El proyecto descrito es apoyado por el premio número 5R01AI116895 del Instituto Nacional de Alergia y Enfermedades Infecciosas. El contenido es responsabilidad exclusiva de los autores y no representa necesariamente las opiniones oficiales del Instituto Nacional de Alergia y Enfermedades Infecciosas o los Institutos Nacionales de Salud.

1. Las muestras de generación <ol><li> El calor activa C. difficile esporas a 65 ° C durante 30 minutos y almacenar en hielo. Nota: C. esporas difficile pueden ser producidos y purificados como se ha descrito previamente 7,9,10. </li><li> Preparar la solución de germinación a X + 1 ml donde X es el número de muestras que deben tomarse durante el ensayo. Nota: La solución de la germinación es específico de las especies de bacterias de esporas que se estudian. Para el ensayo de C. difficile germinación de esporas, se utilizó una solución de Tris 10 mM (pH 7,5), NaCl 150 mM, glicina 100 mM y 10 mM de ácido taurocólico en agua desionizada. </li><li> Antes de comenzar el ensayo, extraer una muestra de la solución de germinación 1,0 ml sin esporas para servir como un espacio en blanco para la detección fragmento de corteza. </li><li> Añadir esporas a un OD 600 de ~ 3,0 a la solución de la germinación y el vórtice rápidamente. Nota: Para nuestros estudios de germinación en B. subtilis, se utilizó la misma OD 600 esporas como para C. difficile. Nota: Esta cantidad de esporas funcionó bien para la liberación fragmento de la corteza durante el seguimiento C. germinación de las esporas difficile. La cantidad de esporas utilizada en este ensayo se debe determinar experimentalmente para cada bacteria o cepa. </li><li> Extraer una muestra 1,2 ml inmediatamente después de la adición de las esporas y se centrifuga a temperatura ambiente durante 1 min a 14.000 x g. Este es el punto de tiempo cero. </li><li> Transferir 1,0 ml del sobrenadante de la muestra centrifugada a un tubo nuevo para el análisis de fragmento de corteza posterior. Nota: En caso de DPA deben ser supervisados, retirar una muestra de 100 l separada para la detección de DPA en la solución utilizando un ensayo basado en fluorescencia terbio 9,18,19. </li><li> Congelar la muestra recogida a -80 ° C. </li><li> Repita los pasos 1.4 y 1.5, a intervalos regulares hasta que todos los puntos de tiempo completadas. Nota: Debido al tiempo requerido para la centrifugación y el muestreo, menos puntos de tiempo THun 2 min aparte no fuera posible durante nuestro procedimiento, por lo que nuestros intervalos de muestreo fueron una vez cada 2 min durante 14 min. </li><li> Como control positivo para la reducción de la detección de azúcar y la cuantificación, incluyen las siguientes cantidades de N acetilglucosamina (nmol): 0, 12,5, 25, 50, 100, 250, 500, y 5000. Preparar estas muestras al mismo tiempo que las muestras de la corteza y de gestión junto con las muestras