The Project described is supported by Award Number 5R01AI116895 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

1. Generating Samples&#160;
<ol>
	<li>Heat activate C. difficile spores at 65 &#176;C for 30 min and store on ice. 
	Note: C. difficile spores can be produced and purified as described previously7,9,10.</li>
	<li>Prepare the germination solution at X + 1 ml&#160;where X is the number of samples to be taken during the assay. 
	Note: The germination solution is specific to the species of bacteria spore being studied. For assaying C. difficile spore germination, we used a solution of 10 mM Tris (pH 7.5), 150 mM NaCl, 100 mM glycine and 10 mM taurocholic acid in deionized water.</li>
	<li>Before beginning the assay, draw a 1.0 ml sample of the germination solution without spores to serve as a blank for cortex fragment detection.</li>
	<li>Add spores to an OD600 of ~3.0 to the germination solution and vortex quickly. 
	Note: For our germination studies in B. subtilis, we used the same OD600 spores as for C. difficile. 
	Note: This amount of spores worked well for monitoring cortex fragment release during C. difficile spore germination. The amount of spores used in this assay should be determined experimentally for each bacterium or strain.</li>
	<li>Remove a 1.2 ml sample immediately after adding the spores and centrifuge at room temperature for 1 min at 14,000 &#215; g. This is the zero time point.</li>
	<li>Transfer 1.0 ml of the supernatant from the centrifuged sample to a fresh tube for subsequent cortex fragment analysis. 
	Note: Should DPA need to be monitored, remove a separate 100 &#956;l sample for the detection of DPA in the solution using an assay based on terbium fluorescence9,18,19.</li>
	<li>Freeze the collected sample at -80 &#176;C.</li>
	<li>Repeat steps 1.4 and 1.5 at regular intervals until all time points completed. 
	Note: Because of time required for centrifugation and sampling, time points less than 2 min apart were not possible during our procedure, so our sampling intervals were once every 2 min for 14 min.</li>
	<li>As a positive control for reducing sugar detection and quantification, include the following amounts of N-acetylglucosamine (nmol): 0, 12.5, 25, 50, 100, 250, 500, and 5,000. Prepare these samples at the same time as cortex samples and run along with the cortex samples so that the standard curve is relevant to the data generated by germinating spores.</li>
	<li>After all time point samples are collected, lyophilize the samples. 
	Note: We lyophilized our samples in 2 ml screw-cap tubes.</li>
</ol>
2. Measuring Cortex Fragments
<ol>
	<li>Resuspend lyophilized samples in 120 &#181;l of 3 N HCl supplemented with 1% phenol and 0.5% &#946;-mercaptoethanol.</li>
	<li>Transfer the resuspended samples to 2-ml screw-cap tubes. 
	Note: For reproducibility, ensure all the lyophilized sample is resuspended and transferred.</li>
	<li>Incubate the samples in a recirculating water bath at 95 &#176;C for 4 hr.</li>
	<li>After incubation, place the samples on ice until they are cool.</li>
	<li>Neutralize the samples with 120 &#181;l of 3 M NaOH.</li>
	<li>Add 80 &#181;l of a saturated sodium bicarbonate solution and 80 &#181;l of a 5% acetic anhydride solution to each sample and vortex.</li>
	<li>Incubate the samples at room temperature for 10 min.</li>
	<li>Transfer the samples back to the 95 &#176;C water bath for EXACTLY 3 min. 
	Note: This step is time sensitive and longer times can affect downstream signal development.</li>
	<li>Remove samples from the water bath and cool on ice.</li>
	<li>Add 400 &#181;l of 6.54% K2B4O7&#183;4H2O to each tube and vortex.</li>
	<li>Incubate the samples in a recirculating water bath at 95 &#176;C for EXACTLY 7 min. 
	Note: This step is time sensitive and longer times can affect downstream signal development.</li>
	<li>Remove the samples and place on ice for 5 min.</li>
</ol>
3. Color Reagent
<ol>
	<li>While samples are on ice (Step 2.12), prepare the color reagent. Dissolve 0.320 g of p-dimethylaminobenzaldehyde (DMAB; Ehrlich&#39;s reagent) in 1.9 ml of glacial acetic acid. 
	Note: The reagent is time, temperature and light sensitive and should not be made in advance.</li>
	<li>After the DMAB completely dissolves, add 100 &#181;l of 10 N HCl and vortex.</li>
	<li>Add 5 ml of glacial acetic acid and vortex.</li>
</ol>
4. Colorimetric Reaction
<ol>
	<li>Transfer 100 &#181;l of each cooled cortex sample to a new 1.5 ml microcentrifuge tube.</li>
	<li>Add 700 &#181;l of the freshly-prepared color solution to each sample.</li>
	<li>Immediately transfer the samples to a 37 &#176;C water bath and incubate for EXACTLY 20 min.</li>
	<li>Transfer 200 &#181;l of each sample to a clear 96-well plate.</li>
	<li>Quantify the samples at 585 nm on a plate reader. Samples should start at a yellow color and a positive reaction will shift to purple. The more cortex present, the deeper the purple color.</li>
</ol>

