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1.生成のサンプル<ol><li>熱はCを活性化させます氷上で30分間、店舗65℃でディフィシレ胞子。 注：C.以前7,9,10説明したようにディフィシレ胞子は生産、精製することができます。 </li><li> Xは、サンプル数がアッセイ中に撮影されるX + 1 mlの発芽溶液を調製します。 注：発芽液が細菌の種に固有のもので検討されて胞子。 C.をアッセイするためのディフィシレ胞子発芽、我々は、10mMトリス（pH7.5）を、150mMのNaCl、脱イオン水中の100 mMグリシンおよび10mMタウロコール酸の溶液を用います。 </li><li>アッセイを開始する前に、皮質の断片を検出するためのブランクとして機能するように胞子をせずに発芽溶液1.0mlサンプルを描きます。 </li><li>すぐに発芽液と渦に〜3.0のOD 600に胞子を追加します。 注：Bの私たちの発芽試験のために枯草菌 、我々は同じODを使用6C.用として00胞子ディフィシル 。 注：胞子のこの量は、Cの間に皮質断片の放出をモニターするためによく働きましたディフィシレ胞子の発芽。このアッセイで使用される胞子の量は、それぞれ細菌または株について実験的に決定されるべきです。 </li><li> 14,000×gで1分間室温で胞子および遠心分離機を追加した直後に1.2ミリリットルのサンプルを削除してください。これは、ゼロ時点です。 </li><li>その後の皮質断片分析のために新しいチューブに遠心分離し、試料からの上清の1.0ミリリットルを転送します。 注：DPAを ​​監視する必要がある場合、テルビウム蛍光9,18,19に基づくアッセイを用いて、溶液中にDPAの検出のための別々の100μlのサンプルを削除します。 </li><li> -80℃で採取した試料を凍結します。 </li><li>全ての時点が完了するまで繰り返して、定期的に1.4と1.5を繰り返します。 ために遠心分離し、サンプリングに必要な時間を、時間点は以下番目注2分は離れて私たちの手順の間には不可能だったので、私たちのサンプリング間隔は、一回14分ごとに2分でした。 </li><li> 0、12.5、25、50、100、250、500、および5000：砂糖の検出および定量化を減少させるためのポジティブコントロールとして、N個の -acetylglucosamine（ナノモル）以下の量が含まれています。皮質サンプルと同時にこれらのサンプルを調製し、標準曲線は、胞子を発芽によって生成されたデータに関連しているように、皮質サンプルと一緒に実行します。 </li><li>すべての時点のサンプルを収集した後、サンプルを凍結乾燥。 注：私たちは、2ミリリットルのスクリューキャップチューブ中で私たちのサンプルを凍結乾燥しました。 </li></ol> 2.皮質断片を測定<ol><li> 1％のフェノール及び0.5％のβメルカプトエタノールを補充した3 N HClを120μlの凍結乾燥試料を再懸濁します。 </li><li> 2 mlのスクリューキャップチューブに再懸濁したサンプルを転送します。 注：再現性のために、すべての凍結乾燥した試料は再懸濁し、転送されていることを確認。 </li> &lt;LI&gt; 4時間95℃で再循環水浴中でサンプルをインキュベートします。 </li><li>彼らはクールになるまでインキュベートした後、氷上にサンプルを置きます。 </li><li> 3 M NaOHを120μlのサンプルを中和します。 </li><li>飽和重炭酸ナトリウム溶液の80μlを、各サンプルと渦に5％無水酢酸溶液の80μLを加えます。 </li><li> 10分間、室温でサンプルをインキュベートします。 </li><li>丁度3分のために戻って95℃の水浴にサンプルを転送します。 注：このステップは敏感で、より長い時間が下り信号の発達に影響を与えることができる時間です。 </li><li>水浴からサンプルを取り出して、氷上で冷却します。 </li><li>各チューブとボルテックスに6.54パーセントのK 2 B 4 O 7・4H 2 Oの400μlのを追加します。 </li><li>丁度7分間95℃の循環水浴中でサンプルをインキュベートします。 注：このステップは敏感で、より長い時間が下り信号の発達に影響を与えることができる時間です。 </li><li>削除します氷上で5分間のサンプルと場所。 </li></ol> 3.色試薬<ol><li>サンプルは氷（ステップ2.12）であるが、発色試薬を準備します。氷酢酸1.9ミリリットルであり、pの -dimethylaminobenzaldehyde（エールリッヒ試薬DMAB）の0.320グラムを溶解させます。 注意：試薬は、時間、温度と光に敏感であり、事前に行われるべきではありません。 </li><li> DMABが完全に溶解した後、10 N HCl及び渦の100μlを添加します。 </li><li>氷酢酸と渦の5ミリリットルを追加します。 </li></ol> 4.比色反応<ol><li>各100μlのを転送新しい1.5 mlのマイクロ遠心チューブに皮質のサンプルを冷却しました。 </li><li>各サンプルに新たに調製した色溶液700μLを加えます。 </li><li>直ちに37℃の水浴にサンプルを転送し、正確に20分間インキュベートします。 </li><li>透明な96ウェルプレートに200μlの各サンプルを転送します。 </li><li>プレート上の585 nmでのサンプルを定量リーダー。サンプルは黄色で開始すべきであると陽性反応が紫にシフトします。より多くの皮質の存在、深い紫色。 </li></ol>

