接合交配测定在接合介入转移蛋白的序列特异性分析
Summary
Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.
Abstract
The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.
Introduction
基因和蛋白质的在微观有机体水平转移起着细菌生存和进化以及感染过程的核心作用。的DNA在细菌之间或细菌和小区之间交换可以通过转化，接合或载体转导来实现。 1,2-共轭是革兰氏阴性细菌如大肠杆菌之间缀合过程中的比较，以在转换和转导独特，DNA转移中的供体-控制的方式，由此复杂的高分子系统连接供体和受体细胞发生。偶联也是其中的细菌细胞与宿主细胞相互作用，以注入基因，蛋白质或化学品中的主机系统的最直接的方式。 3很多时候，这些药剂的转移在主机上拥有显着的效果，从发病机理和致癌主办进化和适应。它已经表明，共轭recombinatioN增加适应3倍的细菌具有高的突变率环境胁迫的条件下的速率。 4此外，缀合是迄今为止通过在细菌菌株的抗生素抗性基因被传播的最常见的途径。 5,6
微生物已经进化专门的分泌系统，以支持跨越细胞膜大分子的转移;目前有9种类型的革兰氏阴性细菌分泌系统（的TSS）的已经被描述：T1SS，T2SS，T3SS，T4SS，T5SS，T6SS，T7SS，以及上述Sec（分泌）和TAT（双精氨酸易位）通路。 7,8-每个型分泌系统的进一步分为不同的亚型，必然因蛋白质的多样性和所涉及的途径的独特性，在不同的细菌菌株。例如，在IV型分泌系统（T4SS），Ti和CAG系统便于执行传输而在F-质粒，R27一第二pKM101 T4SSs便于接合质粒转移。 7,9,10由生物体及其组成蛋白质组装各自的分泌系统和共享细胞内容与收件人或其周围环境的机制进行了详细的了解是有针对性的发展战略，以打击病原微生物的过程中的一个重要因素细胞感染。
以下在大肠杆菌中由莱德伯格＆塔特姆，11初始识别接合大量的移动和接合质粒已被鉴定和表征。 12这种移动质粒显示出相当大的范围是尺寸（从1至200千碱基（kb的）），但是所有移动质粒含有一个relaxase，其识别传送（ORIT）从而使质粒的传输的起源。接合质粒进一步编码基因的功能性T4SS的装配以及一型四耦合蛋白。 12例如大肠杆菌的100 kb的˚F质粒编码33.3 KB转移（TRA）区域内的所有的共轭基因。 13时接合质粒转移有利于形成菌毛所有的蛋白质，交配对形成（MPF），DNA转移和排除功能的F质粒编码的TRA区域的基因。 10,14,15知识的显著体可用于接合T4SSs，但详细介绍了共轭蛋白质和复合物的结构研究只是最近变得可用。 16 – 28
为了组装工艺接合的全面视图，需要突变接合转移蛋白分析详细的结构研究的耦合。这可以通过接合交配试验来实现。为F质粒的TRA区域内编码的每个蛋白质起着的F -介导的共作用njugation;因此，敲除/缺失转印基因将废除细胞（ 图1）的共轭的能力。而较小的移动质粒是更有利于标准的删除程序，对于较大接合质粒如F，基因敲除更容易通过同源重组，其中靶基因被替换为一个传送一个不同的抗生素抗性基因来实现的。在目前的协议中，我们采用同源重组，以取代在55 kb的质粒˚F衍生pOX38锝氯霉素乙酰转移酶（CAT）的兴趣转移基因; 29,30所得敲除质粒pOX38锝Δgene::厘米，有利于耐氯霉素中生长培养基的存在下（厘米）。供体细胞窝藏pOX38锝Δgene::厘米不能影响通过使用检测交配观察接合DNA转移/交配;一个pOX38锝Δgene::厘米施主小区的配合效率和正常RECIPient将减少，或者更多的时候，被废除。在pOX38锝Δgene::厘米质粒的接合转移可以通过一个小复苏质粒窝藏转移靶向基因来恢复。此恢复的质粒可以是一个能够提供组成型表达，如质粒pK184（pK184基因），31个或一个，只要该质粒正确靶向基因到细胞（细胞质或周质）内的正确位置提供了诱导型表达。因此，在这种新的供体之间的配合测定（窝藏pOX38锝Δgene::厘米+ pK184基因质粒） 和受体细胞，匹配效率有望恢复到接近一个正常的供受体匹配检测的。这个系统使得一个通过一系列pK184基因构建体（缺失或点突变）的产生来探测敲除基因的功能和测试每个构建体的还原pOX38锝Δgene::厘米窝藏的配合能力的能力供体细胞秒。
Protocol
Representative Results
Discussion
接合方法提供通过该细菌可以传播基因在挑战性的环境，如抗生素抗性标记的蔓延生长提供进化优势的装置。因为许多接合型质粒的是如此之大，在通过靶基因的突变对接合型质粒本身涉及的传送装置的组件中的蛋白12的功能的研究是非常笨重。本文详述的协议提供了通过其中一个可以更容易地通过使用更小，更容易管理的表达质粒（ 图1）的评估的目的靶基因的方法。我们?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是由来自加拿大的自然科学与工程理事会授予发现（NSERC）的支持。
Materials
| GeneJet Plasmid Mini-Prep Kit | Fisher Scientific | K0503 | |
| GeneJet Gel Extraction Kit | Fisher Scientific | K0692 | |
| GeneJet PCR Purification Kit | Fisher Scientific | K0702 | |
| Q5 Site-Directed Mutagenesis Kit | New England Biolabs | E0554S | |
| Broad Range DNA Ladder | New England Biolabs | N0303A | |
| Petri Dishes | Fisher Scientific | FB0875713 | |
| Electroporator | Eppendorf | 4309000027 | |
| Electroporation cuvettes | Fisher Scientific | FB101 | Cuvettes have a 1 mm gap. |
| Enzymes | |||
| AvaI | New England Biolabs | R0152S | |
| EcoRI | New England Biolabs | R0101S | |
| HindIII | New England Biolabs | R0104L | |
| NdeI | New England Biolabs | R0111S | |
| Phusion DNA Polymerase | New England Biolabs | M0530L | |
| T4 DNA Ligase | New England Biolabs | M0202S | |
| DpnI | New England Biolabs | R0176S | |
| Antibiotics | Final Concentrations | ||
| Chloramphenicol (Cm) | Fisher Scientific | BP904-100 | 20 µg/mL |
| Kanamycin (Km) | BioBasic Inc. | DB0286 | 50 µg/mL |
| Nalidixic acid (Nal) | Sigma-Aldrich | N8878-25G | 10 µg/mL |
| Rifampicin (Rif) | Calbiochem | 557303 | 20 µg/mL |
| Tetracycline (Tc) | Fisher Scientific | BP912-100 | 10 µg/mL |
| Streptomycin (Sm) | Fisher Scientific | BP910-50 | 50 µg/mL |
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