Name: conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation
Uploaded: 2017-01-04T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

この研究は、自然科学からディスカバリーグラント＆カナダのエンジニアリング評議会（NSERC）によってサポートされていました。

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

細菌接合プロセスは、細菌は、抗生物質耐性マーカーの普及など厳しい環境での成長のための進化上の利点を提供する遺伝子を広げることができる手段を提供します。接合性プラスミドの多くは非常に大きいので、接合プラスミド自体に標的遺伝子の突然変異によって転写装置の組み立てに関与するタンパク質上の12機能的研究は扱いにくいです。本明細書で詳細なプロトコルは、一つにはより容易に、より小さく扱い発現プラスミド（ 図1）を使用することによって、目的の標的遺伝子を評価することができる手段を提供します。我々は、Fプラスミド媒介性の結合を研究するためにFプラスミドの誘導体pOX38-TC（ 表1）を使用します 。他の接合性プラスミドはここで詳述プロトコルと適切な派生プラスミドを使用して研究することができます。概説交配アッセイは、いくつかのmodificatiとフロストや同僚、42から適応されていますアドオン。以前の研究では、pOX38-TCΔgene:: Cmを構築物の8,33,42作成は、適切な制限酵素を用いて目的の遺伝子を切断し、pOX38-TCに増幅CATカセットを挿入することによって達成されました。現在の方法では42、我々は、標的遺伝子をノックアウトし、CATカセットと交換するリコンビニアリング大腸菌株 DY330R 33,34における相同組換えを使用します。 F-T4SSはpK184遺伝子回復プラスミドを用いた転写の回収を介して接合伝達を媒介外これはpOX38-TCΔgene:: Cmは、遺伝子特異的ノックアウトのための対照として作用するように構成保有得DY330R株を可能にするという利点を有します。それは野菜室-Cas9方法論を用いてノックアウトを生成することが可能かもしれないが、43は、現時点ではこの可能性を調査していません。 プロセスは、F派生プラスミドpOX38-TC（Figurに遺伝子ノックアウトの世代で始まりますE 1）。これはDY330R細胞（ 表1）中での相同組換えを介して達成され、例えば、DY329、DY331およびDY378と同様の機能を有する他の株も使用することができます。プライマーは、最初pBAD33プラスミド32からCATカセットを増幅し、標的遺伝子（ 表2）に特異的なオーバーハング塩基を含むPCRように設計されています。 PCR産物を、次いでpOX38-TCを保有DY330R細胞にエレクトロポレーションされます。相同組換えは、CATのカセットがCm耐性を提供しながら、効果的にT4SSアセンブリおよび結合を破壊し、標的遺伝子内にインフレームで挿入されているノックアウトプラスミドpOX38-TCΔgene:: Cmとを生成します。これと同時に、標的遺伝子はpOX38-TCからPCR増幅し、小発現プラスミドに挿入し、このプロトコルでは、我々は、必要に応じてしかし1は、標的遺伝子の誘導発現とプラスミドを選んだ可能性があり、構成的発現のためにpK184を使用します。 