Research was supported by the National Science Foundation under grant number 2009522. NMR analysis of masarimycin was supported by the National Science Foundation major research instrumentation program award under grant number 1919644. Any opinions, findings, and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

1. General methods
NOTE: All compounds were purchased from standard suppliers and used without further purification.
<ol>
	<li>Carry out thin-layer chromatography (TLC) on an aluminum plate precoated with silica gel XG F254. Detect spots under a UV lamp, by immersion in p-anisaldehyde stain, or by exposing to I2 vapor.</li>
	<li>Record all nuclear magnetic resonance (NMR) spectra on a 400 MHz spectrometer. 
	NOTE: 1H- NMR and 13C-NMR spectra were referenced to residual solvent peaks. Coupling constants are given in [Hz] and chemical shifts in [ppm].</li>
	<li>Record atmospheric pressure chemical ionization (APCI) mass spectrometry spectra of masarimycin on a compact mass spectrometer equipped with an atmospheric solids analysis probe.</li>
</ol>
2. General procedure for preparation of masarimycin
NOTE: Perform the below steps in a fume hood.
<ol>
	<li>Prepare a 0.1 M solution in methanol of each reactant: cyclohexylamine, cyclohexyl carboxaldehyde, o-iodobenzoic acid, and cyclohexyl isocyanide35. 
	CAUTION: Cyclohexylamine, cyclohexyl isocyanide, and cyclohexyl carboxaldehyde are flammable. They can cause skin corrosion and induce oral, dermal, respiratory, or reproductive toxicity. Keep compounds away from open flames, hot surfaces, and ignition sources. Wear appropriate skin and eye protection, work in a well-ventilated area and avoid inhalation of vapors or mist. For storage, keep bottles tightly closed and store them in a cool, dry place. Store cyclohexyl carboxaldehyde in a desiccator under an N2 atmosphere.</li>
	<li>Mix 5 mL of cyclohexylamine (0.1 M solution in methanol) and 5 mL of cyclohexyl carboxaldehyde (0.1 M in methanol) in a capped round bottom flask and stir the solution using a magnetic stir bar on a stir/hot plate for 30 min at 40 &#176;C in a sand bath. Monitor temperature using a thermometer placed approximately 1 cm below the sand surface.</li>
	<li>After 30 min, add 5 mL of cyclohexyl isocyanide (0.1 M solution in methanol) to the solution from step 2.2 and stir for an additional 20 min at 50 &#176;C. Lastly, add 5 mL of o-iodobenzoic acid (0.1 M solution in methanol) to the reaction mixture and continue stirring at 55 &#176;C for 3-5 h.</li>
	<li>Monitor the progress of the reaction periodically by TLC approximately every hour after the above reaction mixture had been stirred for 3 h.</li>
	<li>Cut a 3 cm x 6 cm strip of aluminum-backed TLC plate. Using a #2 pencil, draw a line approximately 1 cm from the bottom. Using a glass microcapillary, spot approximately 5 &#181;L of the reaction mixture onto the TLC plate and allow it to dry.</li>
	<li>To a 150 mL beaker, add enough mobile phase (90:10 hexane: isopropanol) to cover the bottom of the beaker. Using a pair of tweezers, carefully place the above TLC plate into the beaker ensuring that the TLC plate enters the mobile phase evenly. Cover the top of the beaker with a piece of tinfoil. 
	NOTE: Ensure that the mobile phase does not cover the line and spotted sample.</li>
	<li>Allow the mobile phase to travel up the TLC plate until it is approximately 1 cm below the top of the plate. Remove the TLC plate and using a pencil, draw a line indicating the distance traveled by the mobile phase. Allow the TLC plate to dry in a fume hood.</li>
	<li>Once dried, place the TLC plate in a beaker containing a small amount of solid I2 and cover the beaker with a piece of tin foil. Monitor the TLC for the development of yellow/brown spots. Once developed, remove the TLC plate and mark the location of the spots using a pencil (Supplementary Figure 1). 
