Este estudo foi apoiado pelo United States Department of Agriculture, Agricultural Research Service. nomes comerciais ou produtos comerciais nesta publicação são mencionados somente para a finalidade de fornecer informações específicas e não implica qualquer recomendação ou conselho pelo Departamento de Agricultura dos Estados Unidos. O USDA é um fornecedor de oportunidades iguais e empregador.

1. O Fringe periférica de origem bacteriana Colony <ol><li> Crescer X. fastidiosa (Xf) Temecula 22 de tipo selvagem, mutante de deleção pilG Xf ΔpliG (usando estratégia de deleção anteriormente descritos 23), e a sua complementar XfΔpliG -C (usando previamente descrito estratégia de complementação genética baseada no cromossoma 24) na PD2 de agar com meio placas 25 a 28 ° C durante 5-7 dias. </li><li> Celofane autoclave (1 x 1 cm2) em água a 121 ° C (249 ° F) durante 15 min. Pegue um pedaço de celofane, drenar a água tocando em um canto do celofane em uma placa de Petri vazia, com cuidado colocar o celofane mais de 15% da superfície de agar e ar seco. </li><li> Pegar indivíduo X. colónias fastidiosa com estéril palitos arredondadas e células no local assepticamente em uma folha esterilizada de celofane sobreposto ao 15% da superfície do agar nas placas de ágar. Incubar a placas a 28 ° C durante 2-3 dias. </li><li> Examinar a morfologia da borda das colónias utilizando um microscópio de dissecação com uma lente objectiva 2X e uma lente ocular 10X. Fotografar a orla periférica em torno de colónias. </li></ol> 2. Microscopia e microfluídicos Fluxo Chambers <ol><li> Fabricar dispositivos microfluídicos utilizando procedimentos de foto-litográfica semelhantes aos descritos anteriormente 5,18. Projetar quatro canais paralelos com software de desenho assistido por computador sobre silício mestre usando métodos de litografia padrão 26. </li><li> Criar as câmaras microfluídicos de mestre de wafer de silício com polidimetilsiloxano (PDMS). Pour PDMS não polimerizados sobre a bolacha de silício mestre e curá-la a 60 ° C durante 1 h. Descole a réplica de PDMS do mestre wafer e aparar a réplica PDMS com uma lâmina em um 22 mm x 40 mm como o mesmo tamanho de uma lamela de vidro. </li><li> Expor a réplica de PDMS, uma lamela de vidro (22 x 40 mm2), e um microscslides ope (51 x 76 mm2) ao plasma de ar a 30 W por 2 min 27. Sanduíche do corpo PDMS entre a lamela de vidro eo vidro de microscópio para construir a câmara uidic fl micro. </li><li> Perfurar um furo (5,5 mm de diâmetro) através do PDMS em cada extremidade do canal modelado. Corte o tubo de borracha de silicone em 12-20 cm de comprimento. Inserir uma extremidade da tubagem de borracha de silicone (5,1 mm de diâmetro externo, 2,1 mm de diâmetro interno, parede de 0,8 mm) em cada extremidade dos canais da réplica de PDMS de abertura, e selá-lo com PDMS não polimerizados a 60 ° C durante 1 h. </li><li> Ligue a outra extremidade do tubo até o fim farpado de conectores luer de plástico. Enrole as câmaras de microfluidos montadas com a folha de alumínio e autoclave los durante 20 min. </li><li> Recolha células de bactérias de X. fastidiosa tipo selvagem, mutante Xf ΔpliG, e complementar XfΔpliG -C através de raspagem, usando inoculação descartáveis ​​laços de placas de meio PD2. Ajustar a densidade celularpara uma densidade óptica de 0,05 a 600 nm em PD2 caldo como descrito anteriormente 23. Recolhe-se o solução da célula bacteriana numa seringa de 1 ml estanque ao gás. </li><li> Montar o dispositivo microfluídico em um palco microscópio invertido. Ligue um tubo de entrada para a seringa estanque ao gás 5 ml contendo PD2 caldo. Montar a 5 ml estanque ao gás seringa com as bombas de seringa. </li><li> Ligue o tubo de saída a um reservatório de resíduos. Manter uma taxa de fluxo de meio de 0,2 mL min -1 durante 30 minutos para estabilizar o sistema. </li><li> Conectar tubos laterais de entrada a uma seringa de 1 ml estanque