Name: detecting cortex fragments during bacterial spore germination
Uploaded: 2016-06-25T13:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Das Projekt beschrieben wird durch Verleihungsnummer 5R01AI116895 aus dem Nationalen Institut für Allergien und Infektionskrankheiten unterstützt. Der Inhalt ist allein in der Verantwortung der Autoren und stellen nicht notwendigerweise die offizielle Position des Nationalen Instituts für Allergien und Infektionskrankheiten oder die National Institutes of Health vertreten.

1. Erstellen von Proben <ol><li> Wärme aktivieren C. difficile Sporen bei 65 ° C für 30 min und auf Eis lagern. Hinweis: C. difficile - Sporen hergestellt und gereinigt werden , wie zuvor beschrieben , 7,9,10. </li><li> Bereiten Sie die Keimung Lösung bei X + 1 ml, wobei X die Anzahl der Abtastwerte ist, die während des Assays entnommen werden. Anmerkung: Die Keimlösung ist spezifisch für die Spezies von Bakterien untersucht-Sporen werden. Für C. Testen difficile Sporenkeimung, verwendeten wir eine Lösung von 10 mM Tris (pH 7,5), 150 mM NaCl, 100 mM Glycin und 10 mM Taurocholsäure in deionisiertem Wasser. </li><li> Vor der Durchführung des Tests, ziehen eine 1,0 ml Probe der Keimungslösung ohne Sporen als Rohling für Cortex-Fragment Erkennung dienen. </li><li> Hinzufügen Sporen zu einer OD 600 von ~ 3,0 auf die Keimung Lösung und vortex schnell. Hinweis: Für unsere Keimung Studien in B. subtilis, wir haben das gleiche OD 600 Sporen als für C. difficile. Hinweis: Diese Menge an Sporen funktionierte gut für die Überwachung der Hirnrinde Fragment während C. difficile Spore Keimung. Die Menge von Sporen in diesem Assay verwendet wird, sollte experimentell für jedes Bakterium oder Belastung bestimmt werden. </li><li> Entfernen, um eine 1,2 ml Probe unmittelbar nach der Sporen Zugabe und Zentrifuge bei Raumtemperatur für 1 min bei 14.000 × g. Dies ist der Zeitpunkt Null. </li><li> Übertragen 1,0 ml des Überstands aus der zentrifugierten Probe in ein frisches Röhrchen zur anschließenden cortex Fragmentanalyse. Hinweis: Sollte DPA muss überwacht werden, eine separate 100 ul - Probe zum Nachweis von DPA in der Lösung zu entfernen unter Verwendung eines Assays auf Basis von Terbium - Fluoreszenz 9,18,19. </li><li> Fixieren Sie die gesammelte Probe bei -80 ° C. </li><li> Wiederholen Sie die Schritte 1.4 und 1.5 in regelmäßigen Abständen, bis alle Zeitpunkte abgeschlossen. Hinweis: Da der Zeitaufwand für die Zentrifugation und Probenentnahmen, Zeitpunkte weniger thein 2 min waren abgesehen nicht möglich während unseres Verfahrens, so dass unsere Abtastintervallen waren einmal alle 2 min für 14 min. </li><li> Als Positivkontrolle für Zucker Nachweis und die Quantifizierung Verringerung umfassen die folgenden Mengen an N Acetylglucosamin (nmol): 0, 12,5, 25, 50, 100, 250, 500 und 5.000. Bereiten