	NOTE: If I2 spots are not marked, the stain will dissipate over time. Spots can also be visualized on the TLC plate by UV-light, p-anisaldehyde staining, or potassium permanganate staining (see Supplementary Information).</li>
	<li>Calculate Rf values for all visualized spots using the following formula: 
	Rf = <img alt="Equation 1" src="/files/ftp_upload/63191/63191eq01.jpg" /></li>
	<li>Consider the reaction complete when only one spot with Rf = 0.3 is visible on the TLC plate. Remove the solvent in a rotatory evaporator under reduced pressure and dry the crude product (obtained as a yellowish-brown oil) under a high vacuum until all methanol is evaporated.</li>
	<li>Dissolve the dried crude product in 30 mL of ethyl acetate and transfer it to a separatory funnel. Extract ethyl acetate sequentially with 1 M HCl (2 x 30 mL), H2O (30 mL), saturated NaHCO3 solution (2 x 30 mL), H2O (30 mL) and saturated NaCl solution (2 x 30 mL). Discard the aqueous layers. 
	NOTE: The ethyl acetate layer is the top layer in each of the extractions. For each extraction, vigorously shake the separatory funnel containing the ethyl acetate and aqueous solution (HCl, H2O, NaHCO3, or NaCl) and allow the layers to fully separate.</li>
	<li>Remove the ethyl acetate layer from the separatory funnel and collect it in an Erlenmeyer flask. Add a spatula full of Na2SO4 (anhydrous) to remove residual water from ethyl acetate. 
	NOTE: The ethyl acetate solution is considered dry when Na2SO4 in the flask runs freely and does not clump. If Na2SO4 is clumping, an additional spatula of Na2SO4 can be added.</li>
	<li>Filter the dried ethyl acetate solution through #1 filter paper to remove Na2SO4. Wash the filter paper with a small amount of ethyl acetate. Place the filtered ethyl acetate solution in a round bottom flask and remove the solvent on a rotatory evaporator under reduced pressure to obtain masarimycin as oil once all the ethyl acetate is removed.</li>
	<li>Dissolve the masarimycin oil obtained above in a minimal amount (1-2 mL) of 9:1 hexane: isopropanol and stir on a magnetic stir plate until all the compound is dissolved.</li>
	<li>Purify the dissolved masarimycin by flash chromatography using a 12 g normal phase silica flash column.
	<ol>
		<li>Equilibrate the flash column with 10 column volumes of mobile phase (99:1 hexane: isopropanol) with the instrument set at a flow rate of 15 mL/min. 
		NOTE: After equilibration is completed, stop the flow and disconnect the top of the column from the system.</li>
		<li>Draw up dissolved masarimycin using a 5 mL syringe. Connect the syringe directly to the top of the equilibrated flash column and inject the solution into the column. Reconnect the loaded column to the flash chromatography system and initiate the gradient elution.</li>
		<li>Elute masarimycin from the column using gradient elution to a final mobile phase concentration of 10:90 hexane: isopropanol over 12 column volumes. Monitor the elution of masarimycin via absorption at 230 and 254 nm.</li>
		<li>Collect the compounds eluted from the column by a fraction collector that collects 20 mL of solvent per fraction. 
		NOTE: If a flash chromatography system is not available, purification of masarimycin can be performed via a gravity silica column with a 3:1 (hexane: ethyl acetate) mobile phase. Fractions containing masarimycin can be identified by TLC using the same mobile phase. Visualization of TLC spots was done with either UV light, I2 vapor, or potassium permanganate staining.</li>
		<li>Identify fractions containing masarimycin by TLC (steps 2.5-2.9) or mass spectrometry on a compact mass spectrometer equipped with an atmospheric solids analysis probe. Dry the final product under vacuum (~0.3 mbar). 
		NOTE: Masarimycin is routinely obtained as a colorless oil or solid with a yield of 55%-70% with respect to mmol of cyclohexyl carboxaldehyde added to the reaction. Calculate the final yield of masarimycin by obtaining the mass of the purified masarimycin and calculating the theoretical yield of the reaction using the following formula: 
