15 results on '"Veerasak Srisuknimit"'
Search Results
2. A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.
- Author
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Patricia D A Rohs, Jackson Buss, Sue I Sim, Georgia R Squyres, Veerasak Srisuknimit, Mandy Smith, Hongbaek Cho, Megan Sjodt, Andrew C Kruse, Ethan C Garner, Suzanne Walker, Daniel E Kahne, and Thomas G Bernhardt more...
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Genetics ,QH426-470 - Abstract
Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery. more...
- Published
- 2018
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Catalog
3. Probing the diversity and regulation of tRNA modifications
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Veerasak Srisuknimit, Satoshi Kimura, and Matthew K. Waldor
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Microbiology (medical) ,0303 health sciences ,Bacteria ,030306 microbiology ,Computational biology ,Biology ,Microbiology ,Article ,03 medical and health sciences ,Infectious Diseases ,Gene Expression Regulation ,RNA, Transfer ,Protein Biosynthesis ,Transfer RNA ,Protein biosynthesis ,RNA Processing, Post-Transcriptional ,030304 developmental biology - Abstract
Transfer RNAs (tRNAs) are non-coding RNAs essential for protein synthesis. tRNAs are heavily decorated with a variety of post-transcriptional modifications (tRNA modifications). Recent methodological advances provide new tools for rapid profiling of tRNA modifications and have led to discoveries of novel modifications and their regulation. Here, we provide an overview of the techniques for investigating tRNA modifications and of the expanding knowledge of their chemistry and regulation. more...
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- 2020
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4. Undecaprenyl phosphate translocases confer conditional microbial fitness
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Brandon Sit, Veerasak Srisuknimit, Emilio Bueno, Franz G. Zingl, Karthik Hullahalli, Felipe Cava, and Matthew K. Waldor
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Multidisciplinary - Abstract
The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2–6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host. more...
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- 2022
5. Candidate undecaprenyl phosphate translocases enable conditional microbial fitness and pathogenesis
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Brandon Sit, Veerasak Srisuknimit, Karthik Hullahalli, Emilio Bueno, Felipe Cava, and Matthew K. Waldor
- Abstract
The mechanisms that enable adaptation of peptidoglycan, the structural unit of the bacterial cell wall, to shifting extracellular conditions such as pH remain largely unknown. Here, we identify a DUF368-containing membrane protein in the cholera pathogen Vibrio cholerae that is critical for pathogenesis and alkaline fitness. V. cholerae and Staphylococcus aureus lacking their cognate DUF368-containing protein have pH-dependent cell wall defects consistent with surface accumulation of undecaprenyl phosphate (C55-P), an essential lipid carrier for the biogenesis of peptidoglycan and other key bacterial cell surface polymers. In both species, DUF368-containing proteins exhibit synthetic genetic interactions with putative transporters from the DedA family, suggesting these proteins represent complementary long-sought C55-P translocases that enable envelope maintenance functions critical for microbial fitness within and outside the host.One-Sentence SummaryDUF368-containing and DedA-family proteins are undecaprenyl phosphate transporter candidates and are required for bacterial alkaline fitness and pathogenesis. more...
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- 2022
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6. Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic
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Mithila Rajagopal, Kathryn A. Coe, Fabienne Hennessen, Timothy C. Meredith, Truc Do, Suzanne Walker, Rolf Müller, Wonsik Lee, Marina Santiago, Veerasak Srisuknimit, and Antoine Abou Fayad
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0301 basic medicine ,Transposable element ,Staphylococcus aureus ,030106 microbiology ,Mutant ,Computational biology ,Peptides, Cyclic ,Article ,Machine Learning ,03 medical and health sciences ,Downregulation and upregulation ,Cell Wall ,medicine ,Molecular Biology ,Gene Library ,Lipid II ,Chemistry ,Computational Biology ,Cell Biology ,Lipids ,Uridine Diphosphate N-Acetylmuramic Acid ,Anti-Bacterial Agents ,Up-Regulation ,030104 developmental biology ,Lysobacter ,Mechanism of action ,Lytic cycle ,Docking (molecular) ,Mutation ,DNA Transposable Elements ,medicine.symptom ,Functional genomics ,Genome, Bacterial - Abstract
Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action. more...
