Bu çalışma ABD Tarım Dairesi, Tarımsal Araştırma Servisi tarafından desteklenmiştir. Bu yayındaki ticari adlar veya ticari ürünler belirli bilgi vermek amacıyla sadece belirtilen ve ABD Tarım Bakanlığı tarafından tavsiye veya onaylandığı anlamına gelmez. USDA bir fırsat eşitliği sağlayıcı ve işverendir.

1. Bakteriyel Colony Periferik Fringe <ol><li> X büyümek fastidiosa (Xf) Temecula yabani tip 22, pilG silme mutant PD2 orta agar plakaları 25 ° 28 ° üzerinde (daha önce kromozom tabanlı genetik tamamlama stratejisi 24 tarif kullanarak) (daha önce açıklanan silme stratejisi 23 kullanarak) Xf ΔpliG ve onun tamamlayıcı XfΔpliG -C 5-7 gün bekletilmiştir. </li><li> 15 dakika boyunca 121 ° C&#39;de (249 ° F) su içinde Otoklav selofan (1 x 1 cm2). , Selofan tek parça Pick up boş bir Petri kabı selofan bir köşesini dokunarak su tahliye dikkatle agar yüzeyi ve kuru hava% 15 üzerinde selofan yatıyordu. </li><li> Bireysel X Pick up aseptik agar plakaları agar yüzeyinin% 15 üst üste selofan bir sterilize levha üzerine yuvarlak kürdan ve spot hücreler steril ile fastidiosa koloniler. plaka inkübe2-3 gün 28 ° C &#39;de s. </li><li> 2X objektif lens ile diseksiyon mikroskobu ve 10X oküler lens kullanarak kolonilerin kenar morfolojisi inceleyin. kolonilerin etrafında çevresel saçak fotoğraf. </li></ol> 2. Mikroskopi ve mikroakışkan Akış Odaları <ol><li> Benzer fotoğraf litografik prosedürler kullanılarak Üretiyor mikroakışkan cihazlar önceden 5,18 nitelendirdi. Standart taşbaskı yöntemler 26 kullanarak ana silikon gofret bilgisayar destekli tasarım yazılımı ile dört paralel kanal tasarlayın. </li><li> polidimetilsiloksan (PDMS) ile silikon gofret ustadan mikroakışkan odaları oluşturun. silikon gofret ana üzerinde unpolymerized PDMS dökün ve 1 saat 60 ° C&#39;de onu tedavi. gofret PDMS kopya soyulabilir ve bir cam lamel aynı boyutu olarak 22 mm x 40 mm bir bıçak ile PDMS çoğaltma kırpın. </li><li> PDMS çoğaltma bir cam lamel (22 x 40 mm2) ve Microsc Açığa2 dakika 27 30 W hava plazmaya ope slayt (51 x 76 mm 2). Sandviç cam lamel ve mikro fl uidic odasını oluşturmak için cam mikroskop arasında PDMS gövde. </li><li> desenli kanal her bir ucunda PDMS bir delik (5.5 mm çap) delin. 12-20 cm uzunluğunda içine silikon kauçuk tüp kesti. PDMS yineleme kanalların her biri açılış ucuna silikon kauçuk hortumun bir ucu (dış çapı 5,1 mm, iç çap 2.1 mm, 0.8 mm duvar) ekleyin ve 1 saat boyunca 60 ° C&#39;de polimerize edilmemiş PDMS ile kapatın. </li><li> plastik luer konnektörleri dikenli sonuna kadar boru başka ucunu. Alüminyum folyo ile monte mikroakışkan odaları sarın ve 20 dakika boyunca her otoklav. </li><li> X bakteriler hücreleri toplayın kazıma yoluyla fastidiosa yabani tip mutant Xf ΔpliG ve tamamlayıcı XfΔpliG -C, tek aşılamak kullanarak PD2 orta plakalar döngüler. Hücre yoğunluğunu ayarlayınPD2 suyu 600 nm&#39;de 0.05 bir optik yoğunluğa kadar daha önce tarif edildiği gibi 23. 1 ml gaz geçirmez şırınga içine bakteriyel hücre çözeltisi toplayın. </li><li> ters bir mikroskop sahnede mikroakışkan cihaz monte edin. PD2 suyu içeren 5 ml gaz geçirmez şırınga bir giriş borusunu bağlayın. şırınga pompaları ile 5 ml Gaz Geçirmez şırınga takın. </li><li> Bir atık haznesine çıkış borusunu bağlayın. 