Sie diese Proben in der gleichen Zeit wie Cortex Proben und führen zusammen mit den Cortex-Proben, so dass die Standard-Kurve an die Daten relevant sind, erzeugt durch Sporen keimen. </li><li> Nachdem alle Zeitpunkt Proben gesammelt werden, lyophilisieren die Proben. Anmerkung: Wir gefriergetrocknet, unsere Proben in 2 ml mit Schraubverschluss Röhrchen. </li></ol> 2. Messung Cortex Fragmente <ol><li> lyophilisierte Proben in 120 ul 3 N HCl supplementiert mit 1% Phenol und 0,5% β-Mercaptoethanol resuspendieren. </li><li> Übertragen Sie die resuspendierten Proben auf 2 ml mit Schraubverschluss Röhrchen. Hinweis: Für die Reproduzierbarkeit, sicherzustellen, dass alle die gefriergetrocknete Probe suspendiert und übertragen wird. </li> &lt;li&gt; Inkubieren der Proben in einem Umlaufwasserbad bei 95 ° C für 4 Stunden. </li><li> Nach der Inkubation, legen Sie die Proben auf Eis, bis sie kühl sind. </li><li> Neutralisieren der Proben mit 120 ul 3 M NaOH. </li><li> Hinzufügen, 80 ul einer gesättigten Natriumhydrogencarbonat-Lösung und 80 ul einer 5% Essigsäureanhydrid-Lösung zu jeder Probe und vortexen. </li><li> Inkubieren der Proben bei Raumtemperatur für 10 min. </li><li> Übertragen Sie die Proben zurück zu dem 95 ° C warmen Wasserbad für genau 3 min. Hinweis: Dieser Schritt ist zeitkritisch und längere Zeiten nachgeschaltete Signalentwicklung beeinflussen können. </li><li> Entfernen Proben aus dem Wasserbad und kühlen auf Eis. </li><li> In 400 ul 6,54% K 2 B 4 O 7 · 4H 2 O in jedes Röhrchen und Wirbel. </li><li> Inkubieren der Proben in einem Umlaufwasserbad bei 95 ° C für genau 7 min. Hinweis: Dieser Schritt ist zeitkritisch und längere Zeiten nachgeschaltete Signalentwicklung beeinflussen können. </li><li> Entfernendie Proben und auf Eis für 5 min. </li></ol> 3. Farbreagenz <ol><li> Während Proben auf Eis (Schritt 2.12) sind, bereiten Sie das Farbreagenz. Man löst 0,320 g p -dimethylaminobenzaldehyde (DMAB; Ehrlich-Reagens) in 1,9 ml Eisessig. Hinweis: Das Reagenz ist Zeit, Temperatur und lichtempfindlich und sollte nicht im Voraus gemacht werden. </li><li> Nachdem der DMAB vollständig auflöst, 100 ul 10 N HCl und Wirbel. </li><li> 5 ml Eisessig und Wirbel. </li></ol> 4. kolorimetrische Reaktion <ol><li> Transfer 100 ul jeder gekühlten Kortex Probe in ein neues 1,5 ml Mikrozentrifugenröhrchen. </li><li> In 700 ul der frisch zubereiteten Farblösung zu jeder Probe. </li><li> Unmittelbar übertragen Sie die Proben auf einem 37 ° C Wasserbad und für genau 20 min inkubiert. </li><li> Übertragen Sie 200 ul jeder Probe auf eine klare 96-Well-Platte. </li><li> Quantifizierung der Proben bei 585 nm auf einer PlatteLeser. Die Proben sollten bei einer gelben Farbe und eine positive Reaktion auf lila verschiebt starten. Je mehr Hirnrinde vorhanden ist, desto tiefer die violette Farbe. </li></ol>

