从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类
Summary
我们介绍了从水生栖息地分离，繁殖和表征碳氢化合物降解细菌的过程。该协议概述了细菌分离，通过16S rRNA方法鉴定以及测试其碳氢化合物降解潜力。本文将帮助研究人员表征环境样本中的微生物生物多样性，特别是筛选具有生物修复潜力的微生物。
Abstract
碳氢化合物污染物不易降解，它们在环境中的积累对所有生命形式都是有毒的。细菌编码许多催化酶，并且自然能够代谢碳氢化合物。科学家利用水生生态系统中的生物多样性来分离具有生物降解和生物修复潜力的细菌。这种来自环境的分离物提供了一套丰富的代谢途径和酶，可以进一步用于在工业规模上扩大降解过程。在本文中，我们概述了从水生栖息地分离，繁殖和鉴定细菌物种的一般过程，并使用简单的技术筛选它们利用碳氢化合物作为 体外 唯一碳源的能力。本协议描述了各种细菌物种的分离以及随后使用16S rRNA分析进行鉴定。该协议还提供了表征细菌分离物的碳氢化合物降解潜力的步骤。该协议对于试图从环境栖息地中分离细菌物种以进行生物技术应用的研究人员很有用。
Introduction
碳氢化合物（HC）被广泛用作燃料和化学应用。苯、甲苯、二甲苯等芳烃被广泛用作溶剂1。乙烯和丙烯等烯烃分别作为聚乙烯和聚丙烯聚合物合成的前体。另一种烃类苯乙烯聚合形成聚苯乙烯。人为活动在生产和运输过程中将碳氢化合物引入环境。土壤和水的碳氢化合物污染对环境和人类健康造成严重关切。微生物通过调节生物地球化学循环和利用包括污染物和异生素在内的各种基质，将其转化为碳和能源，在维持生态系统方面发挥着重要作用。微生物对环境污染物进行解毒的过程称为生物修复3,4,5,6,7。
在水生和土壤栖息地中发现了具有降解碳氢化合物能力的微生物8,9,10。已经鉴定出许多具有降解烷烃和芳香族HC潜力的细菌，例如假单胞菌，不动杆菌，红球菌，马里诺杆菌和Oleibacter11。技术先进的培养独立方法的开发有助于发现新的HC降解微生物群落12。直接从源样品中分离的基因组材料通过下一代测序（NGS）等高通量方法进行扩增和测序，然后进行分析，无需培养微生物。NGS方法，如宏基因组分析，价格昂贵，并且存在与扩增过程相关的缺点13。靶向分离碳氢化合物降解微生物的选择性富集培养物14等培养技术仍然有用，因为它们允许研究人员探测和操纵细菌分离物中的代谢途径。
基因组DNA分离和随后的基因组材料测序揭示了有关任何生物体的宝贵信息。全基因组测序有助于鉴定编码抗生素耐药性的基因、潜在药物靶点、毒力因子、转运蛋白、异生代谢酶等15,16,17。16SrRNA编码基因的测序已被证明是鉴定细菌系统发育的可靠技术。多年来基因序列和功能的保存使其成为识别未知细菌并将分离株与最接近的物种进行比较的可靠工具。此外，该基因的长度最适合生物信息学分析18。所有这些特点，以及使用通用引物进行基因扩增的便利性和基因测序技术的改进，使其成为微生物鉴定的黄金标准。
在这里，我们描述了一种从环境样品中回收具有HC降解潜力的可培养微生物的程序。下面描述的方法概述了HC降解细菌的收集和鉴定，分为五个部分：（1）从水样中收集细菌，（2）分离纯培养物，（3）探索细菌分离株的HC降解能力（4）基因组DNA分离，以及（5）基于16S rRNA基因测序和BLAST分析的鉴定。该程序可以适用于分离细菌以进行许多不同的生物技术应用。
Protocol
1. 样品采集、处理和分析 注意：在这里，我们提出了一种从水生栖息地分离细菌的方案。一些分离物可能具有致病性，因此，在使用前后戴上手套并对工作区域进行消毒。 从水体的不同位置收集 500 mL 水样在五个无菌玻璃瓶中。分别使用pH计和温度计测量每个样品的pH和温度。注意：该方案不是针对特定地点的，也可以很容易地适应从碳氢化合物污染的…
Representative Results
图1概述了从水生栖息地分离和筛选细菌以及随后通过16S rRNA分析鉴定细菌的整个过程的示意图。来自印度达德里湿地的水样被收集在无菌玻璃瓶中，并立即带到实验室进行处理。样品通过孔径为0.22 μm的滤片，滤纸与不同的培养基板保持接触。2小时后，除去滤纸，并将板在30°C下孵育过夜以形成菌落（图2）。第二天，选择单个细菌菌落并?…
Discussion
众所周知，地球上只有大约1%的细菌可以在实验室中轻松培养6。即使在可培养的细菌中，许多细菌仍未被描述。分子方法的改进为细菌群落的分析和评估提供了新的维度。然而，这些技术确实有局限性，但它们不会使培养分析变得多余。分离单个细菌种类的纯培养技术仍然是表征生理特性的主要机制。土壤和水生栖息地蕴藏着许多具有新型酶和途径的细菌，这些细菌可用于生物?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Karthik Krishnan博士和RP实验室成员的有益意见和建议。DS得到了SNU-Doctor奖学金和地球观察研究所印度奖学金的支持。RP实验室得到了Shiv Nadar大学的CSIR-EMR拨款和启动资金的支持。
Materials
| Agarose | Sigma-Aldrich | A4718 | Gel electrophoresis |
| Ammonium chloride (NH4Cl) | Sigma-Aldrich | A9434 | Growth medium component |
| Ammonium sulphate | Sigma-Aldrich | A4418 | Growth medium component |
| Bacto-Agar | Millipore | 1016141000 | Solid media preparation |
| Calcium chloride (CaCl2) | MERCK | C4901-500G | Growth medium component |