0.2 ul dk orta akış hızını korumak -1 30 dakika sistemini stabilize etmek için. </li><li> bakteriyel hücre çözeltisi ihtiva eden bir 1 ml&#39;lik gaz geçirmez şırınga ile yandan giriş tüpleri bağlayın. Kanal ulaşılana kadar kauçuk tüp aracılığıyla bakteriyel hücre çözümü yıkayın. Bölmeden önce, görüntü yakalama, bağlanmamış hücreleri temizlemek için 0.2 ul dk&#39;lık bir araç akış hızı -1 30 dakika boyunca muhafaza edilir. </li><li> ışık kontrol etmek mikroskop alan düzeltilmiş bölümü altında mikroskop deklanşör monte edin. deklanşör co deklanşör bağlayınntrol sistemi ve bilgisayara kapı kontrol sistemini bağlayın. </li><li> mikroskop video portuna dijital bir kamera monte ve bilgisayara bağlayın. Menüden &quot;deklanşör&quot; işlevini seçin, time-lapse kayıt yazılımını çalıştırın ve yazılım ile çekim bağlantı kurmak için yazılımda otomatik olarak varsayılan olarak yüklenir deklanşör tanır. </li><li> yazılım otomatik olarak varsayılan yakalama aygıtı olarak dijital fotoğraf makinesi tanımak ve yazılım ile dijital kamera ile iletişim kurmak için time-lapse kayıt yazılımı menüden &quot;dijital fotoğraf makinesi&quot; işlevini seçin. </li><li> 20X faz-kontrast optikler kullanılarak kanalların birinde bakteri hücreleri bulup görüntü yakalama önce 40X objektif lens geçin. </li><li> mikroskop görüntüleri elde etmek için menüden varsayılan parametreleri kullanarak &quot;görüntü elde etme&quot; işlevini seçin, time-lapse kayıt yazılımı çalıştırın.Sonra, &quot;time-lapse Edinme&quot; fonksiyonunu açın ve 6-24 saat X&#39;in seğirmesi hareketliliğini gözlemlemek için gerekli deney bağlı süresince 30 sn 5,18,28 zaman aralığını ayarlamak fastidiosa 5,18,28. time-lapse kayıt başlatmak için &quot;Tamam&quot; a tıklayın. seçmek kayıt bittikten sonra bilgisayardaki hedef klasördeki görüntüleri yığını &quot;farklı kaydet&quot; menüden &quot;Yığın fonksiyonunu&quot; tıklayın. </li><li> birden fazla kanal için, 6 saat boyunca birinci kanaldan her 30 saniye zaman atlamalı çekim. hedef hücreleri bulmak için bir sonraki kanala mikroskop objektif lens taşıyın. Deney dört kanal kullanmak üzere ayarlanmış ise dört kanal sırayla her görüntü yakalamak için yukarıda anlatıldığı gibi zaman atlamalı fonksiyonunu tekrarlayın. sürece üç ardışık gün boyunca zaman atlamalı görüntü yakalama devam edin. Bütün deneyler oda sıcaklığında (23 ± 2 ° C) gerçekleştirilmiştir. </li><li> Bir içine zaman atlamalı görüntüleri derlemektime-lapse kayıt görüntüleme yazılımı kullanarak video dosyası. Run time-lapse kayıt yazılımı, menüden &quot;Yığın işlevi&quot; tıklatın ve bilgisayardan yığılmış dosyaları açmak için &quot;openstack işlevini&quot; seçeneğini seçin. </li><li> tüm görüntüleri seçip &quot;AVI&quot; çıkış biçimi seçerek, yığın modülünden &quot;filmi olun&quot; fonksiyonunu başlatın. bilgisayardaki Hedef klasördeki video dosyasını saklamak için &quot;farklı kaydet&quot; seçeneğini tıklayın. </li><li> bilgisayardaki hedef klasörün derlenen film seçin ve onları oynatmak. Sonra, oluşturulan video dosyaları bakteri hücrelerinin seğirmesi motilite aktivitesinin ortaya çıkan görselleştirme yoluyla tek hücre hareketliliğini gözlemlemek. </li></ol>