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The authors have nothing to disclose.

Upon stimulation, spores undergoing the process of germination lose their resistance properties without the need for macromolecular synthesis. When spore germination is triggered, the spore core releases DPA in exchange for water2. Due to the high DPA content of the dormant spore, the spore core is under intense osmotic pressure and the specialized peptidoglycan, cortex, helps prevent the core from swelling by acting, presumably, as a barrier to expansion2.
The method described above uses Ehrlich&#39;s reagent (a yellow solution) to detect the presence of reducing sugars as a measurable shift to a purple tint. During cortex hydrolysis, NAG and NAM sugars are liberated (depending on the extent of hydrolyzing enzymes, the sugars can be liberated as disaccharides or polysaccharides). Hydrolysis of the glycosidic linkages by HCl (Step 2.1) generates monosaccharides with free reducing ends7. After neutralizing the acid with an equal amount of sodium hydroxide (Step 2.5), the alcohols surrounding the NAG and NAM monosaccharides are acylated by acetic anhydride (Step 2.6)13. Subsequently, unreacted acetic anhydride is converted to acetic acid by heating the sample (Step 2.8). As hypothesized by Morgan and Elson20, the terminal aldehyde spontaneously converts to the enol form. Under alkaline conditions (Step 2.10), this enol form can react with the adjacent acetyl to generate an oxazole derivative which reacts with Ehrlich&#39;s reagent (Step 4.3) to generate a purple color15,20.
This method described above yields consistent, reliable results for the detection of cortex fragments from germinating spores, both B. subtilis and C. difficile9,16,17. The assay requires special attention to ensure the reaction times, temperatures and procedures are performed with precision. Without proper attention to detail, it is easy for the reaction conditions to fail or not reproduce between experiments13. However, the inclusion of a NAG standard aids in the application of the assay between laboratory environments. The NAG positive control standard included in every experiment controls for any inter-experimental variability (e.g., slight variations in reaction times or effectiveness of the color reagent). Reactions using only buffer conditions and reactions with spores incapable of hydrolyzing cortex both yielded no change in color.
The most common problem with this method is inter-experimental variability or complete lack of signal. To control for the variability, the spore preparations must be free of any vegetative cell debris. This cell debris will contain peptidoglycan fragments (NAG and NAM) and will result in a high background signal for the germinating spores. To ensure a pure spore preparation (&#62;99.9% spores), refer to the literature for methods to prepare pure spore suspensions in the organism to be studied7,9,21-23.
If the assay is not yielding any detectable signal, we observed that the lyophilizing step can introduce variability. Due to the force with which the vacuum is released, we observed that the dried sample powder can be blown out of the sample tube. To reduce/prevent this from occurring, use a small syringe needle to poke holes in the tops of sample tubes. The small area of the introduced holes will reduce the likelihood of the lyophilized sample (now in powder form) from being disturbed. Finally, as noted in the methods above, the several incubation steps are very time sensitive. Ensure that all timed steps are monitored carefully. An important consideration for this assay is that the cortex fragments must be released from the germinating spore in order to be detected. If the cortex is hydrolyzed into large fragments, these fragments may be too large to be released through the spore coat. Though an important consideration when troubleshooting an experiment that does not yield a signal, we recommend to confirm that the conditions used to germinate most of the spores in the solution and the other troubleshooting options listed here prior to considering cortex fragment size as a potential problem in the assay.
Not all germination buffers/conditions are suitable for this assay. Because this assay detects the presence of reducing sugars liberated during the germination process13, any germination buffer that contains reducing sugars or any germinant that is, itself, a reducing sugar (e.g., glucose) will result in a strong signal in this assay. If this is the case, high pressure liquid chromatography (HPLC) can be used to detect the presence of cortex fragments24-26. However, because not all laboratories are equipped with such instrumentation, the method described herein can be used by most laboratories and offers simple and easily quantifiable results.
Germination by bacterial spores has largely been studied in Bacillus and a few Clostridium species2,8. The process is generally conserved, however differences exist. Importantly, not all organisms germinate using mechanisms of spore germination as observed in the model spore-formers9,27. Because germination is becoming studied in more diverse organisms, the assay described above can be applied to these organisms to detect cortex fragments during spore germination. By applying this method to these newly studied organisms, researchers can determine the role various germination proteins play in cortex hydrolysis.