pK184-遺伝子プラスミドは、その後、形質転換されますDY330R pOX38-TCΔgene:: Cmを細胞に、これらの細胞は、次いでpOX38-TCΔgene:: Cmは交配アッセイのためXK1200細胞への結合を介して構築転送するためのドナーとして使用されます。交配アッセイでは、ドナーとレシピエント細胞は、それぞれXK1200 pOX38-TCΔgene:: CMとMC4100、です。 pK184遺伝子回収プラスミド、ならびに標的遺伝子（欠失または点変異）の一連の変異体は、MC4100のレシピエント細胞（ 図2を用いて、交配を復元し、その効率を決定する能力を評価するために、ドナー細胞に提供されます。 ;表3）。 手順にとって重要適切なドナーおよびレシピエント株（ 表1）、および使用されるプライマーの設計を使用することです。設計された各プライマーについて、従うべき一般的なガイドラインがあります。我々は厳密に40から60パーセントのGC含量に遵守しようとするが、これは常に可能ではないかもしれません。このような場合には、プライマーWをテストするために実験者の判断でありますわずかにこの範囲を下回るまたは上記i番目のGC含量。順方向および逆方向プライマーの融解温度（T m）は類似していなければならない、アニール温度が（T a）の値は常にPCR 2-5゜CによるT mよりも低くなければなりません。ホモおよびヘテロ二量体化のT m &#39;はsは（より-10キロジュールと30°C以下）非常に低くなければならない一方でヘアピンが開発できるようにするために利用可能な自由エネルギーは、Tよりもはるかに低くする必要があります。プライマーは、所望のように欠失、挿入または点変異を調べるために設計することができます。なお、得られた遺伝子構築物が増幅され、それが適切に発現されるようにインフレームで回収プラスミドに連結されることが当然重要です。コンピテント細胞の形質転換は、それぞれ、エレクトロまたは化学的コンピテント細胞を用いてエレクトロポレーションまたは熱ショックを介して行うことができます。私たちは、エレクトロポレーションは、このようなpOX38-TCと相同組換えoligonucleとして大きな構成のためのより効率的であることがわかりotide、例えばpK184-TRAFとしてより小さい発現プラスミドは、容易に熱ショック方法を用いて、細胞に形質転換することができるが。最後に、両方のドナーとレシピエント株は接合およびリカバリのプラスミド上で使用するものとは異なる別の抵抗を必要とするように、プロトコル全体で使用されている複数の抗生物質が存在するであろうことを覚えておくことが重要です。 別にここで説明交配アッセイ技術から、彼らのアプローチで若干変動する細菌接合を研究するために使用されている他の方法が、あります。水平遺伝子伝達は、細菌が種間の受信者を含む、レシピエント細胞へのプラスミドを転送するプロセスです。 44例えばダールバーグと同僚44による研究は、種間の水平遺伝子伝達の程度を決定するために、細菌接合を使用していました。それらは、KT2442細胞にクローン化したプラスミドに、緑色蛍光タンパク質（GFP）の取り込みを使用しました。染色体リットルKT2442細胞における交流I q遺伝子は、GFPの発現を抑制する。 GFPを担持するプラスミドをlacリI q遺伝子なし種に転送されると、蛍光が観察されます。 44は、種々の種におけるタンパク質の特定の機能を提供するために、その制限にもかかわらず、種間の結合実験は、おそらく異なる種間のタンパク質-タンパク質相互作用のための進化の予測を行うためにここに提示されたプロトコルを用いて結合することができます。