de la corteza de manera que la curva estándar es relevante para los datos generados por la germinación de esporas. </li><li> Después se recogen todas las muestras puntuales tiempo, liofilizar las muestras. Nota: Nos liofilizado nuestras muestras en tubos de 2 ml con tapón de rosca. </li></ol> 2. Medición de la corteza Fragmentos <ol><li> Resuspender muestras liofilizadas en 120 l de HCl 3 N suplementado con 1% de fenol y 0,5% β-mercaptoetanol. </li><li> La transferencia de las muestras resuspendidas a 2 ml tubos con tapa de rosca. Nota: Para la reproducibilidad, garantizan toda la muestra liofilizada se suspende de nuevo y se transfiere. </li> &lt;li&gt; Incubar las muestras en un baño de agua de recirculación a 95 ° C durante 4 horas. </li><li> Después de la incubación, colocar las muestras en hielo hasta que se hayan enfriado. </li><li> Neutralizar las muestras con 120 l de NaOH 3 M. </li><li> Añadir 80 l de una solución saturada de bicarbonato sódico y 80 l de una solución de anhídrido acético al 5% a cada muestra y vórtice. </li><li> Se incuban las muestras a temperatura ambiente durante 10 min. </li><li> La transferencia de las muestras al baño de agua a 95 ° durante exactamente 3 minutos. Nota: Este paso es el tiempo más largos tiempos sensibles y pueden afectar el desarrollo señal descendente. </li><li> Extraer muestras del baño de agua y enfriar en hielo. </li><li> Añadir 400 l de 6,54% K 2 B 4 O 7 · 4H 2 O a cada tubo y vórtice. </li><li> Se incuban las muestras en un baño de agua de recirculación a 95 ° C durante exactamente 7 min. Nota: Este paso es el tiempo más largos tiempos sensibles y pueden afectar el desarrollo señal descendente. </li><li> retirarlas muestras y colocar en hielo durante 5 min. </li></ol> 3. Reactivo de color <ol><li> Mientras que las muestras están en hielo (paso 2,12), preparar el reactivo de color. Disolver 0,320 g de p -dimethylaminobenzaldehyde (DMAB; el reactivo de Ehrlich) en 1,9 ml de ácido acético glacial. Nota: El reactivo es el tiempo, la temperatura y la sensible a la luz y no debe hacerse por adelantado. </li><li> Después de la DMAB se disuelve completamente, añadir 100 l de HCl y agitar 10 N. </li><li> Añadir 5 ml de ácido acético glacial y vórtice. </li></ol> 4. Reacción colorimétrica <ol><li> Transferir 100 l de cada muestra de corteza se enfrió a un nuevo tubo de microcentrífuga de 1,5 ml. </li><li> Añadir 700 l de la solución de color recién preparada a cada muestra. </li><li> Inmediatamente transferir las muestras a un baño de agua a 37 ° e incubar durante exactamente 20 minutos. </li><li> Transferir 200 l de cada muestra a una placa de 96 pocillos clara. </li><li> Cuantificar las muestras a 585 nm en un platolector. Las muestras deben comenzar en un color amarillo y una reacción positiva se desplazarán a púrpura. La corteza más presente, más profundo es el color púrpura. </li></ol>