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- 2018
7. A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery
- Author
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Megan Sjodt, Thomas G. Bernhardt, Mandy D. Smith, Daniel Kahne, Georgia R. Squyres, Sue I Sim, Suzanne Walker, Hongbaek Cho, Jackson Buss, Ethan C. Garner, Veerasak Srisuknimit, Andrew C. Kruse, and Patricia D. A. Rohs more...
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0301 basic medicine ,Cancer Research ,Polymers ,MreB ,Biochemistry ,Polymerases ,Physical Chemistry ,Bacterial cell structure ,Polymerization ,Cell Fusion ,chemistry.chemical_compound ,Mathematical and Statistical Techniques ,Cell Wall ,Morphogenesis ,Cross-Linking ,Materials ,Genetics (clinical) ,Cytoskeleton ,Cell fusion ,Transfer Functions ,biology ,Escherichia coli Proteins ,Cell Cycle ,Chemical Reactions ,Cell cycle ,Cell biology ,Chemistry ,Macromolecules ,Physical Sciences ,Cellular Structures and Organelles ,Research Article ,Glycan ,Cell Physiology ,lcsh:QH426-470 ,Imaging Techniques ,030106 microbiology ,Materials Science ,Peptidoglycan ,Research and Analysis Methods ,Cell wall ,03 medical and health sciences ,Cell Walls ,Bacterial Proteins ,Polysaccharides ,DNA-binding proteins ,Fluorescence Imaging ,Genetics ,Escherichia coli ,Penicillin-Binding Proteins ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cytokinesis ,Biology and life sciences ,Chemical Bonding ,Membrane Proteins ,Proteins ,Cell Biology ,Peptidoglycans ,biochemical phenomena, metabolism, and nutrition ,Polymer Chemistry ,Actins ,lcsh:Genetics ,030104 developmental biology ,chemistry ,biology.protein ,Mathematical Functions - Abstract
Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery., Author summary The cell wall of bacteria determines their shape and protects them from osmotic lysis. Two enzymatic activities are required for cell wall synthesis: glycan polymerization and crosslinking. A major new family of glycan polymerases was recently discovered and was proposed to work in complex with crosslinking enzymes called penicillin-binding proteins (PBPs). How the activities of these enzymes are coordinated to prevent the toxic generation of uncrosslinked glycans has remained unknown. Our analysis of the cell elongation system of Escherichia coli has revealed that this coupling is mediated by changes in the PBP that activate glycan chain synthesis by the polymerase. Furthermore, we present genetic evidence that this activation event is mediated by a component of the elongation machinery with a previously unknown function. Discovery of this activation pathway provides new mechanistic insight into the cell wall biogenesis process and identifies a new avenue to disrupt it for antibiotic development. more...
- Published
- 2018
8. Self-Assembly, Guest Capture, and NMR Spectroscopy of a Metal–Organic Cage in Water
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Veerasak Srisuknimit, David A. Vosburg, Stephanie L. Cheng, and Eun Bin Go
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Green chemistry ,Chemistry ,Chemical shift ,05 social sciences ,Supramolecular chemistry ,050301 education ,macromolecular substances ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Education ,Molecular recognition ,Polymer chemistry ,Proton NMR ,Molecule ,Physical chemistry ,Self-assembly ,0503 education - Abstract
A green organic–inorganic laboratory experiment has been developed in which students prepare a self-assembling iron cage in D2O at room temperature. The tetrahedral cage captures a small, neutral molecule such as cyclohexane or tetrahydrofuran. 1H NMR analysis distinguishes captured and free guests through diagnostic chemical shifts, splitting patterns, diffusion coefficients (using DOSY), and the appearance of captured hydrophobic molecules in D2O even when free guests are insoluble in water. Students are invited to test their hypotheses about guest binding and to perform control and competition experiments. All of the reagents are commercial, the 23-component self-assembly is complete in 1 week at room temperature (requiring neither workup nor purification), and the product solution is intensely purple. In their laboratory reports, students effectively related organic chemistry with molecular self-assembly, supramolecular chemistry, and host–guest interactions. more...