ミクロ生物レベルでの遺伝子やタンパク質の転送は、細菌の生存と発展だけでなく、感染の過程において中心的な役割を果たしています。細菌の間、または細菌と細胞間のDNAの交換は、形質転換、接合またはベクターの形質導入によって達成することができます。 1,2結合は、複雑な高分子システムがドナーとレシピエント細胞を接続することによりDNAの転移がドナー・制御された様式で発生し、 大腸菌などのグラム陰性菌間の共役中にその中の形質転換および形質導入と比較してユニークです。結合はまた、細菌細胞は、ホストシステムの中での遺伝子、タンパク質または化学物質を注入するために、宿主細胞と相互作用する最も直接的な方法です。 図3は、かなり頻繁に、このような薬剤の転送は、進化と適応をホストするために発病し、発癌に至るまで、ホスト上の顕著な効果を持っています。その接合recombinatioが示されていますnは環境ストレスの条件下で高い突然変異率を持つ細菌に適応3倍の速度を増加させます。 4さらに、結合は、はるかに菌株における抗生物質耐性遺伝子が拡散され、それを通して、最も一般的な経路です。 5,6 微生物は細胞膜を横切る巨大分子の転送をサポートするために特殊な分泌系を進化させてきました。 T1SS、T2SS、T3SS、T4SS、T5SS、T6SS、T7SS、ならびに秒（分泌）およびTat（2-アルギニン転座）：記載されているグラム陰性菌における分泌系（のTSS）の9種類が現在あります経路。分泌系の7,8各タイプは、さらに、異なるサブタイプに起因するタンパク質の多様性と異なる細菌株で、関与する経路の特殊性への必要性を分割されています。例えば、IV型分泌系（T4SS）で、TiおよびCAGシステムはF-プラスミド、R27 Aに対し、エフェクター輸送を促進しますND pKM101 T4SSsは、接合性プラスミドの伝達を促進します。生物はその構成タンパク質からそれぞれの分泌系を組み立てると、受信者またはその周囲の環境と細胞内容を共有するメカニズムの7,9,10詳細な理解は、病原性微生物やプロセスに対抗するための目標と戦略の開発における重要な因子であります携帯感染。 レーダーバーグ＆テイタムによって大腸菌での最初の同定細菌接合に続いて、モバイルおよび接合性プラスミドの11多数同定され、特徴付けられています。 12このようなモバイルプラスミドは、かなりの範囲は（1から200以上のキロベース（キロバイト）まで）のサイズであることを示す、しかしすべてのモバイルプラスミドは、それによって、プラスミドの伝達を可能にする転送（oriTを）の原点を認識しrelaxaseを、含まれています。機能T4SSの組み立てだけでなく、タイプの接合プラスミドさらにエンコード遺伝子IV結合タンパク質。 12例えば、 大腸菌の100キロバイトFプラスミドは33.3 kBの転送（TRA）領域内のすべての接合の遺伝子をコードしています。 13ペア形成（MPF）、DNA転移と除外機能を交配線毛形成を促進する全てのタンパク質、接合性プラスミドの転送中にFプラスミドエンコードのTRA領域における遺伝子。 10,14,15は、知識の重要な身体は、接合T4SSsのために利用可能である、しかし、唯一のより最近利用可能になってきている共役タンパク質との複合体の構造研究を詳述しました。 16から28 接合プロセスの包括的なビューを組み立てるためには、接合伝達タンパク質の変異分析に詳細な構造研究のカップリングが必要です。これは、接合交配アッセイを介して達成することができます。 Fプラスミドは、TRA領域内にコードされる各タンパク質は、F媒介コにおいて役割を果たすnjugation;従って、転写遺伝子のノックアウト/欠失は、細胞の接合容量（ 図1）を廃止します。小さい携帯プラスミドは、標準的な削除の手続きをより助長しているが、このようなFのようなより大きな接合プラスミドのために、遺伝子ノックアウトは、より容易に標的遺伝子は明確な抗生物質耐性遺伝子を伝えるものに交換され、相同組換えを介して達成されます。現在のプロトコルでは、我々は55キロバイトFプラスミドの誘導体pOX38-TCでクロラムフェニコールアセチルトランスフェラーゼ（CAT）と利益の伝達遺伝子を置き換えるために、相同組換えを利用します。 29,30、得られたノックアウトプラスミド、pOX38-TCΔgene:: Cmが、増殖培地中のクロラムフェニコール（CM）の存在に対する抵抗性を促進します。 pOX38-TCΔgene:: Cmとを保有するドナー細胞は交配アッセイの使用を介して観察されるように接合DNA転送/交配に影響を与えることができません。 pOX38-TCΔgene:: Cmをドナー細胞と正常RECIPの接合効率ientは廃止され、より多くの場合、減少またはます。 pOX38-TCΔgene:: Cmをプラスミドの接合伝達を標的に転送遺伝子を保有する小型の回復プラスミドを経由して復元することができます。この回復プラスミドは、プラスミドpK184（pK184遺伝子）、31または限り、そのプラスミドが適切細胞（細胞質またはペリプラズム）内の正しい場所に遺伝子を標的として誘導性の発現を提供するものとして、構成的発現を提供するものであることができます。したがって、この新しいドナー間の交配アッセイ（pOX38-TCΔgene:: Cmを+ pK184遺伝子のプラスミドを有します） そして、レシピエント細胞は、接合効率はほぼその正常ドナー・レシピエントの交配アッセイのに復元することが期待されます。このシステムは、CM曲庇:: pK184-遺伝子構築物（欠失または点変異）の一連の生成を介してとpOX38-TCΔgeneの交配能力を回復するために各構築物の能力を試験するノックアウト遺伝子の機能を調べるために1を可能にしますドナー細胞秒。