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The authors have nothing to disclose.

Tras la estimulación, las esporas se someten el proceso de germinación pierden sus propiedades de resistencia sin la necesidad de la síntesis macromolecular. Cuando se activa la germinación de esporas, el núcleo de esporas de prensa DPA, a cambio de agua 2. Debido al alto contenido de DPA de la espora latente, el núcleo de esporas está bajo presión osmótica intenso y el peptidoglicano especializada, corteza, ayuda a prevenir el núcleo de la hinchazón actuando, presumiblemente, como una barrera a la expansión 2. El método descrito anteriormente utiliza el reactivo de Ehrlich (una solución amarilla) para detectar la presencia de azúcares reductores como un cambio medible a un tinte púrpura. Durante la hidrólisis corteza, NAG y NAM azúcares se liberan (dependiendo de la extensión de las enzimas que hidrolizan, los azúcares pueden liberarse como disacáridos o polisacáridos). La hidrólisis de los enlaces glucosídicos por HCl (Paso 2.1) genera monosacáridos con extremos reductores libres 7. después neutralizing del ácido con una cantidad igual de hidróxido de sodio (paso 2.5), los alcoholes que rodean la NAG y monosacáridos NAM están acilados por anhídrido acético (Paso 2.6) 13. Posteriormente, anhídrido acético sin reaccionar se convierte en ácido acético por calentamiento de la muestra (paso 2.8). Como hipótesis por Morgan y Elson 20, el aldehído terminal de convierte espontáneamente a la forma enol. En condiciones alcalinas (paso 2,10), esta forma enol puede reaccionar con la acetil adyacente para generar un derivado de oxazol que reacciona con el reactivo de Ehrlich (Paso 4.3) para generar un color púrpura 15,20. Este método se ha descrito anteriormente produce resultados consistentes y fiables para la detección de fragmentos de la corteza de la germinación de las esporas, tanto B. subtilis y C. difficile 9,16,17. El ensayo requiere especial atención para asegurar la reacción tiempos, temperaturas y procedimientos se realizan con precisión. Sin la debida atenciónal detalle, es fácil para las condiciones de reacción que fallen o no reproducir entre los experimentos 13. Sin embargo, la inclusión de un NAG ayudas estándar en la aplicación del ensayo entre los entornos de laboratorio. El estándar de control positivo NAG incluido en cada experimento controles para cualquier variabilidad inter-experimental (por ejemplo, ligeras variaciones en los tiempos de reacción o la eficacia del reactivo de color). Las reacciones que utilizan sólo las condiciones del tampón y reacciones con esporas incapaces de hidrolizar tanto corteza dado ningún cambio en el color. El problema más común con este método es la variabilidad inter-experimental o completa falta de señal. Para el control de la variabilidad, las preparaciones de esporas deben estar libres de cualquier residuo célula vegetativa. Esta restos celulares contendrá fragmentos de peptidoglicano (NAG y NAM) y dará lugar a una alta señal de fondo de las esporas germinan. Para asegurar una preparación de esporas puro (&gt; 99,9% esporas), consulte la Literature para los métodos para preparar suspensiones de esporas puras en el organismo a estudiar 7,9,21-23. Si el ensayo no está dando ninguna señal detectable, se observó que la etapa de liofilización puede introducir variabilidad. Debido a la fuerza con la que se libera el vacío, se observó que el polvo de la muestra seca puede ser soplado hacia fuera del tubo de muestra. Para reducir / evitar que esto ocurra, utilice una pequeña aguja de la jeringa para hacer agujeros en la parte superior de los tubos de muestra. La pequeña área de los agujeros introducidos reducirá la probabilidad de la muestra liofilizada (ahora en forma de polvo) de ser molestado. Por último, como se indica en los métodos anteriores, los varios pasos de incubación son muy sensibles al tiempo. Asegúrese de que todos los pasos cronometrados son monitoreados cuidadosamente. Una consideración importante para este ensayo es que los fragmentos de la corteza debe ser liberado de la espora de germinación a fin de ser detectado. Si la corteza se hidroliza en grandes fragmentos, estos fragmentos pueden ser demasiado grandes para ser releobre la base a través de la capa de la espora. Aunque una consideración importante para solucionar problemas de un experimento que no produce una señal, se recomienda confirmar que las condiciones utilizadas para germinar la mayor parte de las esporas en la solución y las otras opciones de solución de problemas enumerados aquí antes de considerar el tamaño del fragmento corteza como un problema potencial en el ensayo. No todos los buffers de germinación / condiciones son adecuadas para este ensayo. Debido a que este ensayo detecta la presencia de azúcares reductores liberados durante el proceso de germinación 13, cualquier tampón germinación que contiene azúcares reductores o cualquier germinantes que es, en sí, un azúcar reductor (por ejemplo, glucosa) dará lugar a una fuerte señal en este ensayo. Si este es el caso, la cromatografía líquida de alta presión (HPLC) se puede utilizar para detectar la presencia de fragmentos de la corteza 24-26. Sin embargo, porque no todos los laboratorios están equipados con tales instrumentación, el método descrito en este documento puede ser utilizado por la mayoríalaboratorios y ofrece resultados simples y fácilmente cuantificables. La germinación de las esporas bacterianas en gran medida se ha estudiado en Bacillus y unas pocas especies de Clostridium 2,8. El proceso generalmente se conserva, sin embargo, existen diferencias. Es importante destacar que no todos los organismos germinan utilizando los mecanismos de la germinación de esporas como se observa en el modelo de formadores de esporas 9,27. Debido a la germinación se está estudió en más diversos organismos, el ensayo descrito anteriormente se puede aplicar a estos organismos para detectar fragmentos de la corteza durante la germinación de esporas. Mediante la aplicación de este método para estos organismos estudiados recientemente, los investigadores pueden determinar el papel de las proteínas juegan varios de germinación en la hidrólisis de la corteza.