| Catechol | Sigma-Aldrich | 135011 | Hydrocarbon degradation assay |
| Cetyltrimethylammonium bromide, CTAB | Sigma-Aldrich | H6269 | Genomic DNA Isolation |
| Chloroform | HIMEDIA | MB109 | Genomic DNA isolation |
| Disodium phosphate (Na2HPO4) | Sigma-Aldrich | S5136 | Growth medium component |
| EDTA | Sigma-Aldrich | E9884 | gDNA buffer component |
| Ferrous sulphate, heptahydrate (FeSO4.7H20) | Sigma-Aldrich | 215422 | Growth medium component |
| Glucose | Sigma-Aldrich | G7021 | Growth medium component |
| Glycerol | Sigma-Aldrich | G5516 | Growth medium component; Glycerol stocks |
| Isopropanol | HIMEDIA | MB063 | Genomic DNA isolation |
| LB Agar | Difco | 244520 | Growth medium |
| Luria-Bertani (LB) | Difco | 244620 | Growth medium |
| Magnesium sulphate (MgSO4) | MERCK | M2643 | Growth medium component |
| Manganese (II) sulfate monohydrate (MnSO4.H20) | Sigma-Aldrich | 221287 | Growth medium component |
| Nutrient Broth (NB) | Merck (Millipore) | 03856-500G | Growth medium |
| Peptone | Merck | 91249-500G | Growth medium component |
| Phenol | Sigma-Aldrich | P1037 | Genomic DNA isolation |
| Potassium phosphate, dibasic (K2HPO4) | Sigma-Aldrich | P3786 | Growth medium component |
| Potassium phosphate, monobasic (KH2PO4) | Sigma-Aldrich | P9791 | Growth medium component |
| Proteinase K | ThermoFisher Scientific | AM2546 | Genomic DNA isolation |
| QIAquick Gel Extraction kit | QIAGEN | 160016235 | DNA purification |
| QIAquick PCR Purification kit | QIAGEN | 163038783 | DNA purification |
| R2A Agar | Millipore | 1004160500 | Growth medium |
| SmartSpec Plus Spectrophotometer | BIO-RAD | 4006221 | Absorbance measurement |
| Sodium acetate | Sigma-Aldrich | S2889 | Genomic DNA isolation |
| Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | Growth medium component |
| Sodium dodecyl sulphate (SDS) | Sigma-Aldrich | L3771 | Genomic DNA isolation |
| Styrene | Sigma-Aldrich | S4972 | Styrene biodegradation |
| Taq DNA Polymerase | NEB | M0273X | 16s rRNA PCR |
| Tris-EDTA (TE) | Sigma-Aldrich | 93283 | Resuspension of genomic DNA |
| Tryptic Soy Broth (TSB) | Merck | 22092-500G | Growth medium |
| Yeast extract | Sigma-Aldrich | Y1625-1KG | Growth medium component |
| Zinc sulfate heptahydrate (ZnSO4.7H20) | Sigma-Aldrich | 221376 | Growth medium component |
References
- Sirotkin, A. V. Reproductive effects of oil-related environmental pollutants. Encyclopedia of Environmental Health. , 493-498 (2019).
