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Abstract
Introduction
Protocol
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Materials
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Immunologie et infection

噬菌体介导的莱姆病螺旋体伯氏疏螺旋体的遗传操作

Published: September 28, 2022
doi:
10.3791/64408
1Department of Biomedical Sciences,Quinnipiac University

Summary

噬菌体在细菌细胞之间移动DNA的能力使其成为细菌宿主遗传操作的有效工具。这里介绍的是一种诱导、恢复和使用 φBB-1（ 伯氏疏螺旋体噬菌体）在莱姆病螺旋体的不同菌株之间转导异源 DNA 的方法。

Abstract

将外源DNA引入伯氏螺旋体伯 氏疏螺旋 体几乎完全通过使用电穿孔的转化来完成。与其他特征更好的革兰氏阴性细菌相比，这一过程在莱姆病螺旋体中的效率显着降低。 转化的成功率高度依赖于来自特定背景的高质量DNA的浓缩量，并且受到显着的菌株间变异性的影响。将外源DNA（即穿梭载体、荧光报告基因和抗生素耐药性标记物）引入 伯氏螺旋体 的替代方法可能是莱姆病螺旋体遗传操作有用工具的重要补充。噬菌体已被公认为DNA在称为转导过程中在细菌之间移动的天然机制。在这项研究中，已经开发出一种使用无处不在的疏螺旋体噬菌体φBB-1在相同和不同遗传背景的 伯氏疏螺旋体 细胞之间转导DNA的方法。转导的DNA包括小穿梭载体形式的疏螺旋体DNA和异源DNA。这一证明表明，噬菌体介导的转导作为电穿孔的补充，用于莱姆病螺旋体的遗传操作。本报告描述了从 伯氏疏螺旋体中诱导和纯化噬菌体φBB-1的方法，该噬菌体在转导测定中的应用，以及潜在转导剂的选择和筛选。

Introduction

螺旋体细菌伯氏疏螺旋体遗传操作工具的开发为理解莱姆病的性质增加了不可估量的价值1，2，3，4。伯氏双歧杆菌具有异常复杂的基因组，由一条小的线性染色体以及线性和环状质粒5，6组成。自发质粒丢失、基因内重排（基因在同一生物体内从一个质粒移动到另一个质粒）和水平基因转移（HGT，两个生物体之间的DNA移动）在伯氏双歧杆菌中引起了令人眼花缭乱的遗传异质性（例如，参见Schutzer等人7）。由此产生的基因型（或“菌株”）都是同一物种的成员，但具有遗传差异，影响它们传播和感染不同哺乳动物宿主的能力8，9，10，11。在本报告中，术语“菌株”将用于指具有特定自然来源遗传背景的伯氏双歧杆菌;术语“克隆”将用于指为特定目的或作为实验操作结果而进行基因改造的菌株。

可用于伯氏芽孢杆菌的分子工具箱包括可选标记、基因报告基因、穿梭载体、转座子诱变、诱导启动子和反选择性标记（有关综述，请参阅Drektrah和Samuels12）。这些方法的有效使用需要人工将异源（外来）DNA引入感兴趣的伯氏疏螺旋体菌株中。在伯氏疏螺旋体中，异源DNA的引入几乎完全是通过电穿孔实现的，电穿孔是一种利用电脉冲使细菌膜瞬时渗透到引入培养基中的小块DNA的方法1。大多数细胞（估计为≥99.5%）被脉冲杀死，但其余细胞保留异源DNA的频率很高13。虽然被认为是将DNA引入细菌的最高效方法之一，但电穿孔到伯氏疏螺旋体的频率非常低（范围从5×104到5×106细胞中的1个转化体）13。实现更高频率转变的障碍似乎是技术和生物方面的。在制备电感受态细胞时，伯氏疏螺旋体成功电穿孔的技术障碍包括所需的 DNA 量 （>10 μg） 和螺旋体在制备电感受态细胞时完全处于正确的生长期（对数中期，在 2 × 10 7 个细胞·mL−1 和 7 × 107 个细胞·mL−1 之间）的要求12，13。然而，这些技术障碍可能比生物障碍更容易克服。