Las endosporas son metabólicamente inactivos formas de bacterias que permiten a las bacterias persisten en ambientes desfavorables. En muchas bacterias formadoras de esporas, la formación de esporas es inducida por la privación de nutrientes, pero este proceso puede ser controlado por los cambios en pH, la exposición a oxígeno o otros factores de estrés 1. Si bien en su forma de esporas metabólicamente inactivos, las bacterias se resisten a la radiación UV, desecación, temperaturas más altas, y la congelación de 2. La mayor parte del conocimiento sobre el proceso de esporulación proviene de estudios en el organismo modelo, Bacillus subtilis. El proceso de esporulación comienza con la replicación del ADN y la formación de un 3,4 filamento axial. Un tabique asimétrico luego se divide la célula en dos compartimientos de tamaño desigual. El compartimiento más grande, la célula madre, envuelve el compartimento más pequeño, el preespora. Tanto la célula madre y preespora coordinar la expresión de genes para madurar la espora y la célula madre, finalmente, lisa, de soltar el Dormant de esporas en el medio ambiente que rodea 1. La estructura y composición de esporas se conserva en muchas especies bacterianas. El núcleo de esporas tiene un contenido de agua inferior a la célula vegetativa y es rica en ácido dipicolínico (DPA) 1,2. Durante la formación de esporas, DPA se bombea en el núcleo de esporas a cambio de agua. Que rodea el núcleo es una membrana de esporas interior donde, en la mayoría de las bacterias formadoras de esporas, los receptores que reconocen las pequeñas moléculas que estimulan la germinación de semillas en germinación (2) se encuentran. Situado a las afueras de la membrana interior de la espora es una capa de peptidoglicano de la pared celular. Una capa de peptidoglicano especializada (corteza) rodea el peptidoglicano de la pared celular y se compone de muchos de los mismos componentes que el peptidoglicano de la pared celular [alternando N-acetilglucosamina (NAG) y ácido N-acetilmurámico (NAM)]. Sin embargo, aproximadamente 50% de los residuos NAM en la corteza se han convertido a murámico-δ-lactama 5,6 </sarriba&gt;. Durante la germinación de esporas, estos residuos murámico-δ-lactámicos son reconocidos por las esporas de la corteza enzimas líticas (SCLEs), permitiendo así que la corteza a degradarse (un proceso esencial para completar la germinación), pero no la pared celular. Que rodea la corteza es una membrana externa y varias capas de proteínas de la cubierta 2. Controlar el proceso de germinación es de importancia crítica para las bacterias formadoras de esporas. La germinación se inicia cuando los receptores germinantes interactúan con sus respectivos semillas en germinación 2. En muchas bacterias formadoras de esporas, estas semillas en germinación son aminoácidos, azúcares, nucleótidos, iones o combinaciones de los mismos 2. C. difficile germinación de las esporas es iniciado por combinaciones de ciertos ácidos biliares, [por ejemplo, ácido taurocólico (TA)] y aminoácidos (por ejemplo, glicina) 7-10. Aunque hay diferencias entre el C. vía la germinación difficile y las vías estudiadas en otra forma esporasbacterias, tales como B. subtilis, común a todos es el requisito absoluto para degradar la corteza de esporas para permitir que una célula vegetativa a crecer a partir de la espora germinada 2,8. La degradación de la corteza se puede lograr por el SCLEs CwlJ / SleB (como se encuentra en B. subtilis) o SLEC (como se encuentra en muchos Clostridia). hidrólisis de la corteza reduce la restricción en la pared celular y la espora. Esto permite una rehidratación completa del núcleo, un paso necesario en la reactivación de muchas de las proteínas necesarias para el metabolismo celular 2. Cuando las esporas germinan, los cambios de esporas inactivas de un estado de fase-brillante a un estado de fase-oscuro y este proceso se pueden medir por un cambio en la densidad óptica (DO) a 600 11 nm. Un informe anterior sugiere que gran parte de este cambio en OD es debido a la liberación de la DPA de la espora 12. Durante nuestra reciente estudio, hemos tratado de comparar el tiempo de C. difficile germinación de esporas y seguimiento de laliberación de la DPA y la corteza fragmentos 9. Para este estudio era fundamental para controlar la liberación de fragmentos de la corteza, ya que comenzó a ser liberado por la germinación de las esporas. El ensayo colorimétrico se utiliza aquí se basa en un método para la detección de azúcares reductores con extremos desarrollaron Ghuysen et al. 13. Debido a que otros han descrito protocolos para la detección de azúcares reductores 14 o han modificado los protocolos 15, la literatura sobre este tema puede ser confuso. Aquí, un método paso a paso que detalle para la detección colorimétrica de azúcares reductores liberados de la germinación de C. esporas difficile. Aunque este estudio utiliza C. esporas difficile, los azúcares reductores liberados durante la germinación de esporas de otras bacterias formadoras de esporas son capaces de ser detectadas con este protocolo 9,16,17.