<table><tbody><tr><td>GeneJet Plasmid Mini-Prep Kit</td><td>Fisher Scientific</td><td>K0503</td><td></td></tr><tr><td>GeneJet Gel Extraction Kit</td><td>Fisher Scientific</td><td>K0692</td><td></td></tr><tr><td>GeneJet PCR Purification Kit</td><td>Fisher Scientific</td><td>K0702</td><td></td></tr><tr><td>Q5 Site-Directed Mutagenesis Kit</td><td>New England Biolabs</td><td>E0554S</td><td></td></tr><tr><td>Broad Range DNA Ladder</td><td>New England Biolabs</td><td>N0303A</td><td></td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>Electroporator</td><td>Eppendorf</td><td>4309000027</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Fisher Scientific</td><td>FB101</td><td>Cuvettes have a 1 mm gap.</td></tr><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AvaI</td><td>New England Biolabs</td><td>R0152S</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>HindIII</td><td>New England Biolabs</td><td>R0104L</td><td></td></tr><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111S</td><td></td></tr><tr><td>Phusion DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176S</td><td></td></tr><tr><td>&lt;strong&gt;Antibiotics&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Final Concentrations&lt;/strong&gt;</td></tr><tr><td>Chloramphenicol (Cm)</td><td>Fisher Scientific</td><td>BP904-100</td><td>20 &amp;micro;g/mL</td></tr><tr><td>Kanamycin (Km)</td><td>BioBasic Inc.</td><td>DB0286</td><td>50 &amp;micro;g/mL</td></tr><tr><td>Nalidixic acid (Nal)</td><td>Sigma-Aldrich</td><td>N8878-25G</td><td>10 &amp;micro;g/mL</td></tr><tr><td>Rifampicin (Rif)</td><td>Calbiochem</td><td>557303</td><td>20 &amp;micro;g/mL</td></tr><tr><td>Tetracycline (Tc)</td><td>Fisher Scientific</td><td>BP912-100</td><td>10 &amp;micro;g/mL</td></tr><tr><td>Streptomycin (Sm)</td><td>Fisher Scientific</td><td>BP910-50</td><td>50 &amp;micro;g/mL</td></tr></tbody></table>

conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation

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.

Fプラスミド駆動型細菌接合プロセスは、線毛の合成と接合DNAの転送を容易にするT4SSを組み立てるF-プラスミドのTRA領域内に伝達タンパク質を伴う協調プロセスです。タンパク質TRAF（GenBankアクセッション番号BAA97961、UniProtのID P14497）は、接合F線毛形成に必要とされます。 10,14,35 -それは、触媒CXXCモチーフを持っていませんが、37タンパク質は、C末端チオレドキシン様ドメインが含まれています。それは、そのN末端ドメインを介してTraHタンパク質と相互作用すると予測されているが35,38、39地域は他にあまりない37は 、タンパク質の構造的特徴について知られている、そのC末端ドメインよりも、よりダイナミックであることが示されました。構造研究と併せてTRAFタンパク質の機能的側面を評価するために、我々はまず、相同経由pOX38-TCにTRAF遺伝子をノックアウト組換え大腸菌 DY330R細胞におけるpOX38-TCΔtraF:: Cmのプラスミド（ 表1）を生成します 。また、生成された33,34は、結合の回復を提供し、タンパク質の配列をプローブ有効にするTRAF特異的プライマー（ 表2）からpK184-TRAFプラスミドでした。 pK184-TRAFがトランスで提供されていたXK1200細胞DY330R細胞から40へpOX38-TCΔtraF:: Cmをプラスミドの転送を示している（のトランスは、10 / mlのTcのおよび20μg/ mLのCmが10μgの/ mLのNalで上に成長させました） pOx38-TCΔtraF:: CMで、その（a）は、TRAFノックアウトは、インフレームCATカセットを提供し、（b）はそのpK184-TRAFは、接合機能を回復させることができます。 TRAF変異体のシリーズはXK1200 pOX38-TCΔtraF:: Cmを+ pK184-TRAFΔXを使用して接合交配アッセイ37を使用して、分析のために生成されましたそれぞれのドナーとレシピエントのような細胞およびMC4100細胞41。一つの代表的な変異体TRAF 55から247（ 表1）、タンパク質の領域はTraHと相互作用することが予測さ削除N末端欠失変異体です。完全長TRAFタンパク質がドナー細胞に提供されている場合、空のプラスミドpK184んではない（ 表3）を提供しながら、接合伝達は、（ 図2）復元されます。プラスミドpK184-TRAF 55から247（ 表3）が設けられたとき同様に、XK1200ドナー細胞における接合機能が復元されません。これは、タンパク質の切断部位は、おそらくTraHとの相互作用を介して、接合装置内のTRAFの機能にとって重要であることを示し、さらに変異解析のために標的化するためにタンパク質の領域を提供します。 <img alt="図1" src="/files/ftp_upload/54854/54854fig1.jpg" /> <strong&gt;図1：接合交配アッセイの模式図。ステップ1において、標的遺伝子は、PCRは、標的遺伝子に相同なオーバーハングを有するCATカセットを生成し使用して、DY330R細胞、効率的な組換え株において相同組換えによりノックアウトされています。結果として得られるDY330Rクローンは、プラスミドノックアウト遺伝子が回復プラスミド（pK184-遺伝子）を介してトランスで提供されていない限り、結合を促進することができませんpOX38-TCΔgene:: Cmとし、保有します。得pOX38-TCΔgene:: CmをプラスミドはMC4100の受信者（工程3）と交配アッセイを用いて遺伝子のさらなる評価のためXK1200株（ステップ2）に転送されます。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54854/54854fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54854/54854fig2.jpg" /> 図2： マット結合中の標的遺伝子の機能を評価するためのアッセイをる。ドナーとレシピエント細胞は、大腸菌 XK1200 pOX38-TCΔtraF:: Cmは、それぞれ、pK184-TRAFとMC4100で形質転換しました。得られたトランス（MC4100 pOX38-TCΔtraF:: Cmが）接合機能の正常な回復を示し、Sm及びCmとを含むプレート上で成長します。実験は、単一の寒天プレート上に二重に行われ、連続希釈を修復遺伝子変異体（ 表2）の接合効率を計算するために使用されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54854/54854fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 細菌株/プラスミド </td><td> 関連する特性 </td><td> 選択マーカー（複数可）† </strong&gt; </td><td> 参照 </td></tr><tr><td> 菌株 </td><td></td><td></td><td></td></tr><tr><td> DY330R </td><td> W3110ΔlacU169gal490λc1857Δ（CRO-のbioA）リフR </td><td>解雇</td><td> 33,34 </td></tr><tr><td> XK1200 </td><td> F - lacΔU124Δ（バイオジャイラattλ灘aroGギャル） </td><td> NAL </td><td> 40 </td></tr><tr><td> MC4100 </td><td> araD139Δ（ARGF-LAC）U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR </td><td> Smの</td><td> 41 </td></tr><tr><td> ベクターおよび構築 </td><td></td><td></td><td></td></tr><tr><td> pBAD33 </td><td> P