- Li, C., Busquets, R., Campos, L. C. Assessment of microplastics in freshwater systems: A review. Science of The Total Environment. 707, 135578 (2020).
- Siddiqa, A., Faisal, M. Microbial degradation of organic pollutants using indigenous bacterial strains. Handbook of Bioremediation. , 625-637 (2021).
- Hossain, F., et al. Bioremediation potential of hydrocarbon degrading bacteria: isolation, characterization, and assessment. Saudi Journal of Biological Sciences. , (2021).
- Wongbunmak, A., Khiawjan, S., Suphantharika, M., Pongtharangkul, T. BTEX biodegradation by Bacillus amyloliquefaciens subsp. plantarum W1 and its proposed BTEX biodegradation pathways. Scientific Reports. 10 (1), 17408 (2020).
- Bodor, A., et al. Challenges of unculturable bacteria: environmental perspectives. Reviews in Environmental Science and Bio/Technology. 19 (1), 1-22 (2020).
- Phale, P. S., Sharma, A., Gautam, K. Microbial degradation of xenobiotics like aromatic pollutants from the terrestrial environments. Pharmaceuticals and Personal Care Products: Waste Management and Treatment Technology. , 259-278 (2019).
- Brooijmans, R. J. W., Pastink, M. I., Siezen, R. J. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Microbial Biotechnology. 2 (6), 587-594 (2009).
- Sorkhoh, N. A., Ghannoum, M. A., Ibrahim, A. S., Stretton, R. J., Radwan, S. S. Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments in Kuwait. Environmental Pollution. 65 (1), 1-17 (1990).
- Chaillan, F., et al. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. Research in Microbiology. 155 (7), 587-595 (2004).
- Bôto, M. L., et al. Harnessing the potential of native microbial communities for bioremediation of oil spills in the Iberian Peninsula NW coast. Frontiers in Microbiology. 12, 879 (2021).
- Wang, Y., et al. A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community. PloS One. 7 (10), 47530 (2012).
- Jovel, J., et al. Characterization of the gut microbiome using 16S or shotgun metagenomics. Frontiers in Microbiology. 7, 459 (2016).
- Spini, G., et al. Molecular and microbiological insights on the enrichment procedures for the isolation of petroleum degrading bacteria and fungi. Frontiers in Microbiology. , 2543 (2018).
- Pightling, A. W., et al. Interpreting whole-genome sequence analyses of foodborne bacteria for regulatory applications and outbreak investigations. Frontiers in Microbiology. , 1482 (2018).
- Salipante, S. J., et al. Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology. Journal of Clinical Microbiology. 53 (4), 1072-1079 (2015).
- Lovley, D. R. Cleaning up with genomics: applying molecular biology to bioremediation. Nature Reviews Microbiology. 1 (1), 35-44 (2003).
- Janda, J. M., Abbott, S. L. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. Journal of Clinical Microbiology. 45 (9), 2761-2764 (2007).
- Poindexter, J. S. Biological properties and classification of the Caulobacter group. Bacteriological Reviews. 28 (3), 231-295 (1964).
- Reasoner, D. J., Geldreich, E. A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology. 49 (1), 1-7 (1985).
- Pardee, A. B. The genetic control and cytoplasmic expression of “Inducibility” in the synthesis of β-galactosidase by E. coli. Journal of Molecular Biology. 1 (2), 165-178 (1959).
