用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定
Summary
细菌编码各种机制,参与细菌间竞争。在这里,我们提出了一个基于培养的协议,用于描述细菌分离物之间的竞争相互作用,以及它们如何影响混合种群的空间结构。
Abstract
本手稿描述了一种基于培养的共生测定,用于检测和描述两种细菌群体之间的竞争相互作用。该方法采用稳定的质粒,使每个种群分别具有不同的抗生素抗性能力和荧光蛋白,用于选择和视觉鉴别。在这里,我们描述了竞争维布里奥菲舍尔菌株的制备和联合孵化、荧光显微镜成像和定量数据分析。这种方法很简单,产生快速的结果,并可用于确定一个种群是杀死还是抑制另一个种群的生长,以及竞争是通过可扩散的分子进行中介的,还是需要直接的细胞-细胞接触。由于每个细菌群体表达不同的荧光蛋白,因此测定允许在混合菌群中对相互竞争的人群进行空间歧视。虽然所述方法使用共生细菌V. 菲舍尔使用针对该物种优化的条件执行,但该协议可以适用于大多数可培养的细菌分离物。
Introduction
本手稿概述了一种基于培养的方法,用于确定两种细菌分离物是否具有竞争性相互作用。在研究混合种群时,评估细菌分离物相互作用的程度非常重要,特别是分离物是否通过干扰机制直接竞争。干扰竞争是指一个人口直接抑制增长或杀死竞争对手1的相互作用。这些相互作用是重要的识别,因为它们可以有深刻的影响微生物群落的结构和功能2,3。
微生物竞争机制已广泛发现来自不同环境的细菌基因组,包括宿主相关细菌和自由生存细菌4、5、6、7 8,9.已经描述了各种竞争策略10,11包括可扩散的机制,如杀菌化学品1,12和分泌抗菌肽13,以及需要细胞-细胞接触将抑制作用剂转移到目标细胞9、14、15、16、17的接触依赖机制,18.
虽然基于培养的共育物在微生物学5、8、19中常用,但本手稿概述了如何使用测定来描述竞争机制,以及适应与其他细菌物种一起使用的协议。此外,此方法还描述了分析和呈现数据的多种方法,以回答有关竞争交互性质的不同问题。虽然本文所述技术以前曾用于识别共生Vibrio Fischeri细菌19共生菌株之间特定竞争背后的细菌间杀灭机制,但它们适用于许多细菌物种,包括环境分离物和人类病原体,可用于评估接触依赖性和可扩散的竞争机制。协议中的步骤可能需要优化其他细菌种类。鉴于更多的模型系统正在扩大他们的研究,从同源生物的使用,包括不同的基因型10,16,20,21,这种方法将是一个宝贵的资源寻求了解竞争如何影响多菌株或多物种系统的研究人员。
Protocol
1. 为联合孵化准备菌株 选择适当的参考菌株,作为联合孵育测定期间细菌竞争的目标。请参阅讨论,了解选择参考应变时的最佳做法以及参考应变将如何影响结果。在此协议中,V.菲舍尔应变 ES114 将作为参考应变。 确定将采用哪些选择和筛选方法来区分共育中的分离物 通常,使用含有不同抗生素耐药性基因(如卡那霉素或氯霉素)的稳定质粒来转?…
Representative Results
为了评估细菌群之间的竞争相互作用,为V.fischeri开发并优化了联合孵育测定方案。该方法利用编码抗生素抗性基因和荧光蛋白的稳定质粒,允许每个菌株的微分选择和视觉鉴别。通过分析从共孵化测定中收集的数据,可以识别相互作用的竞争结果和相互作用的机制。例如,使用V.菲舍尔基分离物进行了以下实验。凝结菌株含有两个稳定的质粒之一:pVSV102表示卡那霉?…
Discussion
上述共育测定为发现细菌间竞争提供了一种强有力的方法。这种方法可以识别V.fischeri分离和描述竞争机制之间的特定竞争19。虽然所述方法针对海洋细菌V.菲舍尔进行了优化,但可以轻松修改,以适应其他细菌种类,包括临床和环境分离物。需要注意的是,竞争机制通常有条件地受到5、6、23、24、25、26、27的制约。<sup c…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢审阅者提供的有用反馈。A.N.S.由戈登和贝蒂·摩尔基金会通过授予GBMF 255.03向生命科学研究基金会提供支持。
Materials
| 1.5 mL Microcentrifuge Tubes | Fisher | 05-408-129 | |
| 10 μL multichannel pipette | |||
| 100 μL multichannel pipette | |||
| 300 μL multichannel pipette | |||
| 10 μL single channel pipette | |||
| 20 μL single channel pipette | |||
| 200 μL single channel pipette | |||
| 1000 μL single channel pipette | |||
| 24-well plates | Fisher | 07-200-84 | sterile with lid |
| 96-well plates | VWR | 10062-900 | sterile with lid |
| Calculator | |||
| Chloramphenicol | Sigma | C0378 | stock (20 mg/mL in Ethanol); final concentration in media (2 μg /mL LBS) |
| Fluorescence dissecting microscope with camera and imaging software | |||
| forceps | Fisher | 08-880 | |
| Kanamycin Sulfate | Fisher | BP906-5 | stock (100 mg/mL in water, filter sterilize); final concentration in media (1 μg/mL LBS) |
| Nitrocellulose membrane (FS MCE, 25MM, NS) | Fisher | SA1J788H5 | 0.22 μm nitrocellulose membrane (pk of 100) |
| petri plates | Fisher | FB0875713 | sterile with lid |
| Spectrophotometer | |||
| Semi-micro cuvettes | VWR | 97000-586 | |
| TipOne 0.1-10 μL starter system | USA Scientific | 1111-3500 | 10 racks |
| TipOne 200 μL starter system | USA Scientific | 1111-500 | 10 racks |
| TipOne 1000 μL starter system | USA Scientific | 1111-2520 | 10 racks |
| Vortex | |||
| Name | Company | Catalog Number | Comments |
| LBS media | |||
| 1M Tris Buffer (pH ~7.5) | 50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base) | ||
| Agar Technical | Fisher | DF0812-17-9 | 15 g (Add only for plates) |
| DI water | 950 mL | ||
| Sodium Chloride | Fisher | S640-3 | 20 g |
| Tryptone | Fisher | BP97265 | 10 g |
| Yeast Extract | Fisher | BP9727-2 | 5 g |
References
- Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. Bacterial competition: surviving and thriving in the microbial jungle. Nature Reviews Microbiology. 8 (1), 15-25 (2010).
- Nyholm, S. V., McFall-Ngai, M. The winnowing: establishing the squid-Vibrio symbiosis. Nature Reviews Microbiology. 2 (8), 632-642 (2004).
- Dörr, N. C. D., Blockesh, M. Bacterial type VI secretion system facilitates niche domination. Proceedings of the National Academy of Sciences of the United States of America. 115 (36), 8855-8857 (2018).
- Maclntyre, D. L., Miyata, S. T., Kitaoka, M., Pukatzki, S. The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proceedings of the National Academy of Sciences of the United States of America. 107 (45), 19520-19524 (2010).
- Salomon, D., Gonzalez, H., Updegraff, B. L., Orth, K. Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2. PloS One. 8 (4), e61086 (2013).
- Sana, T. G., et al. Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proceedings of the National Academy of Sciences of the United States of America. 113 (34), E5044-E5051 (2016).
- Schwarz, S., et al. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathogens. 6 (8), e1001068 (2010).
- Wenren, L. M., Sullivan, N. L., Cardarelli, L., Septer, A. N., Gibbs, K. A. Two independent pathways for self-recognition in Proteus mirabilis are linked by type VI-dependent export. MBio. 4 (4), (2013).
- García-Bayona, L., Guo, M. S., Laub, M. T. J. E. Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins. Elife. 6, 24869 (2017).
- Stubbendieck, R. M., Straight, P. D. Multifaceted interfaces of bacterial competition. Journal of bacteriology. 198 (16), 2145-2155 (2016).
- Cornforth, D. M., Foster, K. R. Antibiotics and the art of bacterial war. Proceedings of the National Academy of Sciences of the United States of America. 112 (35), 10827-10828 (2015).
- Shank, E. A., Kolter, R. New developments in microbial interspecies signaling. Current Opinion in Microbiology. 12 (2), 205-214 (2009).
- Roelofs, K. G., Coyne, M. J., Gentyala, R. R., Chatzidaki-Livanis, M., Comstock, L. E. Bacteroidales secreted antimicrobial proteins target surface molecules necessary for gut colonization and mediate competition in vivo. MBio. 7 (4), e01055-e01016 (2016).
- Dey, A., Vassallo, C. N., Conklin, A. C., Pathak, D. T., Troselj, V., Wall, D. Sibling rivalry in Myxococcus xanthus is mediated by kin recognition and a polyploid prophage. Journal of bacteriology. 198 (6), (2016).
- Danka, E. S., Garcia, E. C., Cotter, P. A. Are CDI systems multicolored, facultative, helping greenbeards?. Trends in Microbiology. 25 (5), 391-401 (2017).
- Willett, J. L., Ruhe, Z. C., Coulding, C. W., Low, D. A., Hayes, C. S. Contact-dependent growth inhibition (CDI) and CdiB/CdiA two-partner secretion proteins. Journal of molecular biology. 427 (23), 3754-3765 (2015).
- Cianfanelli, F. R., Monlezun, L., Coulthurst, S. J. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends in Microbiology. 24 (1), 51-62 (2016).
- Joshi, A., Kostiuk, B., Rogers, A., Teschler, J., Pukatzki, S., Yildiz, F. H. Rules of engagement: the type VI secretion system in Vibrio cholerae. Trends in microbiology. 25 (4), 267-279 (2017).
- Speare, L., et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proceedings of the National Academy of Sciences of the United States of America. 115 (36), E8528-E8537 (2018).
- Shank, E. A. Using coculture to detect chemically mediated interspecies interactions. Journal of Visualized Experiments. (80), (2013).
- Long, R. A., Rowley, D. C., Zamora, E., Liu, J., Bartlett, D. H., Azam, F. Antagonistic interactions among marine bacteria impede the proliferation of Vibrio cholerae. Applied and Environmental Microbiology. 71 (12), 8531-8536 (2005).
- Dunn, A. K., Millikan, D. S., Adin, D. M., Bose, J. L., Stabb, E. V. New rfp-and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Applied and Environmental Microbiology. 72 (1), 802-810 (2006).
- Sana, T. G., et al. The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells. Journal of Biological Chemistry. 287 (32), 27095-27105 (2012).
- Bachmann, V., Kostiuk, B., Unterweger, D., Diaz-Satizabal, L., Ogg, S., Pukatzki, S. Bile salts modulate the mucin-activated type VI secretion system of pandemic Vibrio cholerae. PLoS. 9 (8), e0004031 (2015).
- Ishikawa, T., Rompikuntal, P. K., Lindmark, B., Milton, D. L., Wai, S. N. Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PloS One. 4 (8), e6734 (2009).
- Ishikawa, T., et al. Pathoadaptive conditional regulation of the type VI secretion system in Vibrio cholerae O1 strains. Infection and immunity. 80 (2), 575-584 (2012).
- Pollack-Berti, A., Wollenberg, M. S., Ruby, E. G. Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environmental Microbiology. 12 (8), 2302-2311 (2010).
- Meibom, K. L., Blockesh, M., Dolganov, N. A., Wu, C. Y., Schoolnik, G. K. Chitin induces natural competence in Vibrio cholerae. Science. 310 (5755), 1824-1827 (2005).
- Borgeaud, S., Metzger, L. C., Scrignari, T., Blockesh, M. The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer. Science. 347 (6217), 63-67 (2015).
- Townsley, L., Mangus, M. P. S., Mehic, S., Yildiz, F. H. Response of Vibrio cholerae to low-temperature shift: CpsV regulates type VI secretion, biofilm formation, and association with zooplankton. Applied and Environmental Microbiology. 82 (14), 00807-00816 (2016).
- Huang, Y., et al. Functional characterization and conditional regulation of the type VI secretion system in Vibrio fluvialis. Frontiers in microbiology. 8, 528 (2017).
Cite This Article
Speare, L., Septer, A. N. Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates. J. Vis. Exp. (149), e59759, doi:10.3791/59759 (2019).