Name: production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization
Uploaded: 2018-08-11T15:00:00
Description: Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Research was supported by National Institute of Allergy and Infectious Diseases (NIH-NIAID) award R15AI079685.

1. Solution Preparation
NOTE: Prepare solutions described in steps 1.1&#8211;1.9 and 1.8&#8211;1.11 in order to produce E. coli spheroplasts and B. megaterium protoplasts, respectively.
<ol>
	<li>Prepare 1 M Tris-Cl, pH 7.8 by dissolving 10.34 g Tris HCl and 4.17 g of Tris OH in 50 mL of dH2O in a 125 mL flask. Sterilize by filtering through a 25 mm syringe filter with a 0.2 &#181;m membrane and store in a conical tube at room temperature.</li>
	<li>Prepare solut...

<ol>
	<li>Baltzer, S. A., Brown, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+promising+alternatives+to+conventional+antibiotics.">Antimicrobial peptides: promising alternatives to conventional antibiotics.</a> Journal of Molecular Microbiology Biotechnology. 20 (4), 228-235 (2011).</li><li>Hancock, R. E., Sahl, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+and+host-defense+peptides+as+new+anti-infective+therapeutic+strategies.">Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.</a> Nature Biotechnology. 24 (12), 1551-1557 (2006).</li><li>Jenssen, H., Hamill, P., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+antimicrobial+agents.">Peptide antimicrobial agents.</a> Clinical Microbiology Reviews. 19 (3), 491-511 (2006).</li><li>Toke, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+new+candidates+in+the+fight+against+bacterial+infections.">Antimicrobial peptides: new candidates in the fight against bacterial infections.</a> Biopolymers. 80 (6), 717-735 (2005).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+in+2014.">Antimicrobial peptides in 2014.</a> Pharmaceuticals. 8 (1), 123-150 (2015).</li><li>Epand, R. M., Vogel, H. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+analysis+of+hipposin,+a+histone-derived+antimicrobial+peptide+consisting+of+membrane+translocating+and+membrane+permeabilizing+fragments.">Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.</a> Biochim Biophys Acta. 1838 (9), 2228-2233 (2014).</li><li>Koo, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relations+of+parasin+I,+a+histone+H2A-derived+antimicrobial+peptide.">Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.</a> Peptides. 29 (7), 1102-1108 (2008).</li><li>Libardo, M. D., Cervantes, J. L., Salazar, J. C., Angeles-Boza, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+bioactivity+of+antimicrobial+peptides+by+addition+of+amino-terminal+copper+and+nickel+(ATCUN)+binding+motifs.">Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.</a> ChemMedChem. 9 (8), 1892-1901 (2014).</li><li>Park, C. B., Kim, H. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Action+of+the+Antimicrobial+Peptide+Buforin+II:+Buforin+II+Kills+Microorganisms+by+Penetrating+the+Cell+Membrane+and+Inhibiting+Cellular+Functions.">Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions.</a> Biochemical and Biophysical Research Communications. , 253-257 (1998).</li><li>Park, C. B., Yi, K. -. S., Matsuzaki, K., Kim, M. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+analysis+of+buforin+II,+a+histone+H2A-derived+antimicrobial+peptide:+The+proline+hinge+is+responsible+for+the+cell-penetrating+ability+of+buforin+II.">Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (15), 8245-8250 (2000).</li><li>Pavia, K. E., Spinella, S. A., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+histone-derived+antimicrobial+peptides+use+different+antimicrobial+mechanisms.">Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms.</a> Biochim Biophys Acta. 1818 (3), 869-876 (2012).</li><li>Wei, L., LaBouyer, M. A., Darling, L. E., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Spheroplasts+as+a+Model+for+Visualizing+Membrane+Translocation+of+Antimicrobial+Peptides.">Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.</a> Antimicrobial Agents and Chemotherapy. 60 (10), 6350-6352 (2016).</li><li>Chassy, B. M., Giuffrida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Lysis+of+Gram-Positive,+Asporogenous+Bacteria+with+Lysozyme.">Method for the Lysis of Gram-Positive, Asporogenous Bacteria with Lysozyme.</a> Appl. Environ. Microbiol. 39 (1), 153-158 (1980).</li><li>Fitz-James, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+and+Chemical+Studies+of+the+Browth+of+Protoplasts+of+Bacillus+megaterium.">Cytological and Chemical Studies of the Browth of Protoplasts of Bacillus megaterium.</a> J. Biophysic. and Biochem. Cytol. 4 (3), 257-266 (1958).</li><li>Martinac, B., Buechner, M., Delcour, A. H., Adler, J., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-sensitive+ion+channel+in+Escherichia+coli.">Pressure-sensitive ion channel in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 2297-2301 (1986).</li><li>Martinac, B., Rohde, P. R., Cranfield, C. G., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Methods Mol Biol. 966, 367-380 (2013).</li><li>Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Experimental Biophysics: Biological Methods for Physical Scientists. , (2016).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+Escherichia+coli+spheroplast+membranes.">Physical properties of Escherichia coli spheroplast membranes.</a> Biophysical Journal. 107 (9), 2082-2090 (2014).</li><li>Branco, P., Viana, T., Albergaria, H., Arneborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+(AMPs)+produced+by+Saccharomyces+cerevisiae+induce+alterations+in+the+intracellular+pH,+membrane+permeability+and+culturability+of+Hanseniaspora+guilliermondii+cells.">Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.</a> Int J Food Microbiol. 205, 112-118 (2015).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Translocation+Mechanism+of+the+Antimicrobial+Peptide+Buforin+2.">Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2.</a> Biochemistry. 43 (49), 15610-15616 (2004).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> J Vis Exp. (59), e3571 (2012).</li><li>van der Kraan, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrampin:+a+novel+antimicrobial+peptide+in+the+N1-domain+of+bovine+lactoferrin.">Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.</a> Peptides. 25 (2), 177-183 (2004).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Biophys J. 111 (1), 132-139 (2016).</li><li>Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proline+position+on+the+antimicrobial+mechanism+of+buforin+II.">Effect of proline position on the antimicrobial mechanism of buforin II.</a> Peptides. 32 (4), 677-682 (2011).</li><li>Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+Novel+Antimicrobial+Peptide+Buforin+2+with+Lipid+Bilayers:+Proline+as+a+Translocation+Promoting+Factor.">Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor.</a> Biochem. 39 (29), 8648-8654 (2000).</li><li>Decad, G. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Gram-Negative+Bacteria+XII.+Molecular-Sieving+Function+of+Cell+Wall.">Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall.</a> J. Bacteriol. 128 (1), 325-336 (1976).</li><li>Choi, H., Yang, Z., Weisshaar, J. 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The protocols presented here make it feasible for researchers to more rapidly obtain larger sample sizes of bacterial images because the enlarged, spherical bacteria are much easier to locate, orient, and image. This enhanced ability to collect data is valuable in several respects. First, it enables a more systematic quantitative analysis of peptide localization patterns. While qualitative trends can be demonstrated from smaller sets of images, only a large sample set of high-quality images reveal more nuanced trends in ...