<ol>
	<li>Al-Hinai, M. A., Jones, S. W., Papoutsakis, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Clostridium+sporulation+programs:+diversity+and+preservation+of+endospore+differentiation.">The Clostridium sporulation programs: diversity and preservation of endospore differentiation.</a> Microbiol Mol Biol Rev. 79, 19-37 (2015).</li><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+of+spores+of+Bacillus+species:+what+we+know+and+do+not+know.">Germination of spores of Bacillus species: what we know and do not know.</a> J Bacteriol. 196, 1297-1305 (2014).</li><li>Tan, I. S., Ramamurthi, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+formation+in+Bacillus+subtilis.">Spore formation in Bacillus subtilis.</a> Environ Microbiol Rep. 6, 212-225 (2014).</li><li>Fimlaid, K. A., Shen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+mechanisms+regulate+sporulation+sigma+factor+activity+in+the+Firmicutes.">Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.</a> Curr Opin Microbiol. 24, 88-95 (2015).</li><li>Popham, D. L., Helin, J., Costello, C. E., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+peptidoglycan+structure+of+Bacillus+subtilis+endospores.">Analysis of the peptidoglycan structure of Bacillus subtilis endospores.</a> J Bacteriol. 178, 6451-6458 (1996).</li><li>Atrih, A., Zollner, P., Allmaier, G., Foster, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+Bacillus+subtilis+168+endospore+peptidoglycan+and+its+role+during+differentiation.">Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.</a> J Bacteriol. 178, 6173-6183 (1996).</li><li>Sorg, J. A., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salts+and+glycine+as+cogerminants+for+Clostridium+difficile+spores.">Bile salts and glycine as cogerminants for Clostridium difficile spores.</a> J. Bacteriol. 190, 2505-2512 (2008).</li><li>Paredes-Sabja, D., Shen, A., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+spore+biology:+sporulation,+germination,+and+spore+structural+proteins.">Clostridium difficile spore biology: sporulation, germination, and spore structural proteins.</a> Trends Microbiol. 22 (7), (2014).</li><li>Francis, M. B., Allen, C. A., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spore+cortex+hydrolysis+precedes+DPA+release+during+Clostridium+difficile+spore+germination.">Spore cortex hydrolysis precedes DPA release during Clostridium difficile spore germination.</a> J Bacteriol. 197 (14), (2015).</li><li>Francis, M. B., Allen, C. A., Shrestha, R., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acid+recognition+by+the+Clostridium+difficile+Germinant+Receptor,+CspC,+is+important+for+establishing+infection.">Bile acid recognition by the Clostridium difficile Germinant Receptor, CspC, is important for establishing infection.</a> PLoS Pathog. 9, e1003356 (2013).</li><li>Moir, A., Smith, 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=The+Genetics+of+bacterial+spore+germination.">The Genetics of bacterial spore germination.</a> Annu. Rev. Microbiol. 44, 531-553 (1990).</li><li>Zhang, P., Kong, L., Wang, G., Setlow, P., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+Raman+tweezers+and+quantitative+differential+interference+contrast+microscopy+for+measurement+of+dynamics+and+heterogeneity+during+the+germination+of+individual+bacterial+spores.">Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores.</a> Journal of biomedical optics. 15, 056010 (2010).</li><li>Ghuysen, J. -. M., Tipper, D. J., Strominger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+that+degrade+bacterial+cell+walls.">Enzymes that degrade bacterial cell walls.</a> Methods Enzymol. 8, 685-699 (1966).</li><li>Park, J. T., Johnson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+submicrodetermination+of+glucose.">A submicrodetermination of glucose.</a> J Biol Chem. 181, 149-151 (1949).