- Published
- 2015
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9. Direct, Biomimetic Synthesis of (+)-Artemone via a Stereoselective, Organocatalytic Cyclization
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Jonathan P. Litz, Brian C. Fielder, Veerasak Srisuknimit, Mary J. Van Vleet, Shannon P. Wetzler, David A. Vosburg, Eric D. Nacsa, and Kim Quach
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Green chemistry ,Addition reaction ,Chemistry ,Biomimetic synthesis ,Organocatalysis ,Organic Chemistry ,Organic chemistry ,Iminium ,Total synthesis ,Stereoselectivity ,Catalysis ,Conjugate - Abstract
We present a four-step synthesis of (+)-artemone from (–)-linalool, featuring iminium organocatalysis of a doubly diastereoselective conjugate addition reaction. The strategy follows a proposed biosynthetic pathway, rapidly generates stereochemical complexity, uses no protecting groups, and minimizes redox manipulations. more...
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- 2015
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10. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis
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Daniel Kahne, Alexander J. Meeske, Debora S. Marks, Genevieve S Dobihal, Andrew C. Kruse, Thomas A. Hopf, Megan Sjodt, Thomas G. Bernhardt, Patricia D. A. Rohs, Kelly P Brock, Suzanne Walker, Veerasak Srisuknimit, David Z. Rudner, and Anna G. Green more...
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0301 basic medicine ,Models, Molecular ,Protein Folding ,Protein family ,Protein domain ,Peptidoglycan ,Crystallography, X-Ray ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Protein Domains ,Cell Wall ,Escherichia coli ,Molecular replacement ,Multidisciplinary ,biology ,Thermus thermophilus ,biology.organism_classification ,Nucleotidyltransferases ,Transmembrane protein ,Cell biology ,Transmembrane domain ,030104 developmental biology ,chemistry ,Biocatalysis ,Protein folding ,Bacillus subtilis - Abstract
The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 A, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function. more...
- Published
- 2017
11. Peptidoglycan Cross-Linking Preferences of Staphylococcus aureus Penicillin-Binding Proteins Have Implications for Treating MRSA Infections
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Suzanne Walker, Veerasak Srisuknimit, Kaitlin Schaefer, Yuan Qiao, and Daniel Kahne
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0301 basic medicine ,Peptidoglycan metabolism ,Methicillin-Resistant Staphylococcus aureus ,Glycan ,Penicillin binding proteins ,030106 microbiology ,Peptidoglycan ,010402 general chemistry ,medicine.disease_cause ,beta-Lactams ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,medicine ,Penicillin-Binding Proteins ,biology ,Molecular Structure ,Extramural ,General Chemistry ,biochemical phenomena, metabolism, and nutrition ,Staphylococcal Infections ,0104 chemical sciences ,Anti-Bacterial Agents ,Cross-Linking Reagents ,chemistry ,Staphylococcus aureus ,Glycine ,Peptidyl Transferases ,biology.protein - Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global public health problem. MRSA strains have acquired a non-native penicillin-binding protein called PBP2a that crosslinks peptidoglycan when the native S. aureus PBPs are inhibited by β-lactams. If assembly of the pentaglycine branch on the cell wall precursor Lipid II is genetically blocked, MRSA strains become susceptible to β-lactams. Therefore, it has been proposed that PBP2a can only crosslink peptidoglycan strands bearing a complete pentaglycine branch. This hypothesis has never been tested because the necessary substrates have not been available. Here, we obtained S. aureus Lipid II variants having shorter glycine branches and have tested whether PBP2a and two other S. aureus transpeptidases, PBP2 and PBP4, can crosslink peptidoglycan strands made from the variants. There are striking differences in enzymatic activity among these enzymes depending on the length of the glycine branch, but we find that PBP2a can, in fact, crosslink glycan strands bearing triglycine. We report experiments in cells that are consistent with our in vitro findings about the crosslinking preferences of these PBPs. In addition to providing insights into the cell wall physiology of a major pathogen, our studies identify the best target for β-lactam potentiators to treat MRSA. more...
