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
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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.
[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.
引文
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[16]Wei FS.Water and Wastewater Monitoring and Analysis Method[M].4th Edition.Beijing:China Environmental Science Press,2002:210-462(in Chinese)魏复盛.水和废水监测分析方法[M].第4版.北京:中国环境科学出版社,2002:210-462
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[22]Whyte LG,Hawari J,Zhou E,et al.Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.[J].Applied and Environmental Microbiology,1998,64(7):2578-2584
[23]Resmi G,Thampi SG,Chandrakaran S.Brevundimonas vesicularis:A novel bio-sorbent for removal of lead from wastewater[J].International Journal of Environmental Research,2010,4(2):281-288
[24]Dumas DP,Caldwell SR,Wild JR,et al.Purification and properties of the phosphotriesterase from Pseudomonas diminuta[J].Journal of Biological Chemistry,1989,264(33):19659-19665
[25]Liu YD,Su XM,Lu L,et al.A novel approach to enhance biological nutrient removal using a culture supernatant from Micrococcus luteus containing resuscitation-promoting factor(Rpf)in SBR process[J].Environmental Science and Pollution Research,2016,23(5):4498-4508
[2]Mukamolova GV,Turapov OA,Young DI,et al.A family of autocrine growth factors in Mycobacterium tuberculosis[J].Molecular Microbiology,2002,46(3):623-635
[3]Hartmann M,Barsch A,Niehaus K,et al.The glycosylated cell surface protein Rpf2,containing a resuscitation-promoting factor motif,is involved in intercellular communication of Corynebacterium glutamicum[J].Archives of Microbiology,2004,182(4):299-312
[4]Ravagnani A,Finan CL,Young M.A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement[J].BMCGenomics,2005,6:39
[5]Schroeckh V,Martin K.Resuscitation-promoting factors:distribution among actinobacteria,synthesis during life-cycle and biological activity[J].Antonie Van Leeuwenhoek,2006,89(3/4):359-365
[6]Ding LX,Zhang PH,Hong HC,et al.Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp.DS471[J].Acta Microbiologica Sinica,2012,52(1):77-82(in Chinese)丁林贤,张萍华,洪华嫦,等.藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用[J].微生物学报,2012,52(1):77-82
[7]Mukamolova GV,Murzin AG,Salina EG,et al.Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation[J].Molecular Microbiology,2006,59(1):84-98
[8]Kana BD,Mizrahi V.Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling[J].FEMSImmunology and Medical Microbiology,2010,58(1):39-50
[9]Li SH,Jin Y,Cheng J,et al.Gordonia jinhuaensis sp.nov.,a novel actinobacterium,isolated from a VBNC(viable but non-culturable)state in pharmaceutical wastewater[J].Antonie Van Leeuwenhoek,2014,106(2):347-356
[10]Su XM,Liu YD,Hashmi MZ,et al.Rhodococcus biphenylivorans sp.nov.,a polychlorinated biphenyl-degrading bacterium[J].Antonie Van Leeuwenhoek,2015,107(1):55-63
[11]Su XM,Shen H,Yao XY,et al.A novel approach to stimulate the biphenyl-degrading potential of bacterial community from PCBs-contaminated soil of e-waste recycling sites[J].Bioresource Technology,2013,146:27-34
[12]Su XM,Zhang Q,Hu JX,et al.Enhanced degradation of biphenyl from PCB-contaminated sediments:the impact of extracellular organic matter from Micrococcus luteus[J].Applied Microbiology&Biotechnology,2015,99(4):1989-2000
[13]Puspita ID,Kitagawa W,Kamagata Y,et al.Increase in bacterial colony formation from a permafrost ice wedge dosed with a Tomitella biformata recombinant resuscitation-promoting factor protein[J].Microbes and Environments,2015,30(2):151-156
[14]Kelley LA,Sternberg MJ.Protein structure prediction on the Web:a case study using the Phyre server[J].Nature Protocols,2009,4(3):363-371
[15]Puspita ID,Uehara M,Katayama T,et al.Resuscitation promoting factor(Rpf)from Tomitella biformata AHU 1821Tpromotes growth and resuscitates non-dividing cells[J].Microbes and Environments,2013,28(1):58-64
[16]Wei FS.Water and Wastewater Monitoring and Analysis Method[M].4th Edition.Beijing:China Environmental Science Press,2002:210-462(in Chinese)魏复盛.水和废水监测分析方法[M].第4版.北京:中国环境科学出版社,2002:210-462
[17]Cohen-Gonsaud M,Keep NH,Davies AP,et al.Resuscitation-promoting factors possess a lysozyme-like domain[J].Trends in Biochemical Sciences,2004,29(1):7-10
[18]Cohen-Gonsaud M,Barthe P,Bagnéris C,et al.The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes[J].Nature Structural and Molecular Biology,2005,12(3):270-273
[19]Wang XB,Chi CQ,Nie Y,et al.Degradation of petroleum hydrocarbons(C6-C40)and crude oil by a novel Dietzia strain[J].Bioresource Technology,2011,102(17):7755-7761
[20]Robertson LA,van Niel EWJ,Torremans RAM,et al.Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha[J].Applied and Environmental Microbiology,1988,54(11):2812-2818
[21]MartínkováL,UhnákováB,Pátek M,et al.Biodegradation potential of the genus Rhodococcus[J].Environment International,2009,35(1):162-177
[22]Whyte LG,Hawari J,Zhou E,et al.Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.[J].Applied and Environmental Microbiology,1998,64(7):2578-2584
[23]Resmi G,Thampi SG,Chandrakaran S.Brevundimonas vesicularis:A novel bio-sorbent for removal of lead from wastewater[J].International Journal of Environmental Research,2010,4(2):281-288
[24]Dumas DP,Caldwell SR,Wild JR,et al.Purification and properties of the phosphotriesterase from Pseudomonas diminuta[J].Journal of Biological Chemistry,1989,264(33):19659-19665
[25]Liu YD,Su XM,Lu L,et al.A novel approach to enhance biological nutrient removal using a culture supernatant from Micrococcus luteus containing resuscitation-promoting factor(Rpf)in SBR process[J].Environmental Science and Pollution Research,2016,23(5):4498-4508