</li><li>Thompson, J. S., Shockman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+the+Park+and+Johnson+reducing+sugar+determination+suitable+for+the+assay+of+insoluble+materials:+its+application+to+bacterial+cell+walls.">A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls.</a> Anal Biochem. 22, 260-268 (1968).</li><li>Paredes-Sabja, D., Setlow, P., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SleC+is+essential+for+cortex+peptidoglycan+hydrolysis+during+germination+of+spores+of+the+pathogenic+bacterium+Clostridium+perfringens.">SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens.</a> J Bacteriol. 191, 2711-2720 (2009).</li><li>Paredes-Sabja, D., Sarker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+cortex-lytic+enzyme+SleC+from+non-food-borne+Clostridium+perfringens+on+the+germination+properties+of+SleC-lacking+spores+of+a+food+poisoning+isolate.">Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate.</a> Can J Microbiol. 56, 952-958 (2010).</li><li>Hindle, A. A., Hall, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipicolinic+acid+(DPA)+assay+revisited+and+appraised+for+spore+detection.">Dipicolinic acid (DPA) assay revisited and appraised for spore detection.</a> The Analyst. 124, 1599-1604 (1999).</li><li>Kort, R., 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=Assessment+of+heat+resistance+of+bacterial+spores+from+food+product+isolates+by+fluorescence+monitoring+of+dipicolinic+acid+release.">Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.</a> Appl Environ Microbiol. 71, 3556-3564 (2005).</li><li>Elson, L. A., Morgan, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+method+for+the+determination+of+glucosamine+and+chondrosamine.">A colorimetric method for the determination of glucosamine and chondrosamine.</a> The Biochemical journal. 27, 1824-1828 (1933).</li><li>Ramirez, N., Abel-Santos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+germination+of+Clostridium+sordellii+spores+in+vitro.">Requirements for germination of Clostridium sordellii spores in vitro.</a> J. Bacteriol. 192, 418-425 (2010).</li><li>Akoachere, M., 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=Indentification+of+an+in+vivo+inhibitor+of+Bacillus+anthracis+spore+germination.">Indentification of an in vivo inhibitor of Bacillus anthracis spore germination.</a> J. Biol. Chem. 282, 12112-12118 (2007).</li><li>Duncan, C. L., Strong, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+medium+for+sporulation+of+Clostridium+perfringens.">Improved medium for sporulation of Clostridium perfringens.</a> Appl Microbiol. 16, 82-89 (1968).</li><li>Heffron, J. D., Lambert, E. A., Sherry, N., Popham, 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=Contributions+of+four+cortex+lytic+enzymes+to+germination+of+Bacillus+anthracis+spores.">Contributions of four cortex lytic enzymes to germination of Bacillus anthracis spores.</a> J Bacteriol. 192, 763-770 (2010).</li><li>Meador-Parton, J., Popham, 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=Structural+analysis+of+Bacillus+subtilis+spore+peptidoglycan+during+sporulation.">Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation.</a> J Bacteriol. 182, 4491-4499 (2000).</li><li>Lambert, E. A., Popham, 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=The+Bacillus+anthracis+SleL+(YaaH)+protein+is+an+N-acetylglucosaminidase+involved+in+spore+cortex+depolymerization.">The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization.</a> J Bacteriol. 190, 7601-7607 (2008).</li><li>Wang, S., Shen, A., Setlow, P., Li, Y. Q. <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+the+Dynamic+Germination+of+Individual+Clostridium+difficile+Spores+Using+Raman+Spectroscopy+and+Differential+Interference+Contrast+Microscopy.">Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy.</a> J Bacteriol. 197, 2361-2373 (2015).</li></ol>