ao gás que contém a solução da célula bacteriana. Lavar a solução de células bacterianas através do tubo de borracha até que o canal seja atingido. Manter uma taxa de fluxo de meio de 0,2 mL min -1 durante mais 30 minutos, a fim de lavar as células não ligadas da câmara antes da captura da imagem. </li><li> Montar o obturador microscópio sob a parte ajustada ao campo do microscópio para controlar a luz. Ligue o obturador para a co obturadorntrol sistema e ligar o sistema de controlo do obturador para o computador. </li><li> Montar uma câmera digital à porta de vídeo do microscópio e conecte-o ao computador. Executar o software de gravação de lapso de tempo, selecione a função &quot;obturador&quot; do menu, e reconhecer automaticamente o obturador instalado como padrão no software para estabelecer conexões com o obturador com o software. </li><li> Seleccione a função &quot;camera digital&quot; a partir do menu do software de gravação de lapso de tempo para reconhecer automaticamente a câmera digital como o dispositivo de captura padrão no software e estabelecer a comunicação com a câmera digital com o software. </li><li> Localizar as células bacterianas em um dos canais usando 20X de contraste de fase óptica e, em seguida mudar para a lente da objectiva de 40X, antes da captura da imagem. </li><li> Executar o software de gravação de lapso de tempo, selecione a função &quot;aquisição de imagem&quot; usando parâmetros padrão a partir do menu para adquirir as imagens do microscópio.Em seguida, abra a função &quot;Adquirir time-lapse&quot; e defina o intervalo de tempo para 30 seg 5,18,28, com duração de 6-24 horas, dependendo experiência necessária para observar se contraindo motilidade dos X. fastidiosa 5,18,28. Clique em &quot;OK&quot; para iniciar a gravação de lapso de tempo. Clique em &quot;função de pilha&quot; a partir do menu, selecione &quot;salvar como&quot; para empilhar as imagens na pasta de destino no computador depois de terminar a gravação. </li><li> Para múltiplos canais, capturar imagens de lapso de tempo a partir do primeiro canal a cada 30 seg durante 6 horas. Mover a lente objectiva do microscópio para o próximo canal para localizar as células alvo. Repita a função de lapso de tempo, como descrito acima para capturar imagens em cada um dos quatro canais sequencialmente se experimento está configurado para utilizar quatro canais. Continue a captura de imagens de lapso de tempo para até três dias consecutivos. Todas as experiências foram realizadas à temperatura ambiente (23 ± 2 ° C). </li><li> Compilar as imagens de lapso de tempo em umarquivo de vídeo usando o software de imagem de gravação de lapso de tempo. Executar o software de gravação de lapso de tempo, clique em &quot;função Stack&quot; no menu e selecione &quot;função de pilha aberta&quot; para abrir os arquivos empilhados a partir do computador. </li><li> Inicie a função de &quot;fazer cinema&quot; do módulo de pilha, a seleção de todas as imagens e escolher o formato de saída &quot;AVI&quot;. Clique em &quot;Salvar como&quot; para armazenar o arquivo de vídeo na pasta de destino no computador. </li><li> Seleccione os filmes compilados a partir da pasta de destino no computador e reproduzi-los. Em seguida, observe a motilidade das células individuais através da visualização resultante da atividade espasmos motilidade de células de bactérias nos arquivos de vídeo gerados. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Neste estudo, caracteriza o comportamento de movimento de X. fastidiosa PilG mutante Xf ΔpilG e suas complementares estirpes XfΔpilG- C em recém-projetado de múltipla nano-canal paralelo micro câmaras fl uidic. As câmaras uidic recém-projetado micro fl pode ter até quatro câmaras paralelas com 100 um nano-canal de largura em comparação com modelos anteriores, com apenas uma única de 50 m de largura do canal 18. A melhoria mais ampla nano-canal facilita ...

