从环境样品中的内生孢子通过无性细胞定向裂解物理隔离
Summary
A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.
Abstract
Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.
Introduction
这项工作的目的是提供一种协议，用于细菌内生孢子的从营养细菌细胞环境样品中分离。细菌内生孢子的形成是一个生存策略，通常由饥饿触发，在多个属于脊索动物门厚壁菌门1细菌群体的发现。形成内生孢子的细菌被很好的研究，主要是因为一些菌株是病原体，因此医学重要（例如， 炭疽芽孢杆菌或艰难梭菌 ）。形成内生孢子菌的环境菌株已分离出几乎所有的环境（土壤，水，沉淀物，空气，冰，人体肠道，内脏动物，和更多）1-3。因此，厚壁菌在培养物保藏4第二个最丰富的门。
由于其耐寒外皮层和保护核心蛋白，内生孢子能够生存极端的环境条件范围FROM干燥高辐射，极端温度和有害化学物质5。这一显着性使它成为一个挑战提取芽孢6-8 DNA。这可能解释了为什么他们被忽视了环境测序研究9,10。其他方法，如环境样品中内生孢子的荧光抗体11靶向，吡啶二羧酸的定量（DPA）中土壤12和沉积物13，流式细胞仪14或巴氏灭菌和随后的培养15,16已被用于在检索或量化生孢子环境样品。在最近几年，优化的DNA提取方法以及具体的分子的引物靶向生孢子特异性基因序列已经被开发10,17-20。这有助于揭示这组细菌21之间更多的生物多样性，并且也导致了内生孢子的检测应用在工业和医药中，例如在奶粉19。
这里介绍的协议是基于到有害的物理化学条件（例如热和洗涤剂）相对于营养细胞细菌内生孢子的电阻的差别。以破坏营养细胞的样品中，我们连续施加热，溶菌酶和低浓度的洗涤剂。这些治疗的时间和强度都得到了优化，以便不破坏孢子，但以裂解所有营养细胞。一些细胞在一个环境室池比其它更耐磨，所以为了提高破坏所有营养细胞的概率，我们应用了三种不同的治疗方法。这种方法的优点和新颖性在于仍保持原样，在处理后的内生孢子可用于进一步下游分析。这些包括DNA提取，定量PCR（qPCR的）和扩增子或宏基因组测序（专门针对组内生孢子的，从而减少ðiversity，同时增加覆盖）。的内生孢子也可以用于下行种植或定量由荧光显微镜，流式细胞仪，或检测DPA的。这种方法的一个重要特征是，通过未处理样品与处理过的样品进行比较，可以推断出内生孢子的数量和多样性环境样品中除了对应于营养细胞的组件。
Protocol
Representative Results
Discussion
内生孢子外部攻击性理化因素 （如温度或清洁剂）的电阻来设计一种方法，从营养细胞环境样品中分离的细菌芽孢。这是第一个全面的方法来隔离环境样品的内生孢子在非破坏性的方式。以前的方法来定量，检测或分析样品中内生孢子是根据特定的代理内生孢子诸如吡啶二羧酸或特定的标记基因的测定。与此相反，与协议这里介绍，比生孢子其他样品组分被去除，所以内生孢子最终仍作为…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者承认瑞士国家科学基金会的批准号：31003A-一分之一十三万二千三百五十八，31003A_152972和No. 151948，和基金会皮埃尔·名士倒拉科学。
Materials
| Whatman nitrocellulose membrane filters, 0,2 um pore size, 47 mm diameter | Sigma-Aldrich | WHA7182004 Aldrich | |
| Tris(hydroxymethyl)aminomethane | Sigma-Aldrich | 252859 Sigma-Aldrich | CAS 77-86-1 |
| EDTA | Sigma-Aldrich | E9884 Sigma-Aldrich | CAS 60-00-4 |
| Lysozyme from chicken egg white | Sigma-Aldrich | 62971 Fluka | powder, CAS 12650-88-3 |
| Ultra-Turrax homogenizer T18 basic | IKA | 3720000 | |
| Glass filter holders for 47 mm membranes, pyrex glass | EMD Millipore | XX10 047 00 | |
| Chemical duty vacuum pump | Millipore | WP6122050 | 220 V/50 Hz |
| Manifold sampling filtration for 25 mm membranes | Millipore | 1225 Sampling Manifold | polypropylene |
| Dnase | New England Biolabs | M0303S | Rnase free |
| NaOH | Sigma-Aldrich | S5881 Sigma-Aldrich | CAS 1310-73-2 |
| Sodium dodecyl sulfphate (SDS) | Sigma-Aldrich | L3771 Sigma | |
| Whatman nitrocellulose membrane filters, 0,2 um pore size, 25 mm diameter | Sigma-Aldrich | WHA7182002 Aldrich | |
Referenzen
- Nicholson, W. L. Roles of Bacillus endospores in the environment. Cell Mol Life Sci. 59 (3), 410-416 (2002).