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- 2017
12. Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams
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Daniel Kahne, Frederick A. Rubino, Yuan Qiao, Suzanne Walker, Natividad Ruiz, Veerasak Srisuknimit, and Kaitlin Schaefer
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0301 basic medicine ,Glycan ,Staphylococcus aureus ,Biology ,010402 general chemistry ,beta-Lactams ,01 natural sciences ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,Bacterial Proteins ,Cell Wall ,Penicillin-Binding Proteins ,Molecular Biology ,chemistry.chemical_classification ,Lipid II ,Molecular Structure ,Cell Biology ,biology.organism_classification ,Lipids ,0104 chemical sciences ,3. Good health ,Cytolysis ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Peptidyl Transferases ,biology.protein ,Peptidoglycan ,Bacteria - Abstract
Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. β-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by β-lactams have been hampered by lack of access to a suitable substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate the utility of this strategy by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2 (PBP2), which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different β-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors. more...
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- 2016
13. Structure and function of the SEDS:bPBP bacterial cell wall synthesis machinery
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Thomas A. Hopf, Anna G. Green, Veerasak Srisuknimit, Thomas G. Bernhardt, Patricia D. A. Rohs, Debora S. Marks, Alexander J. Meeske, Genevieve S Dobihal, Andrew C. Kruse, Daniel Kahne, Megan Sjodt, David Z. Rudner, Kelly P Brock, and Suzanne Walker more...
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0301 basic medicine ,010407 polymers ,Chemistry ,Condensed Matter Physics ,01 natural sciences ,Biochemistry ,Bacterial cell structure ,0104 chemical sciences ,Structure and function ,Inorganic Chemistry ,03 medical and health sciences ,030104 developmental biology ,Structural Biology ,Biophysics ,General Materials Science ,Physical and Theoretical Chemistry - Published
- 2018
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14. ChemInform Abstract: Direct, Biomimetic Synthesis of (+)-Artemone (I) via a Stereoselective, Organocatalytic Cyclization
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Eric D. Nacsa, Mary J. Van Vleet, Veerasak Srisuknimit, David A. Vosburg, Jonathan P. Litz, Brian C. Fielder, Shannon P. Wetzler, and Kim Quach
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Terpene ,Chemistry ,Biomimetic synthesis ,Organic chemistry ,Stereoselectivity ,General Medicine - Published
- 2016
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15. The Mechanism of Action of Lysobactin
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Rolf Müller, Veerasak Srisuknimit, Yuan Qiao, Kaitlin Schaefer, Suzanne Walker, Daniel Kahne, Heinrich Steinmetz, and Wonsik Lee
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0301 basic medicine ,Staphylococcus aureus ,Teixobactin ,Nanotechnology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,Microscopy, Electron, Transmission ,Depsipeptides ,medicine ,Teichoic acid ,Lipid II ,General Chemistry ,Ramoplanin ,0104 chemical sciences ,030104 developmental biology ,Streptococcus pneumoniae ,chemistry ,Mechanism of action ,Peptidoglycan ,medicine.symptom ,Cell envelope ,medicine.drug - Abstract
Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid II(A)(WTA), substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin's cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage. more...
- Published
- 2015
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