The authors have nothing to disclose.

刺激を受けると、発芽の過程を経て胞子は巨大分子の合成を必要とせずに、それらの抵抗特性を失います。胞子の発芽がトリガされると、胞子コアは水2と引き換えにDPAを ​​解放します。休眠胞子の高いDPA内容に、胞子コアは、拡張2に対するバリアとして、おそらく、作用することにより、膨潤コアを防ぐことができます強烈な浸透圧や専門ペプチドグリカン、皮質下にあります。 上述の方法は、紫の色合いの測定シフトと還元糖の存在を検出するためにエールリッヒ試薬（黄色溶液）を使用します。皮質の加水分解の間、NAGとNAM糖が遊離する（加水分解酵素の程度に応じて、糖は、二糖類または多糖類として遊離させることができます）。塩酸（ステップ2.1）によるグリコシド結合の加水分解は、自由還元末端7で単糖を生成します。 neutraliz後水酸化ナトリウム（ステップ2.5）の等量酸る、NAGとNAM単糖類を囲むアルコールは無水酢酸（ステップ2.6）13によりアシル化されます。その後、未反応の無水酢酸を試料（ステップ2.8）を加熱して酢酸に変換されます。モーガンとエルソン20によって仮説を立てたように、末端アルデヒドは、自発的にエノール型に変換します。アルカリ性条件（ステップ2.10）の下で、このエノール形態は紫色15,20を生成するために、エールリッヒ試薬（ステップ4.3）と反応し、オキサゾール誘導体を生成するために、隣接アセチルと反応することができます。 上記のこの方法は、胞子を発芽から皮質の断片を検出するための一貫性のある、信頼性の高い結果が得られ、両方のB.枯草菌およびC.ディフィシル 9,16,17。アッセイは、反応時間、温度および手順を精密に行われることを保証するために特別な注意が必要です。適切な配慮なし反応条件は、実験13との間で再現失敗するかしないために詳細に、それは簡単です。しかし、実験室環境間アッセイのアプリケーションでNAG標準補助を含めます。 NAG陽性対照標準は、任意の実験間のばらつき（ 例えば 、反応時間で、わずかな変動や色試薬の有効性）のため、すべての実験のコントロールに含まれています。緩衝液のみの条件と皮質の両方を加水分解することができないの胞子との反応を用いて、反応は、色に変化は生じませんでした。 この方法の最も一般的な問題は、実験間のばらつきや信号の完全な欠如です。変動を制御するために、胞子調製物は、任意の栄養細胞破片の自由でなければなりません。この細胞破片はペプチドグリカンフラグメント（NAGとNAM）を含有し、発芽胞子のための高いバックグラウンドシグナルになります。純粋な胞子製剤（&gt; 99.9％の胞子）を確保するために、literatuを参照してください。再7,9,21-23を研究する生物に純粋な胞子懸濁液を調製するための方法のために。 アッセイは、検出可能なシグナルが得られていない場合は、凍結乾燥工程は、変動性を導入することができることを観察しました。真空が解放される力のために、我々は、乾燥した試料粉体を試料管から噴出することができることを観察しました。減らすために/試料管の上部の穴を突くために小さな注射針を使用し、これを防ぎます。導入孔の小領域が乱されるから（現在粉末形態の）凍結乾燥されたサンプルの可能性を減少させます。上記の方法で述べたように最後に、いくつかのインキュベーションステップは非常に時間に敏感です。すべての時限の手順を注意深く監視していることを確認してください。このアッセイの重要な考慮事項は、皮質断片を検出するために発芽胞子から放出されなければならないということです。皮質は、大きな断片に加水分解されている場合は、これらの断片はRELEであるには余りにも大きくなることがあり胞子殻を通してASED。信号が得られない実験のトラブルシューティングを行うときに考慮すべき重要なことが、私たちは前に潜在的な問題のように皮質のフラグメントサイズを考慮に条件がここに記載されている溶液中の胞子およびその他のトラブルシューティングオプションのほとんどを発芽するために使用されることを確認することをお勧めしますアッセイ。 全て発芽バッファは/条件は、このアッセイに適しています。このアッセイは発芽過程13の間に遊離還元糖の存在、糖または、それ自体が任意の発芽を減らす含む任意の発芽・バッファを検出するので、還元糖（ 例えば 、グルコース）は、このアッセイで強いシグナルをもたらすであろう。この場合、高圧液体クロマトグラフィー（HPLC）は、皮質断片24-26の存在を検出することができます。すべての研究所は、このような計測機器が装備されていないので、しかし、本明細書に記載される方法は、ほとんどの地域で使用されることができます研究所や、シンプルで簡単に定量化可能な結果を​​提供しています。 細菌胞子によって発芽は、主としてバチルスといくつかのクロストリジウム属の種2,8に研究されてきました。差異が存在するが、プロセスは、一般的に、保存されています。モデルの胞子形成剤9,27に観察されるように重要なことは、すべての生物は、胞子の発芽のメカニズムを使用して発芽しません。発芽は、より多様な生物において研究になっているので、上述のアッセイは、胞子発芽の間皮質断片を検出するために、これらの生物に適用することができます。これらの新たに研究の生物にこの方法を適用することにより、研究者はさまざまな発芽タンパク質が皮質の加水分解において果たす役割を決定することができます。

