环境中微生物原位检测方法研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2017-0550

摘要/Abstract

摘要： 微生物是生态系统物质循环和能量流动的主要参与者，在生态系统中起着重要的作用。但在现有技术条件下可培养微生物所占比例极小，限制了微生物资源的开发利用。目前有许多方法，可以避开微生物不可培养的问题，直接对微生物进行原位检测。对此，将前人关于微生物生态的原位检测研究方法进行了综述，方便以后对这些方法的合理应用。分别从DNA水平、RNA水平和蛋白质水平，介绍了对应的原位微生物检测方法（如BrdU标记、DNA-SIP、FISH和环境转录物组等），比较了它们的优缺点，并介绍了如何将这些方法与目前流行的高通量测序、单细胞测序等技术相结合来捕获原位活性微生物组等。同时，还对这些方法的特点进行了比较，使得人们可以更清楚地了解在不同场景下对不同方法的选择。这些经过改进的新兴方法及其与其它方法的结合使用将有助于解决微生物生态学研究中出现过或即将出现的很多问题。地球上的各种生态系统复杂而庞大，包含的微生物种群也各有差异。各种原位检测方法对微生物生理生态做出了更加真实有效的描述，必将成为研究微生物生态的有力手段。

关键词: 微生物原位检测, BrdU标记, DNA-SIP, 荧光原位杂交, 宏基因组, 转录物组, 生物正交反应

Abstract: As major participants in ecosystem material cycle and energy flow，microorganisms play an important role in the ecosystem. However，the proportion of the cultivable microorganism under the existing technology is very small，which limits the exploit of microbial resources. At present，there are a number of approaches that can avoid the problem of uncultured microorganisms，which are designed to study in situ microbial activity. Regarding this，we summarized some research methods of studying in situ microbial ecology，allowing it convenient to reasonably use these methods in the future. This article introduces the corresponding microbial detection methods of BrdU-labeling，DNA-SIP，fluorescence in situ hybridization（FISH），and environmental transcriptome from DNA level，RNA level and protein level，respectively，and compares their advantages and disadvantages. It also introduces how to apply these methods combined with popular high-throughput sequencing and single cell sequencing technology to capture the in situ activity of microbial groups. At the same time，comparing the characteristics of these methods，so that we can more clearly understand the choice of different methods under different scenarios. These modified methods combined with other methods will be conducive to solve many have-been or will-happen problems in the study of microbial ecology. The ecosystems on the earth are complex and huge，in which the microbial populations vary. A variety of in situ detection methods have made a more realistic and effective description for the physiology and ecology of microorganisms，which will become a powerful tool for the study of microorganisms.

Key words: microbial detection in sit, BrdU-labeling, DNA-SIP, fluorescence in situ hybridization, metagenome, transcriptome, BONCAT

宋伟凤, 李明聪, 高峥. 环境中微生物原位检测方法研究进展[J]. 生物技术通报, 2017, 33(10): 26-32.

SONG Wei-feng, LI Ming-cong, GAO Zheng. Research Progress on in situ Detection Methods of Microorganisms[J]. Biotechnology Bulletin, 2017, 33(10): 26-32.