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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Bu çalışmada, biz X&#39;in hareket davranışını karakterize fastidiosa PilG mutant Xf ΔpilG ve yeni tasarlanmış çoklu paralel nano-kanal mikro fl uidic odalarında tamamlayıcı XfΔpilG- C suşları. Yeni tasarlanan mikro fl uidic odaları tek bir 50 mikron genişliğinde kanal 18 ile daha önceki tasarımlara göre genişliği 100 mikron nano-kanal ile en fazla dört paralel odaları olabilir. geliştirilmiş geniş nano-kanal medyanın akan ba...

Xylella fastidiosa Grapevine Pierce hastalığı (Vitis vinifera L.), 1,2, 3 dahil olmak üzere ekonomik olarak önemli bitkisel hastalıkların bir dizi neden olan bir Gram-negatif olmayan kamçılı, patojen bir bakteridir. Bu bakteri su ileten ksilem sınırlıdır gemiler. Asmanın Enfeksiyon ksilem gemi ve su stresi sonuçları ve beslenme yetersizlikleri 3 tıkanmasına neden olur. Başarılı kolonizasyon tesisi 3 geri kalanına enfeksiyon ilk siteden taşımak için bakterinin yeteneğine bağlıdır. X özelliği olan polar tip IV Pili 4 uzatılması, bağlanma ve geri çekme yoluyla flagellar bağımsız bakteriyel hareketi için bir araç motilitesini olan seğirmesi fastidiosa 5,6,7. Seğirmesi motilite lazer cımbız ve atomik kuvvet mikroskobu (AFM) 8,9,10 tarafından gözlenmiştir. Bu teknikler kullanılarak, twitching motiliteler N. tip IV pilus tarafından oluşturulan gonorrhoeae ve S. aeruginosa fl uorescently etiketleme pili ve mikroskopik hareketlerini yakalayarak ile karakterize edildi. Her iki yöntem bireysel bakterilerin yapışkan kuvveti ayrıntılı olmasına rağmen, prosedürler karmaşık ve zaman 9,10 alıcıdır. Mikro fl uidic odalar uzun mesafe, tek tek hücrelerin göçünü ve bakteri hücreleri 5,6 küçük agregalar gözlemlemek için kullanılmıştır. Bu odalar bir time-lapse görüntü kayıt sistemi 11,12,13,14 ile entegre bir tabak içinde bir microfabricated-nano-kanal olarak dizayn edilmiştir. Mikro uidic odacık cihazlar bakteri hareketi davranışı ve hücre-hücre etkileşimleri incelemek için çeşitli avantajlar sunar fl: (i) bir çok kanallı yetenekleri ile entegre bir platform sağlar; (Ii) hareketler ve bakteri nano ölçekli özellikleri tek hücre toplamlar inceleyebilirsiniz; (Iii) doğrudan m sağlarbakteriyel hücrelerin ve zaman atlamalı analizi icroscopic görüntü kaydı, (iv) bir mikro-ortamda bakterilerin tek tek ve / veya popülasyonlarının uzun süreli mekansal ve zamansal gözlemler sağlar; (V) bir kanalda kültür ortamının akış hızı hassas bir şekilde kontrol edilebilir ve kültür ortamında (vi) sadece çok küçük bir hacmi (1 mi), her bir deney için gereklidir. Son zamanlarda, mikro fl uidic akış sistemi çeşitli mikroçevrelerde 14,15,16 altında bakteri hücrelerinin davranışlarını araştırmak için istihdam edilmiştir. Yapışkanlık ve E. yüzey eki E. coli, 15 X fastidiosa 16 ve Acidovorax cam yüzeylere 14 mikro fl uidic odaları kullanılarak değerlendirildi citrulli. Acidovorax citrulli tipi IV pili aracılığıyla toplama ve biyo fi lm oluşumu 14 analiz edildi. A. Ayrıca, hareket citrulli akış c altında gözlenenşartlarından Tip IV pili kolonizasyon önemli roller oynayabileceğini göstermiştir ve A. yayıldı sap akış koşulları altında ksilem damarlarında citrulli. Pseudomonas aeruginosa ve X&#39;in seğirmesi motiliteler fastidiosa hücreleri başarılı bir şekilde mikrofabrike akış