内生胞子は、細菌が不利な環境で存続することを可能にする細菌の代謝的に休止状態の形態です。多くの胞子形成細菌は、胞子形成は、栄養欠乏によって誘導されるが、このプロセスは、pH、酸素への曝露または他のストレス1に変更することによって制御することができます。それらの代謝休眠胞子の形で、一方、細菌は、UV照射、乾燥、高温、および2の凍結に耐えます。胞子形成過程に関する知識のほとんどは、モデル生物での研究、 枯草菌（Bacillus subtilis）から来ています。胞子形成のプロセスは、DNA複製および軸方向フィラメント3,4の形成から始まります。非対称隔壁は、2つの不均等サイズの区画にセルを分割します。大きなコンパートメントは、母細胞は、小さな区画、前胞子を飲み込みます。母細胞とドーマンを解放し、胞子、最終的に溶解する母細胞を成熟するために遺伝子発現を調整する前胞子の両方周囲の環境1へのT胞子。 胞子の構造と組成は、多くの細菌種間で保存されています。胞子コアは栄養細胞よりも低い含水量を有しており、ジピコリン酸（DPA）1,2と豊富です。胞子形成中、DPAは、水と引き換えに胞子コアに圧送されます。コアを囲むことは、ほとんどの芽胞形成菌で、発芽を刺激する小分子を認識する受容体（germinants）が2に位置している、内側の胞子膜です。ちょうど胞子の内膜の外側に位置する細胞壁ペプチドグリカンの層です。特殊なペプチドグリカン層（皮質）は、細胞壁のペプチドグリカンを囲み、[N-アセチルグルコサミン（NAG）およびN-アセチルムラミン酸（NAM）交互】細胞壁ペプチドグリカンと同じ構成要素の多くで構成されています。しかし、皮質におけるNAM残基の約​​50％は、ムラミン-δラクタム5,6に変換されています</s&gt;アップ。胞子発芽時には、これらのムラミン-δラクタム残基は、このように皮質はなく、細胞壁（発芽を完了するのに必須のプロセス）を分解することができるように、胞子皮質溶​​解酵素（SCLEs）によって認識されます。皮質の周囲には外膜とコートタンパク質2のいくつかの層です。 発芽のプロセスを制御することは芽胞形成菌のために非常に重要です。発芽受容体は、それぞれのgerminants 2を操作する際に発芽が開始されます。多くの胞子形成細菌では、これらのgerminants 2そのアミノ酸、糖、ヌクレオチド、イオンまたはこれらの組み合わせである。C.ディフィシレ胞子の発芽は、特定の胆汁酸[ 例えば 、タウロコール酸（TA）]およびアミノ酸（ 例えば 、グリシン）7-10の組み合わせによって開始されます。 Cの相違はありますがディフィシル発芽経路および他の胞子形成に研究経路このようなB.などの細菌、 枯草菌は、すべてに共通の栄養細胞が発芽胞子2,8から成長できるようにするために胞子皮質を分解するための絶対条件です。 （多くのクロストリジウムに見られるような）皮質の劣化がSCLEs CwlJ / SleB（ 枯草菌に見られるような）またはSLECによって達成することができます。皮質の加水分解は、細胞壁と胞子上の制約を軽減します。これは完全なコアの再水和、細胞代謝2のために必要なタンパク質の多くを再活性化に必要なステップを可能にします。 胞子が発芽したときに、位相暗状態と、このプロセスのフェーズ明状態から休眠胞子の変更は600での光学密度（OD）の変化によって測定することができます。 11 nmの。前回のレポートでは、ODの変化の多くは胞子12からのDPAのリリースによるものであることを示唆しています。我々の最近の研究の間、我々はCのタイミングを比較しようとしましたディフィシレ胞子の発芽・監視DPAおよび皮質の断片9のリリース。彼らは胞子を発芽によって解放されるようになったように、この研究のためには、皮質の断片の放出を監視するために重要でした。 ここで使用される比色アッセイはGhuysen らが開発した還元末端を有する糖を検出するための方法に基づいていた。13。他の人が還元糖14の検出のためのプロトコルを記述しているか、それらのプロトコル15を変更したので、このテーマに関する文献は混乱することができます。ここでは、詳細C.発芽から遊離した還元糖の比色検出のためのステップバイステップ方法ディフィシレ胞子。この研究では、Cを使用しますがディフィシレ胞子は、他の芽胞形成菌からの胞子の発芽中に放出された還元糖は、このプロトコル9,16,17で検出することができます。