		% yield = <img alt="Equation 2" src="/files/ftp_upload/63191/63191eq02.jpg" /> x 100%</li>
	</ol>
	</li>
	<li>Confirm the structure of masarimycin by NMR.
	<ol>
		<li>Dissolve ~10 mg of masarimycin sample in 0.5 mL of CDCl3. Using a Pasteur pipet, transfer the solution to a 5 mm NMR tube and cap the tube. Place the NMR tube in the spectrometer.</li>
		<li>Acquire 1H and 13C NMR spectra using manufacturer preset experiments. Chemical shift assignments and representative spectra are provided in Supplementary Figures 3-4.</li>
	</ol>
	</li>
	<li>Store masarimycin dry or dissolved in DMSO (25 mM final concentration) at -20 &#176;C until use.</li>
</ol>
3. Microwave procedure for preparation of masarimycin
<ol>
	<li>Prepare 0.6 M solutions of cyclohexylamine, cyclohexyl carboxaldehyde, cyclohexyl isocyanide, and o-iodobenzoic acid in acetonitrile.</li>
	<li>Add a stir bar and 10 mL of acetonitrile to a glass microwave reaction vial.</li>
	<li>Add 2 mL of cyclohexylamine (0.6 M in acetonitrile), 2 mL of cyclohexyl carboxaldehyde (0.6 M in acetonitrile), and 7 mL of acetonitrile to the vial.</li>
	<li>Place the microwave reaction vial in the microwave carousel. Stir the mixture, heat it for 30 min at 50&#176;C at a power setting of 400 W, and allow it to cool to room temperature.</li>
	<li>Add 2 mL of o-iodobenzoic acid (0.6 M in methanol) and 2 mL of cyclohexyl isocyanide (0.6 M in acetonitrile) to the vial. Stir the mixture, heat it to 100 &#176;C in the microwave for 40 min at a power setting of 400 W and allow it to cool to room temperature.</li>
	<li>Monitor the progress of the reaction by TLC (90:10 hexane: isopropanol) using I2 vapor&#160;after the completion of step 3.5. 
	NOTE: If TLC shows that the reaction is incomplete (i.e., multiple spots on TLC), place the reaction vial back in the microwave and set the microwave conditions described in step 3.5.</li>
	<li>Once the reaction is complete, pour the solution into a 100 mL round-bottom flask and evaporate it to dryness using a rotary evaporator.</li>
	<li>Follow steps 2.6-2.16 above to complete the aqueous workup, purification, and characterization of masarimycin.</li>
</ol>
4. Synergy and antagonism assay
<ol>
	<li>Grow Streptococcus pneumoniae R6 on Mueller-Hinton (MH) agar plates containing 5% (v/v) sheep blood at 37 &#176;C under anaerobic conditions. In all experiments, use second passage cells grown in 5 mL of MH broth at 37 &#176;C under anaerobic conditions until OD600 is ~0.4.</li>
	<li>Subject the inhibitors masarimycin and optochin to serial 1:2 dilutions in respective solvents, with the resulting concentrations flanking the minimum inhibitory concentration (MIC) values of each inhibitor.
	<ol>
		<li>Make the initial dilution of masarimycin in dimethyl sulfoxide (DMSO) until a concentration of 100 &#181;M is reached. From this point, make masarimycin dilutions in MH broth. Prepare optochin stock solution (3.5 mM) by dissolving commercially available optochin (see Table of Materials) in sterile MH broth. 
		NOTE: Masarimycin stock solutions were made at 25 mM in DMSO.</li>
	</ol>
	</li>
	<li>To a sterile 96-well microtitre plate, add 2 &#181;L aliquots of each optochin dilution to each row of the plate. To the same plate, add 2 &#181;L aliquots of each masarimycin dilution to each column to create an array of optochin and masarimycin concentrations on the plate (Figure 2).</li>
	<li>Add sterile MH broth (93 &#181;L) to each well containing the above inhibitors. Inoculate the microtitre plates with 5 &#181;L of culture (OD600 ~0.4) from step 4.1. 
	NOTE: Inoculation of the 96-well plate is typically done under anaerobic conditions in an anaerobic workstation. The final volume in the well is 100 &#181;L.</li>