Antimicrobial peptides (AMPs) have gained attention due to their potential use as alternatives to conventional antibiotics1,2,3,4,5. AMPs kill bacteria by either translocating across the cell membrane and interacting with intracellular components such as nucleic acids or by permeabilizing the membrane causing leakage of cell contents6. In addition to their use as antibiotics, translocating AMPs may be adapted for drug delivery applications because they can non-disruptively cross the impermeable cell membrane7,8. We, therefore, seek to understand fundamental AMP mechanisms of action to lay the foundation for their use in drug design.
Confocal microscopy offers a way to assess localization patterns of fluorescently labeled AMPs in bacterial cells providing insights into their mechanism of action9,10,11,12,13,14. By labeling the membrane of the bacteria, one can determine if a fluorescently labeled peptide localizes to the membrane or the intracellular space of a bacterial cell. However, this technique is limited by the small size and rod shape of bacteria, which can make imaging challenging due to the resolution limits of conventional light microscopes and the variable orientation of the bacteria on the slide15.
The goal of the presented method is to enable enhanced visualization of the fluorescently labeled peptide localization patterns using confocal microscopy. Visualization is enhanced by turning the small, thin, rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) bacteria into enlarged, spherical forms referred to as spheroplasts (for gram-negative strains) and protoplasts (for gram-positive strains)16,17,18,19,20,21. Spheroplasts and protoplasts are easier to image because of both their increased size and their symmetric shape, which makes the orientation of a bacterium on a slide irrelevant for its imaging. In addition, we present a systematic approach to quantitatively analyze confocal microscopy data in order to characterize AMPs as either membrane localizing or translocating. Applying these methods makes it easier to distinguish fluorescently labeled peptide localization patterns. The protocols presented here can be used to assess the localization of a variety of membrane-active agents other than AMPs, including cell-penetrating peptides.
One distinct advantage of this technique is that it provides insights into the mechanism of action of AMPs on a single cell level, which may reveal cell-to-cell heterogeneity15, as opposed to other fluorescence assays commonly used to identify the mechanisms of action of AMPs, which only provide bulk estimates9,22,23,24,25. The use of spheroplasts and protoplasts in order to assess AMP cell entry is particular useful26&#160;because they are more physiologically relevant15&#160;than other models used for assessing cell entry, such as lipid vesicles24.

<table>
	<tbody>
		<tr>
			<td>Trizma hydrocloride (Tris HCl)</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris OH)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma</td>
			<td>106361</td>
			<td>Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement</td>
		</tr>
		<tr>
			<td>Cephalexin hydrate</td>
			<td>Sigma</td>
			<td>C4895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Trypticase soy broth</td>
			<td>Fisher Scientific</td>
			<td>B11768</td>
			<td></td>
		</tr>
		<tr>
			<td>BF2 P11A FITC</td>
			<td>NeoScientific</td>
			<td>Custom ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>di-8-ANEPPS</td>
			<td>Biotium</td>
			<td>61012</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869</td>
			<td>Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc 25 mm Syringe Filter w/ 0.2 &#956;m HT Tuffryn Membrane</td>
			<td>Pall Corporation</td>
			<td>4192</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5 II</td>
			<td>For image acquisition</td>
		</tr>
		<tr>
			<td>Leica Application Suite, Advanced Fluorescence</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>For image processing</td>
		</tr>
	</tbody>
</table>

production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

By enlarging bacteria and making them spherical, we can easily distinguish whether peptides localize to the bacterial membrane or readily translocate across the bacterial membrane. The resolution limits of conventional light microscopes make it challenging to distinguish whether peptide signals arise from the membrane or intracellular space in normal bacteria because signals localized to the membrane will appear to overlap with the intracellular space (Figure 3A</stro...

Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

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