のaraBADから下を発現するプラスミド</td><td> Cm </td><td> 32 </td></tr><tr><td> pK184 </td><td>ベクトル、P15Aレプリコンをクローニング2.4キロバイト</td><td>キロ</td><td> 31 </td></tr><tr><td> pK184-TRAF‡&lt;/ TD&gt; <td> pK184でpOX38からF TRAF </td><td>キロ</td><td> 35 </td></tr><tr><td> pK184-TRAF 56から247 </td><td> pK184-TRAFからF TRAFアミノ酸56から247 </td><td>キロ</td><td></td></tr><tr><td> 接合プラスミド </td><td></td><td></td><td></td></tr><tr><td> pOX38-TC </td><td> IncFI、トラ+、RepFIA +、F、ミニのTnのF1 HindIII断片</td><td> Tcの</td><td> 29,30 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> CATとpOX38-TcはTRAFに挿入します</td><td> TcのCmを</td><td> 35 </td></tr><tr><td colspan="4"> †Cmを、クロラムフェニコール;キロ、カナマイシン。 Nalで、ナリジクス酸;リフ、リファンピシン; Smを、ストレプトマイシン; Tcを、テトラサイクリン</td></tr><tr><td colspan="4"> ‡すべてのTRAF構築物は、細胞膜周辺腔への局在化を確実にするために、19残基のリーダー配列を含んでいます。 </td></tr></tbody></table> 表1： <st栄&gt; E。 coli 株およびプラスミド本 研究で使用しました。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 構築します </td><td> プライマー* </td></tr><tr><td> TRAF-CM-について</td><td> 5&#39;- GATCGAGGCTGGCAGTGGTATAACGAGAAAATAAATCCGAAGGA - CTGTGACG GAAGATCACTTC -3 &#39; </td></tr><tr><td> TRAF-CM-REV </td><td> 5&#39;- TCTTCAGAAACGTTCAGGAACTGTTTTGCCAGGTCGTCCT - CTTATTCAGGCGT AGCACCAG -3 &#39; </td></tr><tr><td> pK184-TRAF-について</td><td> 5&#39;- TTTTTT GAATTC TATGAATAAAGCATTACTGCCAC -3 &#39; </td></tr><tr><td> pK184-TRAF-REV </td><td> 5&#39;- TTTTTT AAGCTT TAAAAATTGGGTTTAAAATCTTCAGAAA -3 &#39; </td></tr><tr><td> pK184-TRAF 55から247 -For </td><td> 5&#39;- TACG CATATG ATGGCCGCACTGCAGACGG -3 &#39; </td></tr><tr><td> pK184-TRAF 55から247 </sub&gt; -rev </td><td> 5&#39;- TACG CATATG TCCTGACGCCGGAAAAATAAAGCAGCAGAGTAA -3 &#39; </td></tr><tr><td colspan="2"> †レントらから適応表。許可を得て、2016年35 </td></tr><tr><td colspan="2"> * TRAF張り出し領域はイタリック体です。 （：AAGCTTをEcoRI：GAATTCをNdeI：CATATG HindIIIで）制限酵素部位には下線が引かれています</td></tr></tbody></table> 表2：本研究で使用したプライマーのリスト。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ドナープラスミド† </td><td> 回復プラスミド </td><td> トランス接合‡§（細胞ミリリットル-1） </td><td> 交尾Efficiency§|| </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td>なし</td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38-TΔtraF:: CmをC </td><td> pK184 </td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> pK184-TRAF </td><td> 5×10 3 </td><td> 0.0167 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> pK184-TRAF 55から247 </td><td> 0 </td><td> 0 </td></tr><tr><td colspan="4"> *レントらから適応表。許可を得て、2016年35 </td></tr><tr><td colspan="4"> †ドナー細胞は、3×10 7細胞mlの平均濃度で、 大腸菌 XK1200た-1 </td></tr><tr><td colspan="4"> ‡レシピエント細胞は、 大腸菌 MC4100ました。ポジティブコントロール用のトランスの数は10-5希釈からです。 0は10-2希釈からのトランス接合体がないことを示します。 </td></tr><tr><td colspan="4"> 2〜4個の交配実験の§An平均は、各構築物について実施しました。 </td></tr><tr><td colspan="4"> ||交尾効率がdefinです100ドナー細胞当たりのトランスとして編。 0交配効率は10-2希釈からのトランス接合体がないことを示します</td></tr></tbody></table> 表3：TRAF 欠失構築物により廃止接合効率。

Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

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.

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

conjugative-mating-assays-for-sequence-specific-analysis-transfer

Nanyang Technological University

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JoVE Journal

Genetics

13.7K ビュー.  York University. 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. 

ビデオ: Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

in vitro転写アッセイおよび創薬への応用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

遺伝学

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

木部形成に関与する遺伝子およびプロモーターを研究するための誘導性体細胞セクター分析（ISSA）の使用とセカンダリステム開発

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

生理的酸素条件に曝露されたヒト細胞におけるキャップ結合タンパク質の解析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

サイト固有の細菌染色体工学：ΦC31インテグラーゼを介したカセット取引所（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

生物工学

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

細菌の活用頻度の高解像度の比較

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

大腸菌における天然接合プラスミドを介した遺伝子水平伝播の検出

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

で水平遺伝子伝達の研究のための方法論<em&gt;黄色ブドウ球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

免疫・感染学

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

シアノゲートモジュラークローニングツールキットを用いたコンジュゲーションによるシアノバクテリアの遺伝子改変

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

生物学

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

高スループットシーケンシングのためのグラム陰性細菌におけるトランスポゾン挿入ライブラリの生成

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
First discovered by Lederberg and Tatum in 1946, conjugation is a form of horizontal gene transfer between bacteria that relies on direct physical contact between two bacterial cells (1). Unlike other forms of gene transfer, such as transformation or transduction, conjugation is a naturally occurring process in which DNA is secreted from a donor cell to a recipient cell in a unidirectional manner. This directionality and the ability for this process to increase the genetic diversity of bacteria has given conjugation the reputation as a form of bacterial &#34;mating,&#34; which is believed to have contributed greatly to the recent rise in antibiotic resistant bacteria (2, 3). By using selective pressures, for example the use of antibiotics, conjugation has been manipulated for use in the laboratory setting, making it a powerful tool for horizontal gene transfer between bacteria, and in some cases from bacteria to yeast, plant, and animal cells (4). Apart from applications in the laboratory, bacterium-eukaryote gene transfer by conjugation is an exciting avenue of DNA transfer with a multitude of possible biotechnical applications and naturally occurring implications (5).
Conjugation is thought to work by a &#34;two-step mechanism&#34; (6). First, before any DNA can be transferred, the donor cell must make direct cell-to-cell contact with the recipient. This process has been characterized best in gram-negative bacteria, the most studied of which is Escherichia coli. Cell-to-cell contact is established by the presence of a complex network of extracellular filaments on the donor known as the sex pilus, a conjugative element encoded for by the transferable gene known as the F (fertility) factor (7, 8). In addition to establishing contact between donor and recipient, several proteins are transported via the sex pilus to the recipient cytoplasm, forming a type IV secretion system (T4SS) conduit between the two cells, a necessary structure for the second step of conjugation, DNA transfer (6). By combining this function of the sex pilus with rolling circle replication of DNA, the donor cell is able to transfer DNA in the form of a transposable element, such as a plasmid or transposon, to the recipient by a &#34;shoot and pump&#34; model (6). In this case, the &#34;shooting&#34; is the transport of the pilot protein, with linked DNA, by the T4SS into the recipient cell, and the &#34;pumping&#34; is the active transport of DNA to the recipient, a process reliant on the T4SS and catalyzed by coupling proteins (6). The machinery used in this this process is composed of an origin of transfer sequence (oriT), which must be provided by the DNA in cis and trans genes, which encode a relaxase, mate pair formation complex, and type IV coupling protein, and can be present in cis or trans (9). This relaxase cleaves the nic site within the oriT sequence and covalently attaches to the 5&#39; end of the transferred strand to produce the relaxosome, a single-stranded DNA-relaxase complex with other auxiliary proteins (9). Once formed, the relaxosome connects to the mating pair formation complex, via the type IV coupling protein, which allows for the transfer of the ssDNA-relaxase complex into recipient cells by the T4SS (10). Once in the cytoplasm of the recipient, the DNA can integrate into the recipient genome or exist separately in the form of a plasmid, either of which allow for the expression of its genes.
In this experiment, the widely used conjugation donor strain E. coli WM3064 was used to transfer the gene encoding for ampicillin resistance to the recipient strain E. coli J53. While both strains of the gram-negative bacteria were resistant to tetracycline, only the donor strain WM3064 had the gene for ampicillin resistance, encoded for in the pWD2-oriT shuttle vector, and was auxotrophic to diaminopimelic acid (DAP) (11-13). This experiment consisted of two main steps, the preparation of donor and recipient strains, followed by the transfer of the ampicillin resistance gene from donor to recipient by conjugation (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10516/10516fig1.jpg" /> 
Figure 1: Conjugation schematic. This schematic shows the successful transfer of a plasmid, only one example of a transposable DNA element, from a donor cell to a recipient cell using conjugation. Upon contact with the recipient cell by the donor cell via the sex pilus, the plasmid replicates by rolling circle replication, moves through the multiprotein complex joining the two cells, and forms a new full-length plasmid in the recipient cell.
By incubating a mixture of donor and recipient cells, then successively plating these cells in the presence of tetracycline and DAP, this allowed for the successful transfer of the ampicillin resistance gene. Successively plating cells grown from this mixture in the presence of tetracycline and ampicillin, removed all donor cells due to the lack of DAP and any recipient cells that may not have gained the ampicillin resistance gene, yielding strictly recipient J53 strain bacteria that acquired ampicillin resistance (Figure 2). Once carried out, the successful transfer of the ampicillin resistance gene was confirmed by PCR. Since conjugation was successful, the J53 strain of E. coli contained pWD2-oriT and was resistant to ampicillin, and the gene encoding for this resistance is detectable by PCR. However, if unsuccessful there would have been no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10516/10516fig2.jpg" /> 
Figure 2: Protocol schematic. This schematic shows an overview of the presented protocol.
<img alt="Figure 3A" class="xfigimg" src="/files/ftp_upload/10516/10516fig3a.jpg" /> 
Figure 3A: The confirmation of successful conjugation by PCR. A) Freezer stocks of the conjugated and negative control samples were streaked out on agar plates and a colony was selected (red) for DNA isolation.

Conjugation: Transferring Ampicillin Resistance in E. coli

結合:アンピシリン耐性をドナーからレシピエント大腸菌に移す方法

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...