	<li>Grow cultures for 18 h at 37 &#176;C under anaerobic conditions, followed by the addition of 30 &#181;L of 0.01% (m/v) solution of resazurin sodium salt. Incubate the plate at room temperature for 15 min to allow the formation and stabilization of color. 
	NOTE: Resazurin solution is prepared by dissolving the compound in distilled water and can be stored at 4 &#176;C for up to two weeks.</li>
	<li>Directly read the concentration values from the plate and assign the lowest inhibitor concentration for which no bacterial growth is observed (blue color) as [X] (see step 4.7.1), i.e., the lowest inhibitory concentration of the drug in the presence of the co-drug. 
	NOTE: Positive bacterial growth is identified in the wells by the resazurin dye turning pink. MIC values for each drug alone (i.e., in the absence of co-drug) are determined in a similar manner using the resazurin MIC assay35 with each drug separately (Supplemental Figure 5). MICs in S. pneumoniae are 7.8 &#181;M and 15.85 &#181;M for masarimycin and optochin, respectively.</li>
	<li>Determine the fractional inhibitory concentration (FIC) and FIC index (FICI) using the following equations.
	<ol>
		<li>FIC= [X]/MICx, where [X] (from step 4.6) is the lowest inhibitory concentration of the drug in the presence of the co-drug, and MICx is the lowest inhibitory concentration of the drug in the absence of the co-drug.</li>
		<li>FICI= FICmasarimycin + FICantibiotic 
		NOTE: FICI &#60; 0.5 = synergistic, 0.5 &#60; FICI &#60; 1 = additive, 1 &#60; FICI &#60; 4 = indifferent, FICI &#62; 4 = antagonistic.</li>
	</ol>
	</li>
</ol>
5. Morphological study
<ol>
	<li>Grow Bacillus subtilis 11774 on Luria-Bertani (LB) agar plates (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) containing 1.5% Bacto agar at 37 &#176;C. In all experiments, use second passage cells grown in 5 mL of LB broth at 37 &#176;C until OD600 = 1. Grow S.pneumoniae in the same manner as in step 4.1.</li>
	<li>After obtaining a cell culture density with OD600nm = 1 for B. subtilis, or OD600nm = 0.4 for S. pneumoniae, add masarimycin using a pipette to the culture tube labeled &#34;treated&#34; to a final concentration of 3.8 &#181;M (0.75x MIC for B.subtilis), or 5.85 &#181;M (0.75x MIC for S.pneumoniae). To the second culture tube labeled &#34;control&#34;, add an equivalent volume of DMSO.</li>
	<li>For B.subtilis, place the samples in an incubator at 37 &#176;C for 90 min with shaking at 150 rpm. For S. pneumoniae, incubate the cells without shaking under anaerobic conditions.</li>
	<li>After 90 min, chemically fix the cultures in a 1:10 mixture (v/v) of culture media and fixing buffer (20 mM HEPES, 1% formaldehyde (pH 6.8)) at 4 &#176;C overnight. After fixing is complete, apply 10-20 &#181;L of samples to glass microscope slides using a pipette and allow them to air dry. Fix the air-dried samples by heating the glass slides using a Bunsen burner.</li>
	<li>After heat-fixing, stain samples with the addition of 100 &#181;L of 0.1% (m/v) methylene blue (solution in 20% (v/v) ethanol). Incubate the stained slides for 10 min and wash away the excess dye with dH2O. Then, gently heat the stained slides to 60 &#176;C in an oven for 15-20 min to bring cells to a common focal plane.</li>
	<li>Seal the stained samples by placing a microscope coverslip over the stained cells. Then, seal the edges using microscope slide cement. Place the sealed microscope slide on the microscope stage and bring the image into focus at 100x magnification using bright-field microscopy.</li>
	<li>Place a drop of immersion oil on the microscope slide and bring the field of view to focus using 1000x magnification. Acquire micrographs using a camera attached to the microscope and its associated software. Acquire images using the auto white balance and aperture settings on the software. 
	NOTE: Alternatively, images can be processed using the open-source ImageJ software.</li>
</ol>