Endospores are metabolically dormant forms of bacteria that allow bacteria to persist in unfavorable environments. In many spore-forming bacteria, spore formation is induced by nutrient deprivation but this process can be controlled by changes to pH, exposure to oxygen or other stresses1. While in their metabolically dormant spore form, bacteria resist UV radiation, desiccation, higher temperatures, and freezing2. Most of the knowledge on the sporulation process comes from studies in the model organism, Bacillus subtilis. The sporulation process begins with DNA replication and the formation of an axial filament3,4. An asymmetric septum then divides the cell into two unequally sized compartments. The larger compartment, the mother cell, engulfs the smaller compartment, the forespore. Both the mother cell and forespore coordinate gene expression to mature the spore and the mother cell eventually lyses, releasing the dormant spore into the surrounding environment1.
The spore structure and composition is conserved across many bacterial species. The spore core has a lower water content than the vegetative cell and is rich with dipicolinic acid (DPA)1,2. During spore formation, DPA is pumped into the spore core in exchange for water. Surrounding the core is an inner spore membrane where, in most spore forming bacteria, the receptors which recognize the small molecules that stimulate germination (germinants) are located2. Located just outside the spore&#39;s inner membrane is a layer of cell wall peptidoglycan. A specialized peptidoglycan layer (cortex) surrounds the cell wall peptidoglycan and is composed of many of the same components as cell wall peptidoglycan [alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)]. However, approximately 50% of the NAM residues in the cortex have been converted to muramic-&#948;-lactam5,6. During spore germination, these muramic-&#948;-lactam residues are recognized by the spore cortex lytic enzymes (SCLEs), thus allowing the cortex to be degraded (a process essential to complete germination) but not the cell wall. Surrounding the cortex is an outer membrane and several layers of coat proteins 2.
Controlling the process of germination is critically important for spore-forming bacteria. Germination is initiated when germinant receptors interact with their respective germinants2. In many spore-forming bacteria, these germinants are amino acids, sugars, nucleotides, ions or combinations thereof2. C. difficile spore germination is initiated by combinations of certain bile acids, [e.g., taurocholic acid (TA)] and amino acids (e.g., glycine)7-10. Though there are differences between the C. difficile germination pathway and the pathways studied in other spore-forming bacteria, such as B. subtilis, common to all is the absolute requirement to degrade the spore cortex to allow a vegetative cell to grow from the germinated spore2,8. Cortex degradation can be accomplished by the SCLEs CwlJ/SleB (as found in B. subtilis) or SleC (as found in many Clostridia). Cortex hydrolysis reduces the constraint on the cell wall and the spore. This allows for full core rehydration, a necessary step in reactivating many of the proteins necessary for cellular metabolism 2.
When spores germinate, the dormant spore changes from a phase-bright state to a phase-dark state and this process can be measured by a change in optical density (OD) at 600 nm11. A previous report suggests that much of this change in OD is due to the release of DPA from the spore12. During our recent study, we sought to compare the timing of C. difficile spore germination and monitored the release of DPA and cortex fragments9. For this study it was critical to monitor the release of cortex fragments as they began to be released by germinating spores.
The colorimetric assay used here was based upon a method for detecting sugars with reducing ends developed Ghuysen et al.13. Because others have described protocols for the detection of reducing sugars14 or have modified those protocols15, the literature on this subject can be confusing. Here, we detail a step-by-step method for the colorimetric detection of reducing sugars liberated from germinating C. difficile spores. Though this study uses C. difficile spores, the reducing sugars released during the germination of spores from other spore-forming bacteria are able to be detected with this protocol9,16,17.

