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><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&#039;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 &quot;The Collegiate&quot;</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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JoVE Journal

Biochemistry

2.3K Views.  Bryant University. Masarimycine is een van de weinige remmers van bacteriële autolysines die de groei van bacteriële cellen stopt. Het kan worden gebruikt om de verschillen tussen chemische en genetische inactivatie van autolysines te onderzoeken en biedt een orthogonale benadering van genetica om bacteriële fysiologie te bestuderen. Synthese van masarimycine kan ontmoedigend lijken.Volg nauwgezet de getoonde stappen. Bij twijfel of een reactiestap heeft gewerkt, bevestig dan door dunnelaagchromatogra...

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

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

Genetics

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...

Chemistry

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Immunology and Infection

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

Biology

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

Identifying Drugs that Affect Polymerization of Bacterial Cytoskeletal Proteins

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

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

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