Reid, C. W. has intellectual property involving specific applications of masarimycin.

Masarimycin is a single micromolar bacteriostatic inhibitor of B. subtilis35 and S. pneumoniae37 growth. In B. subtilis, masarimycin has been shown to inhibit the GlcNAcase LytG35, while the precise molecular target in the cell wall of S. pneumoniae has not been identified37. Synthesis of masarimycin using either the classical organic synthesis or microwave procedure provides the inhibitor in good yi...

Peptidoglycan (PG) is a mesh-like polymer that delineates cell shape and structure in both Gram-positive and Gram-negative bacteria1,2. This heteropolymer is a matrix of amino sugars cross-linked by short peptides3,4,5,6 with a backbone composed of &#946;-(1,4)-linked alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues (Figure 1)1. Attached to the C-3 lactyl moiety of MurNAc is the stem peptide. The metabolism of PG involves a tightly coordinated system of biosynthetic and degradative enzymes to incorporate new material into the cell wall7,8. Degradation of PG is carried out by enzymes collectively referred to as autolysins9 and further classified based on the specificity of the bond cleaved. Autolysins participate in many cellular processes including cell growth, cell division, motility, PG maturation, chemotaxis, protein secretion, genetic competence, differentiation, and pathogenicity10,11. Unraveling the specific biological functions of individual autolysins can be daunting, due in part to functional redundancy. However, recent biophysical8,12,13 and computational studies12 have provided new insight into their roles in PG metabolism. In addition, recent reports have provided further insight into the synthesis14 and membrane-mediated15,16,17 steps in PG metabolism. A thorough understanding of the relationship between degradative and synthetic pathways of PG metabolism could give rise to previously untapped antibiotic targets.
While there have been significant advances in methodology to study glycobiology in eukaryotes, bacterial glycobiology and, in particular, PG metabolism has not advanced at a similar rate. Current chemical approaches to study PG metabolism include fluorescently labeled antibiotics18, fluorescent probes19,20, and metabolic labeling21,22,23,24. These new approaches are providing new ways to interrogate bacterial cell wall metabolism. While some of these strategies are capable of labeling PG in vivo, they can be species-specific19, or only work in strains lacking a particular autolysin25. Many PG labeling strategies are intended for use with isolated cell walls26 or with in vitro reconstituted PG biosynthesis pathways20,27,28. The use of fluorescently labeled antibiotics is currently limited to biosynthetic steps and transpeptidation18.
The current knowledge of bacterial autolysins and their role in cell wall metabolism comes from genetic and in vitro biochemical analysis11,29,30,31,32. While these approaches have provided a wealth of information on this important class of enzymes, deciphering their biological role can be challenging. For instance, due to functional redundancy33, deletion of an autolysin in most cases does not result in halting bacterial growth. This is despite their implied role in cell growth and division7,12. Another complication is that genetic deletion of bacterial autolysins can give rise to meta-phenotypes34. Meta-phenotypes arise from the complex interplay between the pathway affected by the genetic deletion and other interconnected pathways. For instance, a meta-phenotype can arise via a direct effect such as the lack of an enzyme, or an indirect effect such as a disruption of regulators.