- Lester, E. D., Satomi, M., Ponce, A. Microflora of extreme arid Atacama Desert soils. Soil Biol Biochem. 39 (2), 704-708 (2007).
- Wilson, M. S., Siering, P. L., White, C. L., Hauser, M. E., Bartles, A. N. Novel archaea and bacteria dominate stable microbial communities in North America’s Largest Hot Spring. Microb Ecol. 56 (2), 292-305 (2008).
- Hugenholtz, P. Exploring prokaryotic diversity in the genomic era. Genome Biol. 3 (2), (2002).
- Onyenwoke, R. U., Brill, J. A., Farahi, K., Wiegel, J. Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes). Arch Microbiol. 182 (2-3), 182-192 (2004).
- Delmont, T. O., et al. Accessing the soil metagenome for studies of microbial diversity. Appl Environ Microbiol. 77 (4), 1315-1324 (2011).
- Ricca, E., Henriques, A. O., Cutting, S. M. . Bacterial spore formers: probiotics and emerging applications. , (2004).
- Kuske, C. R., et al. Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil. Appl Environ Microbiol. 64 (7), 2463-2472 (1998).
- von Mering, C., et al. Quantitative phylogenetic assessment of microbial communities in diverse environments. Science. 315 (8515), 1126-1130 (2007).
- Wunderlin, T., Junier, T., Roussel-Delif, L., Jeanneret, N., Junier, P. Stage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes. Environ Microbiol Rep. 5 (6), 911-924 (2013).
- Siala, A., Hill, I. R., Gray, T. R. G. Populations of spore-forming bacteria in an acid forest soil, with special reference to Bacillus subtilis. J General Microbiol. 81 (1), 183-190 (1974).
- Brandes Ammann, A., Kolle, L., Brandl, H. Detection of bacterial endospores in soil by terbium fluorescence. Int J Microbiol. , 435281 (2011).
- Fichtel, J., Koster, J., Rullkotter, J., Saas, H. High variations in endospore numbers within tidal flat sediments revealed by quantification of dipicolinic acid. Geomicrobiol J. 25 (7-8), (2008).
- Cronin, U. P., Wilkinson, M. G. The potential of flow cytometry in the study of Bacillus cereus. J Appl Microbiol. 108 (1), 1-16 (2010).
- de Rezende, J. R., et al. Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years. ISME J. 7 (1), 72-84 (2013).
- Hubert, C., et al. Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature. Environ Microbiol. 12 (4), 1089-1104 (2010).
- Garbeva, P., van Veen, J. A., van Elsas, J. D. Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR-DGGE. Microb Ecol. 45 (3), 302-316 (2003).
- Brill, J. A., Wiegel, J. Differentiation between spore-forming and asporogenic bacteria using a PCR and southern hybridization based method. J Microbiol Methods. 31 (1-2), 29-36 (1997).
- Rueckert, A., Ronimus, R. S., Morgan, H. W. Development of a real-time PCR assay targeting the sporulation gene, spo0A., for the enumeration of thermophilic bacilli in milk powder. Food Microbiol. 23 (3), 220-230 (2006).
- Bueche, M., et al. Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A. Appl Environ Microbiol. 79 (17), 5302-5312 (2013).
- Wunderlin, T., Junier, T., Roussel-Delif, L., Jeanneret, N., Junier, P. Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments. Environ Microbiol Rep. 6 (6), 631-639 (2014).
- Nicholson, W. L., Law, J. F. Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168. J Microbiol Methods. 35 (1), 13-21 (1999).
