细菌结合频率的高分辨率比较
Summary
为了了解不同条件下各种细菌结合 dna 元素的行为, 我们描述了一种检测共轭频率差异的协议, 具有较高的分辨率, 以估计供体细菌的效率启动共轭。
Abstract
细菌结合是通过共轭 dna 元素水平转移抗生素耐药基因的重要步骤。需要对不同条件下的共轭频率进行深入的比较, 以了解共轭元素在自然界中的传播情况。然而, 传统的比较共轭频率的方法不适合进行深入的比较, 因为选择性板上出现额外的共轭事件会导致高背景。我们成功地减少了背景, 引入了最可能的数字 (mpn) 方法和更高浓度的抗生素, 以防止进一步共轭在选择性液体介质。此外, 我们还开发了一个协议, 通过荧光激活细胞分选 (facs) 将单个供体细胞分化为受体池, 估计供体细胞启动共轭的概率。利用 pbp136 和 pcar1 这两种质粒, 可以在不同搅拌速率的液体介质中检测到假单胞菌细胞的共轭频率差异。pbp136 的共轭起始频率高于 pcar1。利用这些结果, 我们可以更好地了解这两个质粒中的共轭特征。
Introduction
流动遗传元素的细菌结合、共轭质粒以及整合和共轭元素 (ice) 对于遗传信息的水平传播具有重要意义。它能促进细菌的快速进化和适应, 传递耐多药基因1,2。共轭频率可能会受到蛋白质编码的共轭元素动员 dna (mob) 和交配对形成 (mpf), 包括性别皮利, 这是根据 mob 和 mpf 类型3,4分类, 5。它也可能受到供体和受体对6以及细胞7、8、9、10、11、12 (生长速率、细胞密度、固体表面或液体介质、温度、养分供应和阳离子的存在)。为了了解共轭元素是如何在细菌中传播的, 有必要对共轭频率进行详细的比较。
交配后供体和受者对之间的共轭频率通常用常规方法估计如下。(一) 首先, 计算捐助者和受援群体的数目;(ii) 然后, 接受共轭元素的接受的殖民地 (= 跨康尼加人) 计数;(iii) 最后, 共轭频率是通过将异位形成单位 (cfu) 除以供体和/或接受者13的菌落形成单位 (cfu) 来计算的。但是, 在使用此方法时, 背景较高, 因为在细胞密度较高时, 用于获取跨共的选择性板上也可能发生额外的共轭事件。因此, 很难检测到频率上的微小差异 (低于10倍差异)。我们最近介绍了一种最可能的数字 (mpn) 方法, 使用含有较高浓度抗生素的液体介质。该方法通过抑制选择性介质中的进一步共轭来减小背景;因此, 可以用更高的分辨率来估计共轭频率。
共轭可分为三个步骤: (1) 受赠方-收件人对的附件 (2) 共轭转移的起始, (3) 对14的离解.在步骤 (1) 和 (3) 中, 供体和接收细胞之间存在物理相互作用;因此, 细胞密度和环境条件会影响这些步骤, 尽管性毛的特征也很重要。步骤 (2) 可能是由参与共轭的几个基因的表达来调节的, 这些基因可能会受到质粒、供体和受体的各种特征的影响。虽然可以使用细胞作为粒子的估计进行物理模拟, 但应该对步骤 (2) 的频率进行实验测量。有一些报告直接观察如何直接观察相比, 多久可以启动共轭 [步骤 (2)] 使用荧光显微镜15,16;但是, 这些方法不是高吞吐量, 因为必须监视大量的单元。因此, 我们开发了一种利用荧光活化细胞分选 (facs) 估计步骤 (2) 发生概率的新方法。我们的方法可以应用于任何质粒, 而无需识别共轭的基本基因。
Protocol
Representative Results
Discussion
在这里, 我们提出了一个高分辨率的协议, 用于检测不同条件下的共轭频率的差异, 使用 mpn 方法来估计跨子的数量。该协议中的一个重要步骤是在交配后稀释捐献者和受赠者的混合物, 直到没有转基因生物生长。另一个步骤是在选择性液体培养基中添加高浓度的抗生素, 以防止进一步的共轭。这些程序可以减少选择性介质中进一步共轭所造成的背景。我们可以成功地检测差异, 即使在捐献者和接受?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢国家传染病研究所 (日本) 的 k. kamachi 博士提供 pbp136, 并感谢东京大学 (日本) h. nojiri 教授提供 pcar1。我们还感谢丹麦技术大学的莫林·索伦教授提供 pjba28。这项工作得到了 jps kakenhi (赠款编号15h05618 和 15H05618) 至 ms (https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15H05618/, https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15KK0278/) 的支持。
Materials
| MoFlo XDP | Beckman-Coulter | ML99030 | FACS |
| IsoFlow | Beckman-Coulter | 8599600 | Sheath solution |
| Fluorospheres (10 μm) | Beckman-Coulter | 6605359 | beads to set up the FACS |
| Incubator | Yamato Scientific Co. Ltd | 211197-IC802 | |
| UV-VIS Spectrophotometer UV-1800 | SIMADZU Corporation | UV-1800 | |
| 96-well plates | NIPPON Genetics Co, Ltd | TR5003 | |
| microplate type Petri dish | AXEL | 1-9668-01 | for validation of sorting |
| membrane filter | ADVANTEC | C045A025A | for filter mating |
| pippettes | Nichiryo CO. Ltd | 00-NPX2-20, 00-NPX2-200, 00-NPX2-1000 |
0.5-10 μL, 20-200 μL, 100-1000 μL |
| multi-channel pippetes | Nichiryo CO. Ltd | 00-NPM-8VP, 00-NPM-8LP |
0.5-10 μL, 20-200 μL |
| Tryptone | BD Difco | 211705 | |
| Yeast extract | BD Difco | 212750 | |
| NaCl | Sigma | S-5886 | |
| Agar | Nakarai tesque | 01162-15 | |
| rifampicin | Wako | 185-01003 | |
| gentamicin | Wako | 077-02974 | |
| kanamycin | Wako | 115-00342 | |
| Petri dish | AXEL | 3-1491-51 | JPND90-15 |
| microtubes | Fukaekasei | 131-815C | |
| 500 mL disposable spinner flask | Corning | CLS3578 |
References
- Cabezon, E., Ripoll-Rozada, J., Pena, A., de la Cruz, F., Arechaga, I. Towards an integrated model of bacterial conjugation. FEMS Microbiology Reviews. 39 (1), 81-95 (2015).