Currently, there are only a few inhibitors of glycosidase autolysins such as N-acetylglucosaminidases (GlcNAcase) and N-acetylmuramidases, which can be used as chemical probes to study the degradation of PG. To address this, the diamide masarimycin (previously termed as fgkc) has been identified and characterized35 as a bacteriostatic inhibitor of Bacillus subtilis growth that targets the GlcNAcase LytG32 (Figure 1). LytG is an exo-acting GlcNAcase36, a member of cluster 2 within glycosyl hydrolase family 73 (GH73). It is the major active GlcNAcase during vegetative growth32. To our knowledge, masarimycin is the first inhibitor of a PG-acting GlcNAcase that inhibits cellular growth. Additional studies of masarimycin with Streptococcus pneumoniae found that masarimycin likely inhibits cell wall metabolism in this organism37. Here, the preparation of masarimycin is reported for use as a chemical biology probe to study physiology in the Gram-positive organisms B. subtilis, and S. pneumoniae. Examples of morphological analysis of sub-minimum inhibitory concentration treatment with masarimycin, as well as a synergy/antagonism assay are presented. Synergy and antagonism assays using antibiotics with well-defined modes of action can be a useful way to explore connections between cellular processes38,39,40.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Iodobenzoic acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>I7675-25G</td>
			<td>corrosive, irritant, light yellow to orange-brown powder</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>109827-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>SIGMA-ALDRICH</td>
			<td>34851-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>Aluminum backed silica plates</td>
			<td>Sorbtech</td>
			<td>4434126</td>
			<td>silica gel XG F254 on aluminum backed plates</td>
		</tr>
		<tr>
			<td>chloroform-d</td>
			<td>SIGMA-ALDRICH</td>
			<td>151823-50G</td>
			<td>solvent for NMR</td>
		</tr>
		<tr>
			<td>Compact Mass Spectrometer</td>
			<td>Advion-Interchim</td>
			<td>Advion CMS</td>
			<td>compact mass spectrometer equiped with APCI source and atmospheric solids analysis probe</td>
		</tr>
		<tr>
			<td>Corning Costar 96 well flat bottom plates-sterile</td>
			<td>fisher chemical</td>
			<td>07-200-90</td>
			<td>for synergy/antagonism assays</td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>fisher chemical</td>
			<td>12-547</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Cyclohexanecarboxaldehyde</td>
			<td>CHEM-IMPEX INT&#39;L INC.</td>
			<td>24451</td>
			<td>flammable, irritant, colorless to pink liquid</td>
		</tr>
		<tr>
			<td>Cyclohexyl isocyanide</td>
			<td>SIGMA-ALDRICH</td>
			<td>133302-5G</td>
			<td>irritant, colorless liquid, extremly unpleasant odor</td>
		</tr>
		<tr>
			<td>Cyclohexylamine</td>
			<td>SIGMA-ALDRICH</td>
			<td>240648-100ML</td>
			<td>corrosive, flammable, irritant, colorless liquid unless contaminated</td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>SIGMA-ALDRICH</td>
			<td>537446-4L</td>
			<td>flammable, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>flash silica cartridge (12g)</td>
			<td>Advion-Interchim</td>
			<td>PF-50SIHP-F0012</td>
			<td>pack of flash silica columns (12g) for purification of masarimycin</td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>F8775-25ML</td>
			<td>fixing agent for microscopy</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA-ALDRICH</td>
			<td>H8651-25G</td>
			<td>buffer for microscopy fixing solution</td>
		</tr>
		<tr>
			<td>Hexane, mixture of isomers</td>
			<td>SIGMA-ALDRICH</td>
			<td>178918-4L</td>
			<td>environmentally damaging, flammable, irritant, health hazard, colorless liquid</td>
		</tr>
		<tr>
			<td>High performance compact mass spectrometer</td>
			<td>Advion</td>
			<td>expression</td>
			<td>Atmospheric Solids Analysis Probe (ASAP), low resolution</td>
		</tr>
		<tr>
			<td>High Vac</td>
			<td>eppendorf</td>
			<td>Vacufuge plus</td>
			<td>vacuum aided by centrifugal force and temperature</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>258148-2.5L</td>
			<td>corrosive, irritant, colorless liquid</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>320331-2.5L</td>
			<td>strong acid</td>
		</tr>
		<tr>
			<td>immersion oil</td>
			<td>fisher chemical</td>
			<td>12-365-19</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Iodine, resublimed crystals</td>
			<td>Alfa Aesar</td>
			<td>41955</td>
			<td>environmentally damaging, irritant, health hazard, dark grey/purple crystals</td>
		</tr>
		<tr>
			<td>Mestre Mnova</td>
			<td>MestreLab Research</td>
			<td></td>