Xylella fastidiosa é uma bactéria patogénica gram-negativas não flagelados, que faz com que um certo número de doenças de culturas economicamente importantes, incluindo a doença de Pierce em videira (Vitis vinifera L.) 1,2, 3. Esta bactéria é limitada ao xilema de condução de água embarcações. Infecção da videira faz com que o bloqueio dos vasos do xilema e resulta em estresse hídrico e deficiências nutricionais 3. Colonização bem sucedida depende da capacidade da bactéria para se mover a partir do local inicial da infecção para o resto da planta 3. Espasmos motilidade é um meio de movimento bacteriana independente de flagelar através da extensão, apego e retração do pili tipo IV polar 4 que tem sido caracterizado X. fastidiosa 5,6,7. A motilidade espasmos tem sido observado por pinças laser e microscopia de força atômica (AFM) 8,9,10. Utilizando estas técnicas, tmotilidades witching gerado pelo pilus de tipo IV de N. gonorrhoeae e P. aeruginosa foram caracterizados por fl pili rotulagem uorescently e capturando os seus movimentos microscopicamente. Embora ambos os métodos tenham detalhado a força adesiva de bactérias individuais, os procedimentos são complicados e demorados 9,10. As câmaras uidic fl micro foram usados ​​para observar a migração de longa distância de células individuais, bem como pequenos agregados de células bacterianas 5,6. Estas câmaras foram concebidas como um-nano-canal microfabricated em uma placa integrada com um lapso de tempo 11,12,13,14 sistema de gravação de imagem. Micro fl dispositivos câmara uidic oferecem várias vantagens para estudar o comportamento movimento e célula-célula interações de bactérias: (i) fornece uma plataforma integrada com múltiplas capacidades de canal; (Ii) que pode examinar os movimentos e agregações de células individuais nas características de nano-escala de bactérias; (Iii) que permite m diretaicroscopic gravação de imagem de células bacterianas e análises de lapso de tempo, (iv) ele fornece observações espaciais e temporais de longo prazo de populações individuais e / ou de bactérias em um micro-ambiente; (V) a taxa de fluxo de meio de cultura em um canal pode ser controlada com precisão e (VI), apenas um muito pequeno volume (1 ml) de meio de cultura é necessário para cada experiência. Recentemente, o micro fl uidic fluxo do sistema foi utilizado para investigar o comportamento de células bacterianas sob diferentes microambientes 14,15,16. A adesividade e o anexo de superfície do E. coli 15, X. fastidiosa 16 e Acidovorax citrulli 14 em superfícies de vidro foram avaliados através de câmaras uidic micro fl. A formação de agregação e biofilme mediada por tipo pili IV do Acidovorax citrulli foram analisados ​​14. Além disso, o movimento de A. citrulli observado sob fl uxo cs condições demonstraram que o pili de tipo IV podem desempenhar papéis importantes na colonização e a propagação de A. citrulli nos vasos do xilema sob seiva fluxo condições. Os motilidades espasmos de Pseudomonas aeruginosa e X. fastidiosa células foram observadas com sucesso contra uma corrente de fluido numa câmara de fluxo microfabricado 5,6,17. Pilus de tipo IV deficiente PilB e pilQ mutantes de X. fastidiosa foram encontrados para alterar profundamente a velocidade da contraindo motilidade nas condições fl uxo em micro dispositivos uidic fl 5,6,18. Os estudos realizados sobre a adesão bacteriana e motilidade em dispositivos uidic fl micro indicaram que as câmaras uidic micro fl são particularmente adequadas para analisar a motilidade contrair-se e a migração das bactérias mediada por pili in vitro. Estes resultados explicam o mecanismo de migração mediada por espasmos que facilita a ligação célula-célula, a agregação e a colonização dentroo anfitrião, eventualmente, levar a infecção sistémica. Pil-Chp operon de X. fastidiosa contém pilG, Pili, pilJ, Comprimido, CHPB e CHPC que a transdução do sinal de codificação vias 20. Os quimiorreceptores transmembranares ligar estímulos químicos no domínio periplasmático e activar uma cascata de sinalização na sua porção citoplasmática para controlar, em última análise da motilidade espasmos bacteriana. No operão Pil-cogeração de X. fastidiosa, uma PilG proteína fosfo-shuttle é um homólogo de CheY. Em E. coli e P. aeruginosa, CheY é o regulador de resposta de quimiotaxia em sistemas que interagem com as proteínas do motor flagelos 19, 21. Embora as contribuições do operon Pil-Chp em ​​direção a virulência em X. fastidiosa foram examinados 20 recentemente, o papel de pilG no operão quimiotaxia em resposta aos sinais ambientais e regulada pili de tipo IV / motor de X. fastidiosa é UNClear. Para elucidar a visão do regulador de quimiotaxia pilG na actividade da motilidade espasmos de X. fastidiosa, uma câmara de micro uidic FL é utilizado para avaliar a motilidade espasmos dos X. fastidiosa. O pilG de X. fastidiosa é caracterizada pela comparação dos fenótipos de um mutante de deleção Xf ΔpliG, a estirpe complementar XfΔpliG -C e seu tipo selvagem in vitro. Os resultados destacam o papel de pilG na motilidade espasmos de X. 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 é uma bactéria não-flagelado Gram negativo que causa uma série de doenças economicamente importantes de plantas. A motilidade espasmos fornece X. fastidiosa um meio para o movimento de longa distância intra-planta ea colonização, contribuindo para patogenicidade em X. fastidiosa. A motilidade espasmos de X. fastidiosa é operado por pili tipo IV. Pili de tipo IV de Xylella fastidiosa são regulados por pilG, um regulador de quimiotaxia em proteínas do operão de codificação Pil-CHP que estão envolvidos com vias de transdução de sinal. Para elucidar os papéis de pilG na motilidade espasmos de X. fastidiosa, uma pilG -deficient mutante Xf ΔpilG e sua estirpe complementar XfΔpilG- C contendo pilG nativa foram desenvolvidos. A câmara microfluídicos integrados com um sistema de gravação de imagens de lapso de tempo foi usado para observar espasmos motilidade em XfΔpIlg, XfΔpilG- C e sua linhagem selvagem. Usando este sistema de gravação, que permite observações espaciais e temporais de longa duração de agregação, a migração de células individuais e as populações de bactérias através de motilidade espasmos X.. fastidiosa tipo selvagem e complementar estirpe XfΔpilG- C mostrou características típicas espasmos motilidade diretamente observados nas câmaras de fluxo microfluídicos, enquanto mutante XfΔpliG exibiu o fenótipo deficiente se contraindo. Este estudo demonstra que pilG contribui para a motilidade espasmos dos X. fastidiosa. A câmara de fluxo microfluidico é utilizado como um meio para a observação de contracções motilidade.

