[1] 王慧荣[1] 梅荣武[1] 李明智[1] 李欲如[1] 丁林贤[2] 苏晓梅 朱瑞芬 ZHANG Yu;WANG Hui-Rong;MEI Rong-Wu;LI Ming-Zhi;LI Yu-Ru;DING Lin-Xian;SU Xiao-Mei;ZHU Rui-Fen(Environmental Science Research and Design Institute of Zhejiang Province,Hangzhou,Zhejiang 310007,China;College of Geography and Environmental Sciences,Zhejiang Normal University,Jinhua,Zhejiang 321004,China;Zhejiang Environment Technology Co.Ltd.,Hangzhou,Zhejiang 311121,China)

机构地区：[1]浙江省环境保护科学设计研究院,浙江杭州310007 [2]浙江师范大学地理与环境科学学院,浙江金华321004 [3]浙江省环境科技有限公司,浙江杭州311121

出　　处：《微生物学通报》2018年第11期2364-2375,共12页Microbiology China

基　　金：浙江省科技计划(2017F30030;2017C33016);浙江省自然科学基金(LY18E080005);浙江省重大科技专项(2015C03007)~~

摘　　要：【背景】复苏促进因子(Resuscitation promoting factor,RPF)是由某些放线菌分泌的一类活性蛋白质,最初在Micrococcusluteus中被发现。它不但能够复苏休眠细菌恢复生长,而且对正常的细菌也有促生长效果。然而关于RPF对于环境样品中除原始宿主以外细菌的复苏促进作用研究还鲜有报道。【目的】以寻找新的菌种资源、开发环境功能菌角度研究了RPF对环境样品中潜在休眠菌群的复苏促进作用。【方法】首先对来源于Micrococcus luteus IAM 14879的RPF(Ml RPF)蛋白序列进行生物信息学分析和同源模建;随后克隆了Ml RPF编码基因并在大肠杆菌中异源表达;最后利用重组Ml RPF对活性污泥样品中潜在的休眠细菌开展了复苏促生长研究。【结果】Ml RPF在进化树上处于单独分支,远离其它RPF家族成员;模建结构分析表明Ml RPF具有与C型溶菌酶相似的催化结构域,这有助于解释RPF家族蛋白具有溶解酶活性;重组Ml RPF在大肠杆菌中获得了可溶性表达,它能够促进休眠态M. luteus IAM 14879生长,保留了其生理活性;利用重组Ml RPF从某污水厂活性污泥样品中分离获得了Dietzia、Paracoccus、Rhodococcus和Brevundimonas菌株。【结论】为环境样品中微生物多样性研究及特殊微生物资源的发掘提供新的方法,也为基于Ml RPF的废水生物强化处理技术研发提供理论依据。[Background] Resuscitation promoting factor（RPF） is an active protein secreted by some different actinobacteria species, which was first reported in Micrococcus luteus. It resuscitates the dormant bacteria and promotes the growth of active bacteria. However, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. [Objective] In order to find novel strains and explore the functional bacteria, we investigated the role of RPF in promoting the latent dormant bacteria from specific environmental sample. [Methods] Firstly, bioinformatics analysis and homology modeling of Micrococcus luteus RPF（Ml RPF） was performed. Subsequently, the Ml RPF encoding gene was cloned and expressed in Escherichia coli; Finally, we used the purified recombinant Ml RPF to study the resuscitation effect toward latent dormant bacteria from an activated sludge sample. [Results] Bioinformatics analysis revealed that the Ml RPF branched separately and was distant from other RPF family members in the phylogenetic tree. Structural analysis of its ＂RPF-domain＂ suggested that Ml RPF has a similar catalytic structure domain as C-type lysozyme, this finding helps explain why RPF family members have lysozyme-like activity; the recombinant Ml RPF was expressed in soluble form in E. coli expression system, could promote the growth of M. luteus IAM 14879 and retain its physiological activity. By used purified recombinant Ml RPF, we isolated Dietzia, Paracoccus, Rhodococcus, and Brevundimonas strains from an activated sludge sample. [Conclusion] These results not only provide a novel method for the study of microbial diversity and the exploration of special microbial resources in environmental samples, but also provide a theoretical basis for the research and development of wastewater bioaugmentation technology based on Ml RPF.

关 键 词：复苏促进因子 藤黄微球菌 异源表达 休眠细菌 活性污泥 

分 类 号：X172[环境科学与工程—环境科学] X703