odasının 5,6,17 bir akışkan akımına karşı gözlendi. Tip IV pilus X. pilB ve pilQ mutantlar yoksun fastidiosa derinden mikro fl uidic cihazlar 5,6,18 içinde akış koşulları altında motilite seğirmesi hızını değiştirmek için bulunmuştur. Mikro fl uidic cihazlarda bakteri yapışma ve motilitesi üzerinde yapılan çalışmalar, mikro fl uidic odaları in vitro pili aracılı bakterilerin seğirmesi motilitesini ve göç analizi için özellikle uygun olduğunu göstermiştir. Bu sonuçlar hücre-hücre eki, agregasyonu ve kolonizasyonu içinde kolaylaştırır seğirmesi aracılı göç mekanizmasını açıklamakEv sahibi, sonuçta sistemik enfeksiyona yol. X. Pil-Chp operon fastidiosa kodlamak sinyal iletimi 20 yolaklarıyla pilG, pili, pilJ, hap, chpB ve chpC içerir. transmembran kemoreseptörlerin periplazmik etki kimyasal uyaranlara bağlamak ve sonuçta bakteriyel seğirmesi hareketliliğini kontrol etme sitoplazmik kısmı bir sinyal kaskad etkinleştirin. X&#39;in Pil-Chp operonunda fastidiosa, fosfo-geçiş proteini PilG Chey bir homologudur. E. E. coli ve P. aeruginosa, Chey kamçı motor proteinlerin 19, 21 ile etkileşim kemotaksis sistemlerinde yanıt düzenleyicisidir. X&#39;de virülansına doğru Pil-Chp operon katkıları olmasına rağmen fastidiosa çevresel sinyallere yanıt olarak ve X&#39;in ayarlı / motor tip IV Pili için, en son kemotaksis operonunda pilG rolü 20 incelenmiştir fastidiosa unc olduğunulear. X&#39;in seğirmesi hareketlilik aktivitesindeki kemotaksis regülatör pilG bir fikir aydınlatmak için fastidiosa, mikro fl uidic odası X&#39;in seğirmesi motilitesini değerlendirmek için kullanılır fastidiosa. X fastidiosa ve pilG bir silme mutantı Xf ΔpliG tamamlayıcı suşu XfΔpliG C ve in vitro olarak vahşi tip fenotipleri karşılaştırılması ile karakterize edilir. Sonuçlar X&#39;in seğirmesi motilite pilG rolünü vurgulamak 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 fastidiosa bitkiler ekonomik açıdan önemli hastalıklara neden olan bir dizi, bir Gram-negatif olmayan kamçılı bir bakteridir. Seğirmesi motilitesi X sağlar fastidiosa uzun mesafe içi bitki hareketi ve kolonizasyonu için bir araç X patojenik doğru katkıda X&#39;in fastidiosa. seğirmesi motilite fastidiosa tip IV pili tarafından işletilmektedir. Xylella fastidiosa ve Tip IV pili pilG, sinyal transdüksiyon geçiş yolları ile ilişkili olan Pil-Chp operon&#39;u şifreleyen proteinler bir kemotaksis regülatörü tarafından düzenlenir. X&#39;in seğirmesi motilitesinde pilG rollerini aydınlatmak için fastidiosa, bir pilG -açıklı mutant Xf ΔpilG ve yerli pilG içeren tamamlayıcı suşu XfΔpilG- C geliştirilmiştir. Time-lapse görüntü kayıt sistemi ile entegre bir mikroakışkan odaları XfΔp içinde seğirmesi hareketliliğini gözlemlemek için kullanılanILG, XfΔpilG- C ve vahşi tip süzün. Bu kayıt sistemi kullanarak, uzun vadeli mekansal ve zamansal toplama gözlemlerini, tek tek hücrelerin ve seğirmesi hareketlilik yoluyla bakteri popülasyonlarının göç izin verir. X Mutant XfΔpliG seğirmesi eksik fenotip sergilenen oysa fastidiosa yabani tip ve tamamlayıcı XfΔpilG- C suşu, doğrudan mikroakışkan akış gözlerinden gözlenen tipik seğirmesi motilite özellikleri gösterdi. Bu çalışma pilG X. seğirmesi hareketliliğin artmasını göstermektedir fastidiosa. mikroakışkan akış odası seğirmesi hareketliliği gözlemlemek için bir araç olarak kullanılmaktadır.