莱姆病研究人员认识到，伯氏疏螺旋体克隆可分为两大类，就其遗传操纵能力而言13，14。高传代、实验室适应的分离株通常很容易转化，但通常已经失去了感染性所必需的质粒，行为异常，并且无法感染哺乳动物宿主或持续存在于蜱载体12，13 中。虽然这些克隆对于在实验室内剖析螺旋体的分子生物学很有用，但它们对于在地方性动物病循环的生物学背景下研究螺旋体几乎没有价值。另一方面，低传代感染分离株以反映感染状态的生理方式表现，可以完成感染周期，但通常难以引入异源DNA，因此难以操作用于研究12，13。转化低传代分离株的困难至少与两个不同的因素有关：（i）低传代分离株通常紧密聚集在一起，特别是在电穿孔所需的高密度条件下，从而阻止许多细胞充分应用电荷或获取培养基中的DNA13，15;（ii）伯氏双歧杆菌编码至少两种不同的质粒携带的限制性限制性修饰（R-M）系统，这些系统可能在高传代分离株14，16中丢失。R-M系统已经进化到允许细菌识别和消除外来DNA17。事实上，对伯氏芽孢杆菌的几项研究表明，当DNA的来源是伯氏疏螺旋体而不是大肠杆菌时，转化效率会提高13，16。不幸的是，从伯氏疏螺旋体获得电穿孔所需的高浓度DNA是一个昂贵且耗时的前景。电泳和选择低传代分离物时的另一个潜在问题是，该过程似乎有利于失去关键毒力相关质粒lp2514，18，19的转化体;因此，通过电穿孔对低传代伯氏疏螺旋体分离株进行遗传操作的行为可能会选择不适合在地方性动物病循环20中进行生物学相关分析的克隆。鉴于这些问题，一个可以将异源DNA电转化为高传代伯氏疏螺旋体克隆，然后通过电穿孔以外的方法转移到低传代感染性分离株中的系统可能是可用于莱姆病螺旋体的越来越多的分子工具集合的受欢迎的补充。

除了转化（裸DNA的摄取）之外，细菌还定期吸收异源DNA的另外两种机制：偶联，这是细菌之间直接物理接触的DNA交换，以及转导，这是由噬菌体介导的DNA交换21。事实上，噬菌体介导HGT的能力已被用作解剖许多细菌系统内分子过程的实验工具22，23，24。B. burgdorferi不能天然地摄取裸DNA的能力，并且几乎没有证据表明B. burgdorferi编码促进成功偶联所需的装置。 然而，以前的报告已经描述了φBB-1的鉴定和初步表征，φBB-1是B. burgdorferi25，26，27，28的温带噬菌体。φBB-1 包装了在伯氏疏螺旋体25 中发现的 30 kb 质粒家族;该家族的成员被指定为CP32。与φBB-1在伯氏疏螺旋体中参与HGT的作用一致，Stevenson等人报告了在两种菌株中发现的相同的cp32，在其他方面不同的cp32s中，表明最近在这两种菌株之间共享该cp32，可能是通过转导29。还有证据表明，在其他相对稳定的基因组30，31，32，33中，cp32s通过HGT进行了显着重组。最后，φBB-1在同一菌株的细胞之间以及两种不同菌株的细胞之间转导cp32s和异源穿梭载体DNA的能力已在先前证明27，28。鉴于这些发现，φBB-1已被提议作为另一种用于解剖伯氏疏螺旋体分子生物学的工具。

本报告的目的是详细介绍一种从 伯氏疏螺旋体中诱导和纯化噬菌体φBB-1的方法，并提供一种在 伯氏疏螺旋体 克隆之间进行转导测定以及选择和筛选潜在转导剂的方案。

Protocol

所有使用重组DNA和BSL-2生物体的实验都经过昆尼皮亚克大学机构生物安全委员会的审查和批准。 1. 伯氏芽孢杆菌 培养物的制备，用于生产φBB-1 准备补充有 6.6% 热灭活正常兔血清 （BSK） 的巴伯-斯托纳-凯利培养基15。对于 1 L 的 1x BSK，将表 1 中列出的组分混合在 900 mL 水中，使用 1 N 氢氧化钠将 pH 调节至 7.6，并在 4 °C ?…