			<td>software for processing NMR spectra</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>439193-4L</td>
			<td>flammable, toxic, health hazard, colorless liquid</td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>SIGMA-ALDRICH</td>
			<td>M9140-25G</td>
			<td>microscopy stain for staining cell walls</td>
		</tr>
		<tr>
			<td>meuller-hinton agar plates + 5% sheep blood</td>
			<td>fisher chemical</td>
			<td>B21176X</td>
			<td>growth media for Streptococcus pneumoniae</td>
		</tr>
		<tr>
			<td>meuller-hinton broth</td>
			<td>fisher chemical</td>
			<td>DF0757-17-6</td>
			<td>growth media for Streptococcus pneumoniae</td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>fisher chemical</td>
			<td>22-310397</td>
			<td>for microscopy</td>
		</tr>
		<tr>
			<td>Microwave Synthesis Labstation</td>
			<td>MILESTONE</td>
			<td>START SYNTH</td>
			<td>device that requires the ventilation of a fume hood, equipped with synthesis carousel</td>
		</tr>
		<tr>
			<td>NMR tubes</td>
			<td>SIGMA-ALDRICH</td>
			<td>Z562769-5EA</td>
			<td>5mm NMR tubes 600 MHz</td>
		</tr>
		<tr>
			<td>Nuclear Magnetic Resonance (NMR)</td>
			<td>Bruker</td>
			<td>Ascend 400</td>
			<td>large superconducting magnet (400MHz)</td>
		</tr>
		<tr>
			<td>optochin</td>
			<td>fisher chemical</td>
			<td>AAB21627MC</td>
			<td>ethylhydrocupreine hydrochloride</td>
		</tr>
		<tr>
			<td>petrie plates</td>
			<td>Celltreat</td>
			<td>229695</td>
			<td>for preparing agar plates for bacterial growth</td>
		</tr>
		<tr>
			<td>Primo Star Bright field/Phase contrast Microscope with ERc5s camera</td>
			<td>Zeiss</td>
			<td></td>
			<td>for morphology studies</td>
		</tr>
		<tr>
			<td>puriFlash</td>
			<td>interchim</td>
			<td>XS520plus</td>
			<td>flash chromatography purification system</td>
		</tr>
		<tr>
			<td>resazurin</td>
			<td>SIGMA-ALDRICH</td>
			<td>R7017-1G</td>
			<td>for synergy/antagonism assays</td>
		</tr>
		<tr>
			<td>Rotary Evaporator</td>
			<td>Heidolph</td>
			<td>Hei-VAP Value &#34;The Collegiate&#34;</td>
			<td>solvent evaporator</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>SIGMA-ALDRICH</td>
			<td>S6014-500G</td>
			<td>irritant, white powder</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>fisher chemical</td>
			<td>S271-1</td>
			<td>crystalline, colorless</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>SIGMA-ALDRICH</td>
			<td>S5886-500G</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Sodium sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>7985592-500G</td>
			<td>anhydrous, granular, white</td>
		</tr>
		<tr>
			<td>tryptone</td>
			<td>fisher chemical</td>
			<td>BP1421-500</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Whitney DG250 Workstation</td>
			<td>Microbiology International</td>
			<td>DG250</td>
			<td>anaerobic workstation. Anaerobic gas mixture used: 5% hydrogen, 10% carbon dioxide, balance nitrogen</td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>fisher chemical</td>
			<td>BP1422-500</td>
			<td>for growth of B.subtilis and preparation of LB media</td>
		</tr>
		<tr>
			<td>Zen Lite (blue) software</td>
			<td>Zeiss</td>
			<td></td>
			<td>for acquiring micrographs</td>
		</tr>
	</tbody>
</table>

synthesis of masarimycin, a small molecule inhibitor of gram-positive bacterial growth

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

Masarimycin is a small molecule bacteriostatic inhibitor of B. subtilis and S. pneumoniae and has been shown to inhibit the exo-acting GlcNAcase LytG in B. subtilis35,37 and target the cell wall in S. pneumoniae37. Masarimycin can be efficiently prepared either by the classical or microwave-assisted organic synthesis with yields in the 55%-70% range. Microwave-assisted synthesis has the advantage of a si...

Watch this Scientific Journal Video about Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth at JoVE.com

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

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Research

JoVE Journal

Biochemistry

2.3K Views.  Bryant University. A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Video: Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...