<table><tbody><tr><td>2.0 ml screw cap tube</td><td>USA scientific</td><td>1420-3700</td><td></td></tr><tr><td>p1000 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p200 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>p20 Eppendorf pipet</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>100 - 1,250 &amp;#956;l pipet tips</td><td>VWR</td><td>89079-486</td><td></td></tr><tr><td>1 - 200 &amp;#956;ll pipet tips</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>N-acetyl-D-glucosamine</td><td>Sigma</td><td>A3286-25G</td><td></td></tr><tr><td>Hydrochloric Acid - 10 N</td><td>BDH-Aristar</td><td>BDH3032-3.8LP</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Alfa Aesar</td><td>L04295</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>BDH</td><td>BDH0280-500G</td><td></td></tr><tr><td>Potassium Tetraborate tetrahydrate</td><td>Alfa Aesar</td><td>39435</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>BDH8019-500G</td><td></td></tr><tr><td>4-(Dimethylamino)benzaldehyde</td><td>Sigma</td><td>156477-100G</td><td></td></tr><tr><td>Saturated Phenol</td><td>Fisher</td><td>BP1750-400</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>SpectraMax M3</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Acetic Acid, Glacial</td><td>BDH</td><td>BDH3098-3.8LP</td><td></td></tr><tr><td>Heated, Circulating Water Bath</td><td>VWR Scientific</td><td>Model 1136</td><td></td></tr><tr><td>Microtest 96</td><td>Falcon</td><td>353072</td><td>96 well clear tissue culture plates</td></tr><tr><td>Culture tubes</td><td>VWR</td><td>89000-506</td><td></td></tr><tr><td>Lyophilizer</td><td></td><td></td><td></td></tr><tr><td>Heat Blocks</td><td></td><td></td><td></td></tr><tr><td>Vortex Machine</td><td></td><td></td><td></td></tr></tbody></table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