Representative Results

使用噬菌体在更容易转化的 伯氏疏螺旋体 菌株或难以电转化的克隆之间移动DNA，代表了对莱姆病决定因素进行持续分子研究的另一种工具。本文描述的转导测定可以根据需要进行修改，以促进DNA在任何感兴趣的克隆之间的移动，使用一种或两种抗生素来选择潜在的转导剂。高传代菌株CA-11.2A克隆与高传代菌株B31克隆和菌株297的低传代毒力克隆之间的噬菌体DNA和异源 大肠杆菌/伯氏…

Discussion

转导的使用可以代表一种克服至少一些与伯氏疏螺旋体1，4，13，37电转化相关的生物学和技术障碍的方法。在许多系统中，噬菌体可以通过广义或特化的转导在细菌细胞之间移动宿主（非噬菌体）DNA22，23，24，49<sup …

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者希望感谢Shawna Reed，D. Scott Samuels和Patrick Secor的有益讨论以及Vareeon （Pam） Chonweerawong的技术援助。这项工作得到了生物医学科学系的支持，并为昆尼皮亚克大学健康科学学院的Christian H. Eggers提供了研究资助。

Materials

1 L filter units (PES, 0.22 µm pore size) Millipore Sigma S2GPU10RE
12 mm x 75 mm tube (dual position cap) (polypropylene) USA Scientific 1450-0810 holds 4 mL with low void volume (for induction)
15 mL conical centrifuge tubes (polypropylene) USA Scientific 5618-8271
1-methyl-3-nitroso-nitroguanidine (MNNG) Millipore Sigma CAUTION: potential carcinogen; no longer readily available, have not tested offered substitute
5.75" Pasteur Pipettes (cotton-plugged/borosilicate glass/non-sterile) Thermo Fisher Scientific 13-678-8A autoclave prior to use
50 mL conical centrifuge tubes (polypropylene) USA Scientific 1500-1211
Absolute ethanol
Agarose LE Dot Scientific inc. AGLE-500
Bacto Neopeptone Gibco DF0119-17-9
Bacto TC Yeastolate Gibco 255772
Bovine serum albumin (serum replacement grade) Gemini Bio-Products 700-104P
Chloroform (for molecular biology) Thermo Fisher Scientific BP1145-1 CAUTION: volatile organic; use only in a chemical fume hood
CMRL-1066 w/o L-Glutamine (powder) US Biological C5900-01 cell culture grade
Erythromycin Research Products International Corp E57000-25.0
Gentamicin reagent solution Gibco 15750-060
Glucose (Dextrose Anhydrous) Thermo Fisher Scientific BP350-500
HEPES Thermo Fisher Scientific BP310-500
Kanamycin sulfate Thermo Fisher Scientific 25389-94-0
Millex-GS (0.22 µM pore size) Millipore Sigma SLGSM33SS to filter sterilize antibiotics and other small volume solutions
Mitomycin C Thermo Fisher Scientific BP25312 CAUTION: potential carcinogen; use only in a chemical fume hood
N-acetyl-D-glucosamine MP Biomedicals, LLC 100068
Oligonucleotides (primers for PCR) IDT DNA
OmniPrep (total genomic extraction kit) G Biosciences 786-136
Petri Dish (100 mm × 15 mm) Thermo Fisher Scientific FB0875712
Petroff-Hausser counting chamber Hausser scientific HS-3900
Petroff-Hausser counting chamber cover glass Hausser scientific HS-5051
Polyethylene glycol 8000 (PEG) Thermo Fisher Scientific BP233-1
Rabbit serum non-sterile trace-hemolyzed young (NRS) Pel-Freez Biologicals 31119-3 heat inactivate as per manufacturer's instructions
Semi-micro UV transparent cuvettes USA Scientific 9750-9150
Sodium bicarbonate Thermo Fisher Scientific BP328-500
Sodium chloride Thermo Fisher Scientific BP358-1
Sodium pyruvate Millipore Sigma P8674-25G
Spectronic Genesys 5 Thermo Fisher Scientific
Streptomycin sulfate solution Millipore Sigma S6501-50G
Trisodium citrate dihydrate Millipore Sigma S1804-500G sodium citrate for BSK
DOWNLOAD MATERIALS LIST

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Keywords: Phage TransductionBorrelia BurgdorferiLyme DiseaseBiologie moléculaireGenetic ManipulationPhage ProductionPEG PrecipitationAntibiotic Selection
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Eggers, C. H. Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi. J. Vis. Exp. (187), e64408, doi:10.3791/64408 (2022).
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