A presença de uma colónia franja periférica indicativo de tipo IV mediada pelo pilus motilidade espasmos, foi observada nas colónias de X. fastidiosa tipo selvagem e complementar estirpe Xf ΔpliG -C (Figura 1). Mutante XfΔpliG, no entanto, não exibiram uma franja ao redor da periferia de colónias (Figura 1). Imagens de lapso de tempo de células bacterianas em câmaras de fluxo nano-microfluídico revelou que c...

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

Neste estudo, uma câmara de fluxo nano-microfluídico foi utilizado para visualizar e funcionalmente caracterizar a motilidade espasmos da Xylella fastidiosa, bactéria que causa a doença de Pierce em videira.

Visualização de Twitching motilidade e Caracterização do papel da<em&gt; PilG</em&gt; in<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

Research

JoVE Journal

Immunology and Infection

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

10.7K visualizações.  University of California, Davis.  Neste estudo, uma câmara de fluxo nano-microfluídico foi utilizado para visualizar e funcionalmente caracterizar a motilidade espasmos da Xylella fastidiosa, bactéria que causa a doença de Pierce em videira. 

Vídeo: 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.

Imagem latente da Live-pilha fluorescência de completa ciclo vegetativo celular do lento crescimento Social bactéria 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 ...

Bioquímica

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.

Estabelecimento de Zebrafish Larval como um modelo Animal para investigar Trypanosoma cruzi motilidade 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 ...

Biologia do Desenvolvimento

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.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

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

Imunologia e Infecção

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.

Comportamento de gravação em multicelulares Myxococcus xanthus Os biofilmes usando lapso de tempo Microcinematography

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

Biologia

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.

Uma abordagem semi-quantitativa para avaliar formação de biofilmes usando o desenvolvimento da colônia enrugado

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.

Visualização cinética de interespécies interespécies unicelulares interespécies

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

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.

Preparação, Imaging, e quantificação de Motilidade Ensaios superfície da bactéria

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.

Caracterização biofísica das funções Flagellar motor

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

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

Imagem de lapso de tempo de enxames bacterianos e a resposta ao estresse coletivo

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

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.

Investigando a Motilidade Impulsionada por Flagella em Escherichia coli aplicando três técnicas estabelecidas em uma série

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

Visualização de Twitching motilidade e Caracterização do papel da

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