- Ely, B. Genetics of Caulobacter crescentus. Methods in Enzymology. 204, 372-384 (1991).
- R, C. Gram staining. Current Protocols in Microbiology. , (2005).
- Green, M. R., Hughes, H., Sambrook, J., MacCallum, P. Molecular cloning: a laboratory manual. Molecular Cloning: A Laboratory Manual. , 1890 (2012).
- Chauhan, D., Agrawal, G., Deshmukh, S., Roy, S. S., Priyadarshini, R. Biofilm formation by Exiguobacterium sp. DR11 and DR14 alter polystyrene surface properties and initiate biodegradation. RSC Advances. 8 (66), 37590-37599 (2018).
- Takeo, M., Nishimura, M., Shirai, M., Takahashi, H., Negoro, S. Purification and characterization of catechol 2,3-Dioxygenase from the aniline degradation pathway of Acinetobacter sp. YAA and its mutant enzyme, which resists substrate inhibition. Bioscience, Biotechnology, Biochemistry. 71, 70079-70080 (2007).
- Nguyen, O. T., Ha, D. D. Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT. Annals of Microbiology. 69 (3), 267-277 (2019).
- Hupert-Kocurek, K., Guzik, U., Wojcieszyńska, D. Characterization of catechol 2, 3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration. Acta Biochimica Polonica. 59 (3), (2012).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1-2), 248-254 (1976).
- Peterson, G. L., et al. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry. 83 (2), 346-356 (1977).
- Chen, W. P., Kuo, T. T. A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Research. 21 (9), 2260 (1993).
- William, S., Feil, H., Copeland, A. Bacterial genomic DNA isolation using CTAB. Sigma. 50 (6876), (2012).
- Frank, J. A., et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology. 74 (8), 2461-2470 (2008).
- Sanger, F., Nicklen, S., Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America. 74 (12), 5463-5467 (1977).
- Johnson, M., et al. NCBI BLAST: a better web interface. Nucleic Acids Research. 36, 5-9 (2008).
- Harayama, S., Rekik, M. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. Journal of Biological Chemistry. 264 (26), 15328-15333 (1989).
- Li, X., et al. Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria. The Journal of International Medical Research. 48 (5), 300060520925594 (2020).
- Coico, R. Gram staining. Current Protocols in Microbiology. , (2005).
- Clarridge, J. E. III Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews. 17 (4), 840 (2004).
- Dereeper, A., et al. Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research. 36, 465-469 (2008).
- Wadowsky, R. M., Wolford, R., McNamara, A. M., Yee, R. B. Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water. Applied and Environmental Microbiology. 49 (5), 1197-1205 (1985).
- du Toit, W. J., Pretorius, I. S., Lonvaud-Funel, A. The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. Journal of Applied Microbiology. 98 (4), 862-871 (2005).
- Johnson, J. S., et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nature Communications. 10 (1), 1-11 (2019).
- Kim, E., et al. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate. BMC Microbiology. 20 (1), 1-14 (2020).
- Poretsky, R., Rodriguez-R, L. M., Luo, C., Tsementzi, D., Konstantinidis, K. T. Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics. PLoS ONE. 9 (4), (2014).
- Johnson, B. H., Hecht, M. H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Bio/Technology. 12 (12), 1357-1360 (1994).
- Rodríguez-Carmona, E., Cano-Garrido, O., Seras-Franzoso, J., Villaverde, A., García-Fruitós, E. Isolation of cell-free bacterial inclusion bodies. Microbial Cell Factories. 9 (1), 71 (2010).
- Hoiczyk, E., Hansel, A. Cyanobacterial cell walls: News from an unusual prokaryotic envelope. Journal of Bacteriology. 182 (5), 1191 (2000).
- Erstad, S. M., Sakuragi, Y. Easy and efficient permeabilization of cyanobacteria for in vivo enzyme assays using B-PER. Bio-Protocol. 8 (1), (2018).
Cite This Article
Sethi, D., Priyadarshini, R. Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats. J. Vis. Exp. (178), e63101, doi:10.3791/63101 (2021).