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The authors have nothing to disclose.

Nach Stimulation unterzogen Sporen den Prozess der Keimung verlieren ihre Beständigkeitseigenschaften ohne die Notwendigkeit einer makromolekularen Synthese. Wenn Sporenauskeimung ausgelöst wird, 2 die Spore Kern freigibt DPA im Austausch für Wasser. Aufgrund der hohen DPA - Gehalt der ruhenden Spore, die sporen Kern unter intensiver osmotischen Druck und dem Fach Peptidoglycan, Hirnrinde, hilft durch Einwirken vermutlich als Barriere für Expansions 2 den Kern aufquellen verhindern. <p clas...

Endospores metabolisch ruhenden Formen von Bakterien, die Bakterien erlauben, in ungünstigen Umgebungen zu bestehen. In vielen sporenbildenden Bakterien, wird die Sporenbildung von Nährstoffmangel induziert aber dieser Prozess kann 1 durch Änderungen der pH - Wert, der Exposition gegenüber Sauerstoff oder anderen Spannungen gesteuert werden. Während in ihrer metabolisch ruhenden Sporenform, wider Bakterien UV - Strahlung, Austrocknung, höhere Temperaturen und Einfrieren 2. Der größte Teil des Wissens über die Sporulation Prozess stammt aus Studien im Modellorganismus Bacillus subtilis. Die Sporulation Prozess beginnt mit der DNA - Replikation und die Bildung eines axialen Glühfaden 3,4. Eine asymmetrische Septum teilt sich dann die Zelle in zwei ungleich große Fächer. Die größere Kammer, die Mutterzelle, verschlingt die kleinere Kammer, die forespore. Sowohl die Mutterzelle und forespore Genexpression koordinieren, um die Sporen und die Mutterzelle zu reifen schließlich lysiert, die Freigabe der dormant Sporen in die Umgebung 1. Die Sporen Struktur und Zusammensetzung ist in vielen Bakterienarten konserviert. Die Spore Kern hat einen geringeren Wassergehalt als die vegetative Zelle und ist reich mit Dipicolinsäure (DPA) 1,2. Während die Sporenbildung wird DPA in die Spore Kern im Austausch für Wasser gepumpt. Den Kern umgibt , ist eine innere Membran sporen wo in den meisten sporenbildenden Bakterien, die Rezeptoren , die die kleinen Moleküle erkennen , die Keimung (Keimungsmittel) stimulieren 2 befindet. Das Hotel liegt etwas außerhalb der inneren Membran der Spore ist eine Schicht aus Zellwandpeptidoglycan. Eine spezialisierte Peptidoglycan Schicht (Cortex) umgibt die Peptidoglycan-Zellwand und viele der gleichen Komponenten wie Zellwandpeptidoglycan [alternierende N-Acetylglucosamin (NAG) und N-Acetylmuraminsäure (NAM)] zusammengesetzt ist. Jedoch etwa 50% der NAM - Reste in der Hirnrinde haben muramic-δ-lactam 5,6 umgewandelt </sup&gt;. Während der Sporenkeimung, diese muramic-δ-Lactam-Reste werden durch die Sporenrinde lytische Enzyme (SCLEs) erkannt, so dass die Rinde ermöglicht abgebaut werden (ein Prozess wesentlich Keimung abzuschließen), aber nicht die Zellwand. Die Rinde umgibt , ist eine äußere Membran und mehrere Schichten von Hüllproteinen 2. den Prozess der Keimung Steuerung ist von entscheidender Bedeutung für die sporenbildende Bakterien. Die Keimung wird eingeleitet , wenn Keimungsinduktor Rezeptoren interagieren mit ihren jeweiligen Keimungsmittel 2. In vielen sporenbildende Bakterien sind diese Keimungsmittel Aminosäuren, Zucker, Nukleotide, Ionen oder Kombinationen davon . 2 C. difficile Sporenkeimung durch Kombinationen bestimmter Gallensäuren, [zB Taurocholsäure (TA)] und Aminosäuren (beispielsweise Glycin) 7-10 eingeleitet. Obwohl es Unterschiede zwischen der C. difficile Keimung Weg und die untersuchten Wege in andere sporenbildendeBakterien, wie B. subtilis, allen gemeinsam ist die absolute Notwendigkeit der Spore Kortex abzubauen eine vegetative Zelle zu ermöglichen , von der gekeimten Spore 2,8 zu wachsen. Cortex Abbau kann durch die SCLEs CwlJ / SLEB erreicht werden oder SLEC (wie in B. subtilis gefunden) (wie in vielen Clostridia gefunden). Cortex Hydrolyse verringert die Beschränkung der Zellwand und der Spore. Dies ermöglicht eine volle Kern Rehydratisierung notwendiger Schritt in 2 viele der Proteine ​​, die für den Zellstoffwechsel zu reaktivieren. Wenn Sporen keimen, die ruhende Sporen ändert sich von einem phasen hellen Zustand in einen Phase-Dunkel-Zustand und dieser Prozess kann durch eine Veränderung der optischen Dichte (OD) bei 600 zu messen nm 11. Ein früherer Bericht legt nahe , dass ein großer Teil dieser Änderung in der OD auf die Freisetzung von DPA aus der Spore 12 zurückzuführen ist. Während unserer Studie haben wir versucht , den Zeitpunkt C zu vergleichen difficile Sporenkeimung und überwacht dieFreisetzung von DPA und Cortex - Fragmente 9. Für diese Studie war es wichtig, die Freisetzung von Cortex-Fragmente zu überwachen, wie sie begann von keimen Sporen freigesetzt werden. Die kolorimetrischen Test hier verwendet wurde , basiert auf einem Verfahren zur Bereitstellung von Zuckern mit reduzierenden Enden entwickelt Ghuysen et al erfasst wird . 13. Weil andere 14 - Protokolle zum Nachweis von reduzierenden Zuckern beschrieben haben oder diese Protokolle 15 modifiziert, kann die Literatur zu diesem Thema verwirrend sein. Hier stellen wir ausführlich die Schritt- für -Schritt - Verfahren zur kolorimetrischen Detektion von Zuckern befreit keimen C. Reduktions difficile - Sporen. Obwohl diese Studie verwendet C. difficile - Sporen sind die reduzierenden Zucker während der Keimung von Sporen von anderen sporenbildenden Bakterien freigesetzt können mit diesem Protokoll 9,16,17 erkannt werden.