表1は、NAG標準を使用した典型的な結果を示しています。データは、標準曲線を生成するために使用される。 表2は、100 mMグリシン及び10 mMのTA（発芽促進条件）、または100mMのグリシンのみ（未発芽状態）を補充した発芽バッファにおける発芽アッセイの代表的な結果を示しています。 TA、Cの非存在下ではディフィシレ胞子は発芽せず、溶液中の皮質の断片の存在はほとんど変化しません。しかし、C.、TAお ​​よびグリシンの両方の存在下で、 ディフィシレ胞子は発芽し溶液中に皮質の断片を放出します。これは、反応した試料の経時的なOD 585の増加として観察されます。このアッセイの間に放出さ皮質断片（ナノモル）の合計量は、 図1Bと同様の標準曲線を用いて計算しました。このアッセイでは、皮質の大部分が加水分解される（または解放）15分後により発芽暴露、胞子の大部分はこの時間枠（ 図2;以前の出版物7と一貫）で発芽していることを示唆しています。 発色試薬でサンプルを開発した後、ほとんどのサンプルは、肉眼に紫色が表示されない場合があります。 図1は、N-アセチルグルコサミンから生成された代表的なサンプルを示しています。サンプルの多くは、目に見える紫色を示していません。しかし、250ナノモル、500ナノモル及び5000 nmolのサンプルは、陰性対照（0ナノモル）と視覚的に異なる表示されます。 OD 585 nmで測定した場合、信号が最高の線形回帰（R 2 = 0.999）をフィット生成しました。 <img alt="図1" src="/files/ftp_upload/54146/54146fig1.jpg" /> 図1： 検出N-アセチルグルコサミン （A）N-アセチルグルコサミンサンプル（0ナノモル、12.5ナノモル、。25ナノモル、50ナノモルを、100ナノモル、250ナノモル、500ナノモル、5,000ナノモル）が記載された手順に従って凍結乾燥して反応させました。 （B）サンプルを、96ウェルプレート、および585 nmで検出した反応物質の量に添加しました。信号は、標準曲線を作成するために使用されました。 <img alt="図2" src="/files/ftp_upload/54146/54146fig2.jpg" /> 図2： 発芽結果のグラフィカルな表現は、 表2の計算されたナノモル皮質は、時間に対してプロットしました。 （●）100 mMグリシン+ 10mMのTA。 （■）100 mMグリシン。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54146/54146fig2large.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> NAG濃度（ナノモル） </td><td> OD 585 </td></tr><tr><td> 0 </td><td> 0.047 </td></tr><tr><td> 12.5</td><td> 0.054 </td></tr><tr><td> 25 </td><td> 0.066 </td></tr><tr><td> 50 </td><td> 0.084 </td></tr><tr><td> 100 </td><td> 0.128 </td></tr><tr><td> 250 </td><td> 0.285 </td></tr><tr><td> 500 </td><td> 0.556 </td></tr><tr><td> 5,000 </td><td> 4.0 </td></tr><tr><td>ブランク= </td><td> 0.046 </td></tr></tbody></table> 表1：NAG標準。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="4"> クロストリジウム・ディフィシレ胞子の発芽</td></tr><tr><td></td><td colspan="4">発芽液（10mMトリス（pH7.5）を、150mMのNaCl） </td></tr><tr><td></td><td colspan="2"> 100mMのグリシン+ 10mMのTA </td><td colspan="2"> 100mMのグリシン</td></tr><tr><td>時間（分） </td><td> OD 585 </td><td>計算された（ナノモル） </td><td> OD 585 </td><td>計算された（ナノモル） </td></tr><tr><td> 0 </td><td> 0.046 </td><td> 0.00 </td><td> 0.047 </td><td> 0.00 </td></tr><tr><td> 2 </td><td> 0.048 </td><td> 2.54 </td><td> 0.048 </td><td> 1.27 </td></tr><tr><td> 4 </td><td> 0.05 </td><td> 5.07 </td><td> 0.049 </td><td> 2.54 </td></tr><tr><td> 6 </td><td> 0.055 </td><td> 11.41 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 8 </td><td> 0.059 </td><td> 16.49 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 10 </td><td> 0.06 </td><td> 17.76 </td><td> 0.051 </td><td> 5.07 </td></tr><tr><td> 12 </td><td> 0.061 </td><td> 19.02 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 14 </td><td> 0.063 </td><td> 21.56 </td><td> 0.051 </td><td> 5.07 </td></tr></tbody></table> 表2：スポアGerminatiサンプル結果に。