参考文献

[1]Canfield DE, Glazer AN, Falkowski PG. The evolution and future of Earth's nitrogen cycle[J]. Science, 2010, 330(6001)：192-196.
[2] Anderson IC, Cairney JWG. Diversity and ecology of soil fungal communities：increased understanding through the application of molecular techniques[J]. Environ Microbiol, 2004, 6(8)：769-779.
[3] Schleifer KH. Microbial diversity：facts, problems and prospects[J]. Systematic and Applied Microbiology, 2004, 27(1)：3-9.
[4]Pace NR, Stahl DA, Lane DJ, et al. Analyzing natural microbial populations by rRNA sequences[J]. ASM American Society for Microbiology News, 1985, 51(1)：4-12.
[5]Tringe SG, Hugenholtz PA renaissance for the pioneering 16S rRNA gene[J]. Current Opinion in Microbiology, 2008, 11(5)：442-446.
[6]Handelsman J. Metagenomics：application of genomics to uncultured microorganisms[J]. Microbiology and Molecular Biol Rev, 2005, 69(1)：195-195.
[7]Hatzenpichler R, Connon SA, Goudeau D, et al. Visualizing in situ translational activity for identifying and sorting slow-growing archaeal- bacterial consortia[J]. Proc Natl Academy of Sciences, 2016, 113(28)：E4069-E4078.
[8]Biggs MJP, Richards RG, Dalby MJ. Using immuno-scanning electron microscopy for the observation of focal adhesion-substratum interactions at the nano-and microscale in S-phase cells[J]. 3D Cell Culture：Methods and Protocols, 2011：53-60.
[9]Urbach E, Vergin KL, Giovannoni SJ. Immunochemical detection and isolation of DNA from metabolically active bacteria[J]. Appl Environ Microbiol, 1999, 65(3)：1207-1213.
[10]Yin B, Crowley D, Sparovek G, et al. Bacterial functional redundancy along a soil reclamation gradient[J]. Appl Environ Microbiol, 2000, 66(10)：4361-4365.
[11]Artursson V, Finlay RD, Jansson JK. Combined bromodeoxyuridine immunocapture and terminal-restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species[J]. Environ Microbiol, 2005, 7(12)：1952-1966.
[12]Allison SD, Czimczik CI, Treseder KK. Microbial activity and soil respiration under nitrogen addition in Alaskan boreal forest[J]. Global Change Biology, 2008, 14(5)：1156-1168.
[13]Goldfarb KC, Karaoz U, Hanson CA, et al. Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance[J]. Frontiers in Microbiology, 2011, 2.
[14]Mou X, Sun S, Edwards RA, et al. Bacterial carbon processing by generalist species in the coastal ocean[J]. Nature, 2008, 451(7179)：708-711.
[15]David MM, Cecillon S, Warne BM, et al. Microbial ecology of chlorinated solvent biodegradation[J]. Environ Microbiol, 2015, 17(12)：4835-4850.
[16]Hamasaki K, Taniguchi A, Tada Y, et al. Active populations of rare microbes in oceanic environments as revealed by bromodeoxyuridine incorporation and 454 tag sequencing[J]. Gene, 2016, 576(2)：650-656.
[17] 贾仲君. 稳定性同位素核酸探针技术 DNA-SIP 原理与应用[J]. 微生物学报, 2011, 51(12)：1585-1594.
[18] Radajewski S, Ineson P, Parekh NR, et al. Stable-isotope probing as a tool in microbial ecology[J]. Nature, 2000, 403(6770)：646-649.
[19] Dumont MG, Pommerenke B, Casper P. Using stable isotope prob-ing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment[J]. Environ Microbiol Reports, 2013, 5(5)：757-764.
[20] Huang WE, Stoecker K, Griffiths R, et al. Raman-FISH：combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function[J]. Environ Microbiol, 2007, 9(8)：1878-1889.
[21] Bauman JG J, Wiegant J, Borst P, et al. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA[J]. Experimental Cell Research, 1980, 128(2)：485-490.
[22]Kubota K. CARD-FISH for environmental microorganisms：technical advancement and future applications[J]. Microbes and Environments, 2013, 28(1)：3-12.
[23]Bobrow MN, Harris TD, Shaughnessy KJ, et al. Catalyzed reporter deposition, a novel method of signal amplification application to immunoassays[J]. Journal of Immunological Methods, 1989, 125(1)：279-285.
[24]Bobrow MN, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification：II. Application to membrane immunoassays[J]. Journal of Immunological Methods, 1991, 137(1)：103-112.
[25] Kerstens HM, Poddighe PJ, Hanselaar AG. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine[J]. Journal of Histochemistry & Cytochemistry, 1995, 43(4)：347-352.
[26]Kawakami S, Kubota K, Imachi H, et al. Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization(Two-Pass TSA-FISH)with single oligonucleotide probes[J]. Microbes and Environments, 2010, 25(1)：15-21.
[27]Kawakami S, Kubota K, Imachi H, et al. Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization(Two-Pass TSA-FISH)with single oligonucleotide probes[J]. Microbes and Environments, 2010, 25(1)：15-21.
[28]Kenzaka T, Ishidoshiro A, Tani K, et al. Scanning electron microscope imaging of bacteria based on DNA sequence[J]. Letters in Applied Microbiology, 2009, 49(6)：796-799.
[29]Handelsman J, Rondon MR, Brady SF, et al. Molecular biological access to the chemistry of unknown soil microbes：a new frontier for natural products[J]. Chemistry & Biology, 1998, 5(10)：R245-R249.
[30]Leininger S, Urich T, Schloter M, et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils[J]. Nature, 2006, 442(7104)：806-809.
[31]Frias-Lopez J, Shi Y, Tyson GW, et al. Microbial community gene expression in ocean surface waters[J]. Proc Natl Academy of Sciences, 2008, 105(10)：3805-3810.
[32]Kalyuzhnaya MG, Lapidus A, Ivanova N, et al. High-resolution metagenomics targets specific functional types in complex microbial communities[J]. Nature Biotechnology, 2008, 26(9)：1029-1034.
[33]Korem T, Zeevi D, Suez J, et al. Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples[J]. Science, 2015, 349(6252)：1101-1106.
[34]Brown CT, Olm MR, Thomas BC, et al. In situ replication rates for uncultivated bacteria in microbial communities[J]. BioRxiv, 2016：057992.
[35]Voorhies AA, Eisenlord SD, Marcus DN, et al. Ecological and genetic interactions between cyanobacteria and viruses in a low-oxygen mat community inferred through metagenomics and metatranscriptomics[J]. Environ Microbiol, 2016, 18(2)：358-371.
[36]De Filippis F, Genovese A, Ferranti P, et al. Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate[J]. Scientific Reports, 2016, 6：21871.
[37]Bagchi S, Lamendella R, Strutt S, et al. Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor[J]. Scientific Reports, 2016, 6：28327.
[38]杨麦云, 陈鹏. 生物正交标记反应研究进展[J]. 化学学报, 2015, 73(8)：783-792.
[39]Hatzenpichler R, Scheller S, Tavormina PL, et al. In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry[J]. Environ Microbiol, 2014, 16(8)：2568-2590.
[40]Borneman J. Culture-independent identification of microorganisms that respond to specified stimuli[J]. Appl Environ Microbiol, 1999, 65(8)：3398-3400.
[41]Radajewski S, McDonald IR, Murrell JC. Stable-isotope probing of nucleic acids：a window to the function of uncultured microorganisms[J]. Current Opinion in Biotechnology, 2003, 14(3)：296-302.
[42]Blazewicz SJ, Barnard RL, Daly RA, et al. Evaluating rRNA as an indicator of microbial activity in environmental communities：limitations and uses[J]. The ISME Journal, 2013, 7(11)：2061-2068.
[43]Hoehler TM, Jorgensen BB. Microbial life under extreme energy limitation[J]. Nature Reviews Microbiology, 2013, 11(2)：83-94.
[44]Paerl HW, Steppe TF. Scaling up：the next challenge in Environ Microbiol[J]. Environ Microbiol, 2003, 5(11)：1025-1038.