<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.

Tabelle 1 zeigt typische Ergebnisse mit NAG - Standards. Daten verwendet werden , um eine Standardkurve zu erzeugen. Die Tabelle 2 zeigt typische Ergebnisse von einem Keimungsassay in Keimung Puffer , ergänzt mit 100 mM Glycin und 10 mM TA (Keimung fördernden Bedingungen) oder 100 mM Glycin allein (nicht-keimende Bedingungen). In Abwesenheit von TA, C. difficile - Sporen keimen nicht , und es gibt wenig Änderung in Gegenwart von cortex Fragme...

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.

Erkennen Cortex Fragmente während der bakteriellen Sporenauskeimung

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

Research

JoVE Journal

Immunology and Infection

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

Serie: 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.

Physische Isolierung von Endospores aus Umweltproben durch gezielte Lyse von vegetativen Zellen

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

Forschung

Umwelt

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).

Nachweis von Bakterien mit fluorogenen DNAzyme

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

Immunologie und Infektion

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.

Verbesserung der Bacillus subtilis-Sporenzählung und Markierungsanalyse in der Durchflusszytometrie

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

Biochemie

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.

Visualisierung von Germinosomes und die innere Membran in Bacillus Subtilis Sporen

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

Biotechnik

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.

Visuelle und Mikroskopische Auswertung von Streptomyces Entwicklungsstörungen Mutanten

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.

Sexuelle Entwicklung und Ascosporenausschleuderung in Fusarium graminearum</em

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

Biologie

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.

Spore-Adsorption als ein Nonrecombinant Display-System für Enzyme und Antigene

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.

Mit Cokultur zur Erkennung chemisch vermittelte Interspezies-Interaktionen

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.

Untersucht die nachteiligen Effekte der Low Druck Plasmasterilisation auf das Überleben von Bacillus Subtilis Sporen mittels Live Cell Mikroskopie

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.

Untersuchung von Teliospore Keimen mit Microrespiration Analyse und Mikrodissektion

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