- Johnson, C. M., Grossman, A. D. Integrative and conjugative elements (ICEs): what they do and how they work. Annual Review of Genetics. 49, 577-601 (2015).
- Garcillán-Barcia, M. P., Alvarado, A., de la Cruz, F. Identification of bacterial plasmids based on mobility and plasmid population biology. FEMS Microbiology Reviews. 35 (5), 936-956 (2011).
- Smillie, C., Garcillán-Barcia, M. P., Francia, M. V., Rocha, E. P., de la Cruz, F. Mobility of plasmids. Microbiology and Molecular Biology Reviews. 74 (3), 434-452 (2010).
- Garcillán-Barcia, M. P., Francia, M. V., de la Cruz, F. The diversity of conjugative relaxases and its application in plasmid classification. FEMS Microbiology Reviews. 33 (3), 657-687 (2009).
- Shintani, M., et al. Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains. Biotechnology Letters. 27 (23-24), 1847-1853 (2005).
- Yanagida, K., et al. Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities. Bioscience, Biotechnology, and Biochemistry. 80 (5), 1020-1023 (2016).
- Schuurmans, J. M., et al. Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains. Plasmid. 72, 1-8 (2014).
- Bradley, D. E., Taylor, D. E., Cohen, D. R. Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12. Journal of Bacteriology. 143 (3), 1466-1470 (1980).
- Nakazawa, S., et al. Different transferability of incompatibility (Inc) P-7 plasmid pCAR1 and IncP-1 plasmid pBP136 in stirring liquid conditions. PLoS One. 12 (10), e0186248 (2017).
- Verma, T., Ramteke, P. W., Garg, S. K. Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent. Biotechnology and Applied Biochemistry. 102 (1-6), 5-20 (2002).
- Sakuda, A., et al. Divalent cations increase the conjugation efficiency of the incompatibility P-7 group plasmid pCAR1 among different Pseudomonas hosts. Microbiology. 164 (1), 20-27 (2018).
- Corliss, T. L., Cohen, P. S., Cabelli, V. J. R-plasmid transfer to and from Escherichia coli strains Isolated from human fecal samples. Applied and Environmental Microbiology. 41 (4), 959-966 (1981).
- Zhong, X., Krol, J. E., Top, E. M., Krone, S. M. Accounting for mating pair formation in plasmid population dynamics. Journal of Theoretical Biology. 262 (4), 711-719 (2010).
- Gilmour, M. W., Lawley, T. D., Taylor, D. E. The cytology of bacterial conjugation. EcoSal Plus. 2004, (2004).
- Minoia, M., et al. Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas. Proceedings of the National Academy of Sciences of the United States of America. 105 (52), 20792-20797 (2008).
- Kamachi, K., et al. Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements. Microbiology. 152 (12), 3477-3484 (2006).
- Simon, R., Priefer, U., Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nature Biotechnology. 1 (9), 784-791 (1983).
- Andersen, J. B., et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Applied and Environmental Microbiology. 64 (6), 2240-2246 (1998).
- Shintani, M., et al. Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation. Applied and Environmental Microbiology. 72 (5), 3206-3216 (2006).
- Shintani, M., et al. Single-cell analyses revealed transfer ranges of IncP-1, IncP-7, and IncP-9 plasmids in a soil bacterial community. Applied and Environmental Microbiology. 80 (1), 138-145 (2014).
- Jarvis, B., Wilrich, C., Wilrich, P. T. Reconsideration of the derivation of most probable numbers, their standard deviations, confidence bounds and rarity values. Journal of Applied Microbiology. 109 (5), 1660-1667 (2010).
- Haagensen, J. A., Hansen, S. K., Johansen, T., Molin, S. In situ detection of horizontal transfer of mobile genetic elements. FEMS Microbiology Ecology. 42 (2), 261-268 (2002).
- Herrero, M., de Lorenzo, V., Timmis, K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. Journal of Bacteriology. 172 (11), 6557-6567 (1990).
- Bagdasarian, M., et al. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene. 16 (1-3), 237-247 (1981).
- Maeda, K., et al. Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676. Journal of Molecular Biology. 326 (1), 21-33 (2003).
- Takahashi, Y., Shintani, M., Yamane, H., Nojiri, H. The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids. Bioscience, Biotechnology, and Biochemistry. 73 (3), 744-746 (2009).