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

細菌胞子の発芽中に皮質断片を検出

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.3K ビュー.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

ビデオ: Detecting Cortex Fragments During Bacterial Spore Germination

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

栄養細胞の標的溶解によって環境サンプルからの内生胞子の物理的な分離

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

環境学

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

蛍光性DNAzymesを使用した細菌の検出

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫・感染学

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

フローサイトメトリーにおける 枯草菌 胞子の計数と標識解析の改善

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

生化学

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Germinosomes と枯草菌胞子の内部の膜の可視化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

生物工学

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

放線菌突然変異体のビジュアルと顕微鏡的評価

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

の性的開発と子嚢胞子放電フザリウムgraminearum種</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

生物学

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

酵素の抗原 Nonrecombinant 表示システムとして胞子吸着

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

化学的に媒介種間相互作用を検出するための共培養を使用して

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

細菌胞子の発芽中に皮質断片を検出

要約

さらに動画を探す

この動画の章

栄養細胞の標的溶解によって環境サンプルからの内生胞子の物理的な分離

蛍光性DNAzymesを使用した細菌の検出

フローサイトメトリーにおける枯草菌胞子の計数と標識解析の改善

Germinosomes と枯草菌胞子の内部の膜の可視化

放線菌突然変異体のビジュアルと顕微鏡的評価

の性的開発と子嚢胞子放電フザリウムgraminearum種

酵素の抗原 Nonrecombinant 表示システムとして胞子吸着

化学的に媒介種間相互作用を検出するための共培養を使用して

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

細菌胞子の発芽中に皮質断片を検出

要約

さらに動画を探す

この動画の章

栄養細胞の標的溶解によって環境サンプルからの内生胞子の物理的な分離

蛍光性DNAzymesを使用した細菌の検出

フローサイトメトリーにおける 枯草菌 胞子の計数と標識解析の改善

Germinosomes と枯草菌胞子の内部の膜の可視化

放線菌突然変異体のビジュアルと顕微鏡的評価

の性的開発と子嚢胞子放電フザリウムgraminearum種

酵素の抗原 Nonrecombinant 表示システムとして胞子吸着

化学的に媒介種間相互作用を検出するための共培養を使用して

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

フローサイトメトリーにおける枯草菌胞子の計数と標識解析の改善