<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.

Table 1 shows typical results using NAG standards. Data are used to generate a standard curve. Table 2 shows typical results from a germination assay in germination buffer supplemented with 100 mM glycine and 10 mM TA (germination-promoting conditions) or 100 mM glycine only (non-germinating conditions). In the absence of TA, C. difficile spores do not germinate and there is little change in the presence of cortex fragments in the solution. However, in the presence of both TA and glycine, C. difficile spores germinate and release cortex fragments into the solution. This is observed as an increase in the OD585 over time of the reacted samples. The total amount of cortex fragments (nmol) released during this assay was calculated using a standard curve similar to Figure 1B. In this assay, most of the cortex is hydrolyzed (or released) by 15 minutes post-germinant exposure, suggesting that most of the spores have germinated in this time frame (Figure 2; consistent with a previous publication7).
After developing the samples in the color reagent, most samples may not show the purple color to the naked eye. Figure 1 shows a representative sample generated from N-acetylglucosamine. Many of the samples do not show a visible purple color. However, the 250 nmol, 500 nmol and 5,000 nmol samples appear visually different from the negative control (0 nmol). When measured at OD585 nm, the signal generated best fits a linear regression (R2 = 0.999).
<img alt="Figure 1" src="/files/ftp_upload/54146/54146fig1.jpg" /> 
Figure 1: Detecting N-acetylglucosamine. (A) N-acetylglucosamine samples (0 nmol, 12.5 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 500 nmol, 5,000 nmol) were lyophilized and reacted according to the described procedure. (B) Samples were added to a 96-well plate and the amount of reacted material detected at 585 nm. The signal was used to generate a standard curve. <img alt="Figure 2" src="/files/ftp_upload/54146/54146fig2.jpg" /> 
Figure 2: Graphical Representation of Germination Results. The calculated nmol cortex in Table 2 was plotted vs. time. (&#9679;) 100 mM glycine + 10 mM TA; (&#9632;) 100 mM glycine. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>NAG Concentration (nmol)</td>
			<td>OD585</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>blank =</td>
			<td>0.046</td>
		</tr>
	</tbody>
</table>
Table 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 Spore Germination</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">Germination Solution (10 mM Tris (pH 7.5), 150 mM NaCl)</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">100 mM Glycine + 10 mM TA</td>
			<td colspan="2">100 mM Glycine&#160;</td>
		</tr>
		<tr>
			<td>Time (minutes)</td>
			<td>OD585</td>
			<td>Calculated (nmol)</td>
			<td>OD585</td>
			<td>Calculated (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>
Table 2: Sample Spore Germination Results.

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.

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 Views.  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.

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

Environment

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).

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

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.

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

Biochemistry

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.

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

Bioengineering

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.

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.

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

Biology

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.

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.

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.

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.

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

Detecting Cortex Fragments During Bacterial Spore Germination

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Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection