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1.細菌コロニーの周辺フリンジ<ol><li> Xを育てます28℃でfastidiosa（ のXf）テメキュラ野生型22、pilG欠失変異体のXfΔpliG（前述削除戦略23を使用）、およびその相補XfΔpliG-C（以前染色体ベースの遺伝的相補戦略24を用いて説明した）PD2培地寒天プレート上で25 5-7日のために、C。 </li><li> 15分間121°C（249°F）で水にオートクレーブセロファン（1×1cm 2の）。 、セロハンの1枚をピックアップ空のペトリ皿にセロハンの一角に触れて水を抜く、慎重に寒天表面と空気乾燥の15％の上にセロハンを築きます。 </li><li>個々のXを拾いますfastidiosaコロニー滅菌丸い楊枝で寒天プレートに寒天表面の15％に重ねセロハンの滅菌シート上に無菌的に細胞を発見。プレートをインキュベート2〜3日間28℃でS。 </li><li> 2X対物レンズと10倍の接眼レンズを解剖顕微鏡を用いて、コロニーのエッジ形態を調べます。コロニーの周辺のフリンジを撮影。 </li></ol> 2.顕微鏡やマイクロ流体フローチャンバー<ol><li>以前5,18に記載したものと同様のフォトリソグラフィ手順を使用したマイクロ流体デバイスを製造します。標準的なリソグラフィ方法26を使用して、マスターのシリコンウェハ上のコンピュータ支援設計ソフトウェアを持つ4つの並列チャネルを設計します。 </li><li>ポリジメチルシロキサン（PDMS）でシリコンウェハマスターからマイクロ流体チャンバを作成します。シリコンウエハーマスター上に未重合PDMSを注いで、1時間60℃でそれを硬化させます。ウェハマスターからPDMSのレプリカをはがし、ガラスカバースリップの同じサイズとして22ミリメートルX 40ミリメートルにブレードとPDMSレプリカをトリミングします。 </li><li> PDMSのレプリカ、ガラスカバースリップ（22×40 mm 2）とし、microscを公開2分27用30 Wで空気プラズマにOPEスライド（51のx 76ミリメートル2）。サンドイッチガラスカバースリップと微小流体室を構築するためのガラス顕微鏡の間にPDMS本体。 </li><li>パターン化チャネルの各端部にPDMS貫通孔​​（5.5ミリメートル直径）ドリル。長い12〜20センチメートルにシリコーンゴムチューブをカットします。 PDMSレプリカのチャネルの各開口端​​部に、シリコーンゴム管（外径5.1ミリメートル、内径2.1 mmのを0.8mmの壁）の一端を挿入し、1時間60℃で重合していないPDMSとそれを密封します。 </li><li>プラスチック製のルアーコネクタのとげの終わりにチューブの他端を接続します。アルミホイルで組み立てられたマイクロ流体チャンバをラップし、20分のためにそれらをオートクレーブ。 </li><li> Xの細菌細胞を収集fastidiosa野生型、変異型のXfΔpliG、および相補XfΔpliG-C PD2培地プレートから使い捨て接種ループを使用して、掻き介して。細胞密度を調整しますPD2ブロス中の600nmで0.05の光学密度を、以前23記載されているように。 1ミリリットルガスタイトシリンジに細菌の細胞溶液を収集します。 </li><li>倒立顕微鏡のステージ上にマイクロ流体デバイスをマウントします。 PD2ブロスを含む5ミリリットルガスタイトシリンジに導入管を接続します。シリンジポンプで5ミリリットルガスタイトシリンジを取り付けます。 </li><li>廃棄物容器に排出管を接続します。 0.2μL分の媒体流量を維持-1で30分間、システムを安定化します。 </li><li>細菌細胞溶液を含む1ミリリットルガスタイトシリンジに側入口管を接続します。チャネルに到達するまで、ゴム管を介して細菌の細胞溶液を洗い流します。チャンバーからの事前の画像キャプチャに結合していない細胞をフラッシュするために0.2μlの分の媒体流量-1別の30分間の維持。 </li><li>光を制御するために、顕微鏡の視野-調整部分の下顕微鏡シャッターをマウントします。シャッター共同にシャッターを接続しますntrolシステムやコンピュータにシャッター制御システムを接続します。 </li><li>顕微鏡のビデオポートにデジタルカメラをマウントし、それをコンピュータに接続します。タイムラプス撮影のソフトウェアを実行して、メニューから「シャッター」機能を選択して、ソフトウェアとシャッターへの接続を確立するためのソフトウェアでデフォルトとして自動的にインストールシャッターを認識しています。 </li><li>自動的にソフトウェアのデフォルトのキャプチャデバイスとしてデジタルカメラを認識し、ソフトウェアとデジタルカメラとの通信を確立するためにタイムラプスレコーディングソフトウェアのメニューから「デジタルカメラ」機能を選択します。 </li><li> 20X位相差光学系を用いて、チャネルのいずれかで細菌細胞を見つけ、その後、画像キャプチャの前に40X対物レンズに切り替えます。 </li><li>顕微鏡から画像を取得するには、メニューからデフォルトパラメータを使用して、「画像取得」機能を​​選択し、タイムラプス録画ソフトウェアを実行します。次に、「タイムラプスを取得」機能を開き、6-24時間は、Xのけいれん運動性を観察するために必要な実験に応じての持続時間30秒5,18,28に時間間隔を設定fastidiosa 5,18,28。タイムラプス撮影を開始するために「OK」をクリックします。メニューから「スタック機能」をクリックして、録音を終えた後、コンピュータ上の保存先フォルダ内の画像をスタックに「名前を付けて保存」を選択します。 </li><li>複数のチャネルについては、6時間、最初のチャンネルから30秒ごとにタイムラプス画像をキャプチャします。標的細胞を見つけるために次のチャネルに顕微鏡の対物レンズを移動します。実験は4つのチャネルを利用するように設定されている場合は、4つのチャネルを順次それぞれの画像をキャプチャするために上記のようにタイムラプス機能を繰り返します。限り3として日間連続タイムラプス画像キャプチャを続けます。全ての実験は、室温（23±2℃）で行いました。 </li><li>タイムラプス画像をコンパイルしますタイムラプス撮影イメージングソフトウェアを使用して、ビデオファイル。タイムラプス撮影のソフトウェアを実行して、メニューから「スタック機能」をクリックして、コンピュータから積み重ねられたファイルを開くには、「オープンスタック機能」を選択します。 </li><li>すべての画像を選択し、「AVI」出力フォーマットを選択し、スタックモジュールから「映画を作る」機能を起動します。コンピュータ上の保存先フォルダ内のビデオファイルを保存するために「名前を付けて保存」をクリックします。 </li><li>コンピュータ上の保存先のフォルダからコンパイルされたムービーを選択し、それらを再生。そして、生成されたビデオファイルの中の細菌細胞の単収縮運動の活動の結果の可視化を通じて単一細胞の運動性を観察します。 </li></ol>

<ol>
	<li>Purcell, A. H., Hopkins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fastidious+xylem-limited+bacterial+plant+pathogens.">Fastidious xylem-limited bacterial plant pathogens.</a> Annu. Rev. Phytopathol. 34, 131-151 (1996).</li><li>Purcell, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylella+fastidiosa,+a+regional+problem+or+global+threat.">Xylella fastidiosa, a regional problem or global threat.</a> J. Plant Pathology. 79, 99-105 (1997).</li><li>Hopkins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylella+fastidiosa:+Xylem-limited+bacterial+pathogen+of+plants.">Xylella fastidiosa: Xylem-limited bacterial pathogen of plants.</a> Annu. Rev. Phytopathol. 27, 271-290 (1989).</li><li>Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+pili+and+twitching+motility.">Type IV pili and twitching motility.</a> Annu. Rev. Microbiol. 56, 289-314 (2002).</li><li>Meng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upstream+migration+of+Xylella+fastidiosa+via+pilus-driven+twitching+motility.">Upstream migration of Xylella fastidiosa via pilus-driven twitching motility.</a> J. Bacteriol. 187, 5560-5567 (2005).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+and+type+IV+pili+of+Xylella+fastidiosa+affect+twitching+motility,+biofilm+formation+and+cell-cell+aggregation.">Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation.</a> Microbiology. 153, 719-726 (2007).</li><li>Simpson, A. J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+the+plant+pathogen+Xylella+fastidiosa.">The genome sequence of the plant pathogen Xylella fastidiosa.</a> Nature. 406, 151-157 (2000).</li><li>Maier, B., Potter, L., So, M., Long, C. D., Seifert, H. S., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+pilus+motor+forces+exceed+100+pN.">Single pilus motor forces exceed 100 pN.</a> Proc. Natl. Acad. Sci. USA. 99, 16012-16017 (2002).</li><li>Touhami, A., Jericho, M. H., Boyd, J. M., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+characterization+and+determination+of+adhesion+forces+of+Pseudomonas+aeruginosa+pili+by+using+atomic+force+microscopy.">Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy.</a> J. Bacteriol. 188, 370-377 (2006).</li><li>Skerker, J. M., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+extension+and+retraction+of+type+IV+pili.">Direct observation of extension and retraction of type IV pili.</a> Proc. Natl. Acad. Sci. USA. 98, 6901-6904 (2001).</li><li>Brown, D. C., Larson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+to+parallel+plate+flow+chambers+to+reduce+reagent+and+cellular+requirements.">Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.</a> BMC Immunol. 2, 9 (2001).</li><li>Thomas, W. E., Nilsson, L. M., Forero, M., Sokurenko, E. V., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-dependent+'stick-and-roll'+adhesion+of+type+1+fimbriated+Escherichia+coli.">Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli.</a> Mol. Microbiol. 53, 1545-1557 (2004).</li><li>Thomas, W. E., Trintchina, E., Forero, M., Vogel, V., Sokurenko, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+to+target+cells+enhanced+by+shear+force.">Bacterial adhesion to target cells enhanced by shear force.</a> Cell. 109, 913-923 (2002).</li><li>Bahar, O., Fuente, D. L., Burdman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+adhesion,+biofilm+formation+and+motility+of+Acidovorax+citrulli+using+microfluidic+flow+chambers.">Assessing adhesion, biofilm formation and motility of Acidovorax citrulli using microfluidic flow chambers.</a> FEMS Microbiol. Lett. 312, 33-39 (2010).</li><li>Thomas, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+laminar+flow+system+to+explain+shear-enhanced+bacterial+adhesion.">Using a laminar flow system to explain shear-enhanced bacterial adhesion.</a> Proceedings of ICMM2005, Third International Conference on Microchannels and Mini-channels. , 751-759 (2005).</li><li>Fuente, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+adhesion+forces+of+type+I+and+type+IV+pili+of+Xylella+fastidiosa+bacteria+by+use+of+a+microfluidic+flow+chamber.">Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber.</a> Appl. Environ. Microbiol. 73, 2690-2696 (2007).</li><li>DeLange, P. A., Collins, T. L., Pierce, G. E., Robinson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PilJ+localizes+to+cell+poles+and+is+required+for+type+IV+pilus+extension+in+Pseudomonas+aeruginosa.">PilJ localizes to cell poles and is required for type IV pilus extension in Pseudomonas aeruginosa.</a> Curr Microbiol. 55, 389-395 (2007).</li><li>Fuente, D. L., Burr, T. J., Hoch, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+type+I+and+type+IV+pilus+biosynthetic+genes+affect+twitching+motility+rates+in+Xylella+fastidiosa.">Mutations in type I and type IV pilus biosynthetic genes affect twitching motility rates in Xylella fastidiosa.</a> J. Bacteriol. 189, 7507-7510 (2007).</li><li>Ferandez, A., Hawkins, A. C., Summerfield, D. T., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cluster+II+che+genes+from+Pseudomonas+aeruginosa+are+required+for+an+optimal+chemotactic+response.">Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response.</a> J. Bacteriol. 184, 4374-4383 (2002).</li><li>Cursino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Operon,+Pil-Chp,+That+Controls+Twitching+Motility+and+Virulence+in+Xylella+fastidiosa.">Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa.</a> Mol. Plant Microbe Interact. 10, 1198-1206 (2011).</li><li>Hazelbauer, G. L., Falke, J. J., Parkinson, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+chemoreceptors:+High-performance+signaling+in+networked+arrays.">Bacterial chemoreceptors: High-performance signaling in networked arrays.</a> Trends Biochem. Sci. 33, 9-19 (2008).</li><li>Costa, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+hosts+of+Xylella+fastidiosa+in+and+near+southern+California+vineyards.">Plant hosts of Xylella fastidiosa in and near southern California vineyards.</a> Plant Dis. 88, 1255-1261 (2004).</li><li>Shi, X. Y., Dumenyo, C. K., Hernandez-Martinez, R., Azad, H., Cooksey, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+regulatory+pathways+in+Xylella+fastidiosa:+genes+and+phenotypes+controlled+by+algU.">Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.</a> Appl. Environ. Microbiol. 73, 6748-6756 (2007).</li><li>Matsumoto, A., Young, G. M., Igo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-Based+Genetic+Complementation+System+for+Xylella+fastidiosa.">Chromosome-Based Genetic Complementation System for Xylella fastidiosa.</a> Appl. Environ. Microbiol. 75, 1679-1687 (2009).</li><li>Davis, M. J., Purcell, A. H., Thomson, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+Media+for+the+Pierce's+Disease+Bacterium.">Isolation Media for the Pierce's Disease Bacterium.</a> Phytopathology. 70, 425-429 (1980).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Annu. Rev. Mater. Sci. 28, 153-184 (1998).</li><li>Chaudhury, M. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interfacial+interactions+between+semispherical+lenses+and+flat+sheets+of+poly-(dimethylsiloxane)+and+their+chemical+derivatives.">Direct measurement of interfacial interactions between semispherical lenses and flat sheets of poly-(dimethylsiloxane) and their chemical derivatives.</a> Langmuir. 7, 1013-1025 (1991).</li><li>Cruz, L. F., Parker, J. K., Cobine, P. A., De La Fuente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-enhanced+twitching+motility+in+Xylella+fastidiosa+is+linked+to+a+single+PilY1+homolog.">Calcium-enhanced twitching motility in Xylella fastidiosa is linked to a single PilY1 homolog.</a> Appl. Environ. Microbiol. 80, 7176-7196 (2014).</li></ol>

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。

本研究では、Xの動きの挙動を特徴付けfastidiosa PilG変異体のXfΔpilGと新たに設計された複数の並列-ナノチャネル微小流体チャンバ内のその相補XfΔpilG-C株。新たに設計された微小流体室は、単一の50μm幅のチャネル18と以前の設計に比べて幅が100μmのナノチャネルと最大4つの並列チャンバを持つことができます。改善され、より広いナノチャネル?...

Xylellaのfastidiosaはブドウでピアース病を含む、経済的に重要な作物の多数の疾患を引き起こすグラム陰性非鞭 ​​毛、病原性細菌である（ ヴィティスヴィニフェラ L.）1,2、3。この細菌は導水木部に限定されています船。ブドウの感染は、水ストレスや栄養不足3における木部船と結果の閉塞を引き起こします。成功植民地化は、植物3の残りの部分に感染の初期のサイトから移動する細菌の能力に依存します。運動性をけいれんすると、鞭毛に依存しない細菌の延長を経て動き、添付ファイル、およびXに特徴付けられている極性IV型線毛4の後退の手段でありますfastidiosa 5,6,7。 単収縮運動性は、レーザーピンセット及び原子間力顕微鏡（AFM）8,9,10により観察されました。これらの技術を用いて、TN.のIV型線毛によって生成された魔術の運動能淋菌およびP.緑膿菌は、FL uorescentlyラベリング線毛と微視的にその動きを捕捉することによって特徴づけられました。両方の方法は、個々の細菌の接着力を詳述しているが、手順が複雑で時間9,10を消費しています。微小流体チャンバは、長距離個 ​​々の細胞の遊走、ならびに細菌細胞5,6の小さな凝集物を観察するために使用しました。これらのチャンバは、タイムラプス画像記録システム11,12,13,14と統合プレートに微細加工・ナノチャンネルとして設計されました。マイクロuidic室デバイスが移動行動や細菌の細胞間相互作用を研究するためのいくつかの利点を提供FL：（ⅰ）それは、複数のチャネル機能を備えた統合プラットフォームを提供します。 （ⅱ）それは細菌のナノスケールの特徴における単一細胞の運動や集計を調べることができます。 （ⅲ）それが直接メートルを可能にしますicroscopic細菌細胞の画像記録とタイムラプス解析、（iv）は、それは微小環境中の細菌の個々のおよび/または集団の長期的、空間的および時間的な観測を提供します。 （V）チャネルにおける培地の流量を正確に制御することができ、（VI）培地の非常に少量（1 ml）を各実験について必要とされます。 最近では、微小流体流動システムは、様々な微小環境14,15,16の下で細菌細胞の挙動を調査するために採用されています。接着性およびE.の表面付着大腸菌 15、X. fastidiosa 16、およびアシドボラックスは、ガラス表面への14の微小流体チャンバーを用いて評価したcitrulli。 アシドボラックスcitrulliのIV型線毛によって媒介凝集し、バイオフィルムの形成は、14を分析しました。 A.のさらに、運動流Cの下で観察citrullionditionsは、IV型線毛は、Aの植民地化と普及に重要な役割を果たしていることを実証しました樹液流条件下での木部船でcitrulli。 緑膿菌およびXのけいれんの運動能fastidiosa細胞が正常に微細加工フローチャンバー5,6,17内の流体流れに逆らって観察しました。 IV型線毛は、XのpilBとpilQ変異体を欠損しましたfastidiosaは深く微小流体デバイス5,6,18における流動条件でけいれん運動の速度を変更することが見出されました。微小流体デバイスにおける細菌の接着および運動性について行った研究は、微小流体チャンバが、インビトロで線毛媒介細菌の単収縮運動性および遊走を分析するのに特に適していることが示されました。これらの結果は、内の細胞 - 細胞付着、凝集・定着を促進するけいれん媒介移行メカニズムを説明しますホストは、最終的には全身感染につながります。 XのPIL-のCHPオペロンfastidiosaは、エンコード信号の伝達が20を経路pilG、線毛、pilJ、ピル、chpBとchpCが含まれています 。膜貫通化学受容体は、ペリプラズムドメイン内の化学的刺激を結合して、最終的には細菌のけいれんの運動性を制御するために、それらの細胞質部分のシグナル伝達カスケードを活性化します。 Xのピル-のCHPオペロン中fastidiosa、ホスホシャトルタンパク質PilGはチェイに相同体です。 E.でcoliおよびP.緑膿菌、崔はべん毛モータータンパク質19、21と相互作用する走化性システムのレスポンスレギュレーターです。 Xの病原性に向けてピル-のCHPオペロンの貢献が、 fastidiosaは 、最近20の環境シグナルへとX.の規制/モータIV型線毛に応答した走化性オペロン中pilGの役割を調べましたfastidiosaは、UNCでありますリア。 Xの攣縮運動の活動に走化性レギュレータpilGの洞察を解明するために、 fastidiosa、微小流体チャンバーはXのけいれんの運動性を評価するために使用されますfastidiosa。 X.のfastidiosaのpilGは、 欠失変異体 のXfΔpliG、補完的な歪みXfΔpliG-Cおよびin vitroでその野生型の表現型を比較することを特徴としています。結果は、Xのけいれん運動中pilGの役割を強調表示しますfastidiosa。

<table><tbody><tr><td>&lt;strong&gt;Biology materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;X. fastidiosa (Xf)&lt;/em&gt; Temecula wild type</td><td></td><td></td><td>Costa, H. S., &lt;em&gt;et al.&lt;/em&gt;, 2004 &lt;sup&gt;22&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;pilG&lt;/em&gt; deletion mutant &lt;em&gt;Xf&amp;Delta;pliG&lt;/em&gt;</td><td></td><td></td><td>Shi, X. Y., &lt;em&gt;et al.&lt;/em&gt;, 2007 &lt;sup&gt;26&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;pilG&lt;/em&gt; complementary strain &lt;em&gt;Xf&amp;Delta;pliG&lt;/em&gt;-C&amp;nbsp;</td><td></td><td></td><td>Davis, M. J., &lt;em&gt;et al.&lt;/em&gt; 1998 &lt;sup&gt;23&lt;/sup&gt;</td></tr><tr><td>&lt;strong&gt;Physical materials and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable inoculating loops</td><td>VWR international, Radnor, PA</td><td>#22-363-607</td><td>quantitative procedures such as bacterial collection</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning Corporation</td><td>#0002709226</td><td>Sylgard 184 silicone Elastomeric Kits</td></tr><tr><td>AmScope MD2000 digital camera</td><td>AmScope, Irvine, CA</td><td>SE305R-AZ-E</td><td>Image, video recording and measurement&amp;nbsp;</td></tr><tr><td>Tubes line</td><td>Edgewood, NY</td><td>#T4300</td><td>Connected to the syringe and microfluidic chamber</td></tr><tr><td>Plastic luer connectors</td><td>Edgewood, NY</td><td></td><td>Connected to the syringe and microfluidic chamber</td></tr><tr><td>Syringe pumps</td><td>Pico Plus, Harvard Apparatus, MA</td><td>#702209</td><td>The flow rate can be adjusted while the pump is running.</td></tr><tr><td>Syringes</td><td>Gastight, Hemilton Company, Reno, NV</td><td>#1005</td><td>Provide the flowing broth</td></tr><tr><td>Inverted Olympus IMT-2 microscope</td><td>Olympus</td><td>IMT-2 FLuoro PHase</td><td>Image observation and recording</td></tr><tr><td>SPOT-RT digital camera</td><td>Diagnostic Instruments, Inc., MI</td><td>RT230</td><td>Image, video recording and measurement</td></tr><tr><td>Microscope Shutter</td><td>The &lt;em&gt;UNIBLITZ, US&lt;/em&gt;</td><td>#LS2T2</td><td>Control camera&amp;rsquo;s exposure time</td></tr><tr><td>Microscope Shutter Control system</td><td>The &lt;em&gt;UNIBLITZ, US&lt;/em&gt;</td><td>VCM-D1</td><td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td></tr><tr><td>MetaMorph Image software</td><td>Universal Imaging Corp., PA</td><td></td><td>Real-time super-resolution image processing&amp;nbsp;</td></tr></tbody></table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylellaのfastidiosaは、植物の経済的に重要な多くの疾患の原因となるグラム陰性非鞭 ​​毛細菌です。けいれん運動性は、Xを提供しますXの病原性に寄与する、長距離イントラ植物の動きと植民地化のための手段をfastidiosa fastidiosa。X.のけいれん運動fastidiosaは、IV型線毛によって運営されています。 XylellaのfastidiosaのIV型線毛はpilG、シグナル伝達経路に関与しているピル-Chpのオペロンをコードするタンパク質における走化性の調節因子によって調節されています。 Xの攣縮運動にpilGの役割を解明するために、 fastidiosa、pilG欠損変異体のXfΔpilGとネイティブpilGを含むその相補株XfΔpilG-Cを開発しました。タイムラプス画像記録システムと統合マイクロ流体チャンバはXfΔpに痙攣運動を観察するために使用されましたILG、XfΔpilG-Cおよびその野生型株。 この記録システムを使用して、長期の空間的および時間的な凝集の観察結果、個々の細胞および単収縮運動を介して細菌の集団の移動を可能にする。X.変異XfΔpliGがけいれん欠損表現型を示し、一方fastidiosa野生型および相補XfΔpilG-C株は、直接的にマイクロ流体フローチャンバーにおいて観察された典型的な単収縮運動の特性を示しました。この研究は、pilGは Xのけいれん運動性に寄与することを実証していますfastidiosa。マイクロ流体フローチャンバーは、単収縮運動性を観察するための手段として使用されます。

IV型線毛によって媒介される単収縮運動を示す周辺コロニー縞の存在は、Xのコロニーが観察されましたfastidiosa野生型および相補性のXfΔpliG-C株（ 図1）。ミュータントXfΔpliGは 、しかし、コロニーの周囲（ 図1）の周りにフリンジを示しませんでした。ナノマイクロ流体フローチャンバー内の細菌細胞のタイ?...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

本研究では、ナノマイクロ流体フローチャンバーは、視覚化し、機能的にXylella fastidiosa、グレイプバインのピアース病を引き起こす細菌の単収縮運動を特徴づけるために使用しました。

の役割の攣縮運動の可視化とキャラクタリゼーション<em&gt; PilG</em&gt;で<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

visualization-twitching-motility-characterization-role-pilg-xylella

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.7K ビュー.  University of California, Davis. 本研究では、ナノマイクロ流体フローチャンバーは、視覚化し、機能的にXylella fastidiosa、グレイプバインのピアース病を引き起こす細菌の単収縮運動を特徴づけるために使用しました。 

ビデオ: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

生化学

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

調査cruzi トリパノソーマの原因運動In Vivo動物モデルとしてのゼブラフィッシュ稚魚の確立

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

発生生物学

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫・感染学

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

で多細胞の動作を記録する Myxococcusザンザス</emタイムラプスMicrocinematographyを使用して>バイオフィルム

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

生物学

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

単細胞間細菌相互作用の運動的可視化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

細菌の運動性における細菌の糖転移酵素の役割を解明するためのヌル変異体の生成

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

準備、イメージング、および細菌表面運動性アッセイの定量化

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

べん毛モーターの機能の生物物理学的特性評価

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

シリーズで3つの確立された技術を適用することにより 、大腸菌 におけるフラゲラ駆動運動性の調査

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

の役割の攣縮運動の可視化とキャラクタリゼーション

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蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

調査cruzi トリパノソーマの原因運動In Vivo動物モデルとしてのゼブラフィッシュ稚魚の確立

発生とのマルチ表現型ハイコンテンツスクリーニング

で多細胞の動作を記録する Myxococcusザンザスバイオフィルム

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

単細胞間細菌相互作用の運動的可視化

細菌の運動性における細菌の糖転移酵素の役割を解明するためのヌル変異体の生成

準備、イメージング、および細菌表面運動性アッセイの定量化

べん毛モーターの機能の生物物理学的特性評価

シリーズで3つの確立された技術を適用することにより、大腸菌におけるフラゲラ駆動運動性の調査

の役割の攣縮運動の可視化とキャラクタリゼーション

要約

さらに動画を探す

この動画の章

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

調査cruzi トリパノソーマの原因運動In Vivo動物モデルとしてのゼブラフィッシュ稚魚の確立

発生とのマルチ表現型ハイコンテンツスクリーニング

で多細胞の動作を記録する Myxococcusザンザスバイオフィルム

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

単細胞間細菌相互作用の運動的可視化

細菌の運動性における細菌の糖転移酵素の役割を解明するためのヌル変異体の生成

準備、イメージング、および細菌表面運動性アッセイの定量化

べん毛モーターの機能の生物物理学的特性評価

シリーズで3つの確立された技術を適用することにより 、大腸菌 におけるフラゲラ駆動運動性の調査

シリーズで3つの確立された技術を適用することにより、大腸菌におけるフラゲラ駆動運動性の調査