<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.

La Tabla 1 muestra los resultados típicos que usan las normas de NAG. Los datos se utilizan para generar una curva estándar. Tabla 2 muestra los resultados típicos de un ensayo de germinación en tampón de germinación suplementado con glicina 100 mM y TA 10 mM (condiciones de germinación de promoción) o glicina 100 mM sólo (condiciones no de germinación). En ausencia de TA, C. esporas difficile no germinan y hay poco cambio en la presencia de fragmentos de la corteza en la solución. Sin embargo, en la presencia de ambos TA y glicina, C. esporas germinan difficile y liberan fragmentos de la corteza en la solución. Esto se observa como un aumento en el OD 585 con el tiempo de las muestras reaccionado. La cantidad total de fragmentos de corteza (nmol) liberados durante este ensayo se calculó utilizando una curva estándar similar a la figura 1B. En este ensayo, la mayor parte de la corteza se hidroliza (o liberado) por 15 minutos posterioresexposición germinantes, lo que sugiere que la mayoría de las esporas han germinado en este marco de tiempo (Figura 2; consistente con una publicación anterior 7). Después de desarrollar las muestras en el reactivo de color, la mayoría de las muestras pueden no mostrar el color púrpura a simple vista. La figura 1 muestra una muestra representativa generado a partir de N-acetilglucosamina. Muchas de las muestras no muestran un color púrpura visible. Sin embargo, los 250 nmol, 500 nmol y 5.000 nmol muestras aparecen visualmente diferente del control negativo (0 nmol). Cuando se mide a una DO de 585 nm, la señal generada mejor se ajuste a una regresión lineal (R2 = 0,999). <img alt="Figura 1" src="/files/ftp_upload/54146/54146fig1.jpg" /> Figura 1: Detección. N-acetilglucosamina muestras (A) N-acetilglucosamina (0 nmol, 12,5 nmol,25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, 5000 nmol) se liofilizaron y reaccionar de acuerdo con el procedimiento descrito. Se añadieron (b) las muestras a una placa de 96 pocillos y la cantidad de material reaccionado detectado en 585 nm. La señal se utiliza para generar una curva estándar. <img alt="Figura 2" src="/files/ftp_upload/54146/54146fig2.jpg" /> Figura 2:. Representación gráfica de los resultados de la germinación La corteza nmol calculada en la Tabla 2 se representó frente al tiempo. (●) mM glicina + 100 mM TA 10; (■) glicina 100 mM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Concentración NAG (nmol) </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> = blanco </td><td> 0,046 </td></tr></tbody></table> Tabla 1: NAG Standard. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="4"> Clostridium difficile germinación de esporas </td></tr><tr><td></td><td colspan="4"> Solución de germinación (mM Tris 10 (pH 7,5), NaCl 150 mM) </td></tr><tr><td></td><td colspan="2"> mM Glicina 100 mM + TA 10 </td><td colspan="2"> La glicina 100 mM </td></tr><tr><td> Tiempo (minutos) </td><td> OD 585 </td><td>Calculado (nmol) </td><td> OD 585 </td><td> Calculado (nmol) </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> Tabla 2: Ejemplo de Spore Germinatisobre los resultados.

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.

La detección de la corteza Fragmentos Durante la germinación de esporas bacterianas

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

Video: 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.

El aislamiento físico de endosporas de muestras ambientales por Targeted lisis de las células vegetativas

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

Investigación

Medio ambiente

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).

La detección de bacterias mediante ADNzimas fluorogénicos

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

Inmunología e Infección

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.

Mejora de la enumeración de esporas de Bacillus subtilis y el análisis de etiquetas en citometría de flujo

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

Bioquímica

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.

Visualización de Germinosomes y la membrana interna de las esporas de Bacillus subtilis

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

Bioingeniería

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.

Evaluación visual y microscópica de mutantes desarrollo de Streptomyces

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.

Desarrollo Sexual y descarga en las ascosporas Fusarium graminearum</em

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

Biología

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.

Adsorción de esporas como un sistema de visualización Nonrecombinant para enzimas y antígenos

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.

Usando Coculture detectar químicamente mediadas por interacciones interespecíficas

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.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

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.

Germinación de esta investigadora mediante el análisis de Microrespiration y microdisección

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