Tip IV pilus-aracılı seğirmesi hareketlilik gösteren bir çevresel koloni saçak varlığı, X kolonileri gözlendi fastidiosa yabani tip ve tamamlayıcı Xf ΔpliG -C suşu (Şekil 1). Mutant XfΔpliG Ancak koloni çevresi etrafında bir saçak (Şekil 1) sergilemedi. Nano-mikroakışkan akış odalarına bakteri hücrelerinin Time-lapse görüntüleme seğirmesi motilite hem vahşi tip X gözlen...

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

Bu çalışmada, nano-mikroakışkan akış odası görselleştirmek ve işlevsel Xylella fastidiosa, dedikodu olarak Pierce hastalığa neden olan bir bakterinin seğirmesi motilitesini karakterize etmek kullanılmıştır.

Rolü seğirmesi Motilitesi görselleştirme ve Karakterizasyonu<em&gt; PilG</em&gt; &#39;de<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 Görüntüleme.  University of California, Davis.  Bu çalışmada, nano-mikroakışkan akış odası görselleştirmek ve işlevsel Xylella fastidiosa, dedikodu olarak Pierce hastalığa neden olan bir bakterinin seğirmesi motilitesini karakterize etmek kullanılmıştır. 

Video: 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.

Floresans Live-hücre görüntüleme, tam bitkisel hücre döngüsü yavaş büyüyen sosyal bakteri 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 ...

Biyokimya

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.

Kuruluş, larva Zebra balığı bir hayvan modeli araştırmak Trypanosoma cruzi hareketliliği In Vivo olarak

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

Gelişim Biyolojisi

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.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

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

İmmünoloji ve Enfeksiyon

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.

Hücreli Davranış kaydetme Myxococcus xanthus</emZaman atlamalı Microcinematography kullanarak> Biyofilmler

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

Biyoloji

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.

Buruşuk Koloni Gelişimi kullanarak Biyofilm Oluşumu değerlendirin Bir Yarı-kantitatif Yaklaşım

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.

Tek Hücreli Türler Arası Bakteri Etkileşimlerinin Kinetik Görselleştirilmesi

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.

Bakteriyel Glikoziltransferazların Bakteriyel Motilitedeki Rolünü Aydınlatmak için Boş Mutantların Üretilmesi

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.

Bakteriyel Yüzey Motilite Tahliller hazırlanması, Görüntüleme ve miktarının belirlenmesi

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.

Kuyruktaki Motor fonksiyonların Biyofiziksel Karakterizasyonu

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.

Bir Seride Üç Yerleşik Teknik Uygulayarak Escherichia coli'de Flagella Odaklı Hareketliliği Araştırmak

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

Rolü seğirmesi Motilitesi görselleştirme ve Karakterizasyonu

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