Mur Enzymes Research Articles

Mini Review: Antimicrobial Agents Based on Natural Compounds: The Key to Solving the Current Crisis

Context: The rapid development of resistance mechanisms by bacteria has led to the loss of effectiveness of many commonly used antibiotics, posing a serious threat to global public health. Therefore, the objective of this study is to provide further insights into combating antibiotic resistance and addressing the urgent need for additional effective antimicrobial treatments as alternatives. Methods: In this narrative review, natural compounds derived from plants, fungi, and other sources were evaluated for their antimicrobial properties based on clinical trials and in vitro studies. These compounds have gained attention due to their potential to serve as alternative or adjunctive therapies. Results: We briefly reviewed and introduced antibacterial agents derived from natural substances and extracts as an alternative solution to the current crisis. Many natural sources, such as antimicrobial peptides (AMPs), nanoparticles, enzyme disruption of acylhomoserine lactones (AHLs), Mur enzymes, lysocine E, plant-derived natural products, polyphenols, nanozymes, antimicrobial peptidomimetics, and antimicrobials of fungal origin (such as penicillin, griseofulvin, amphotericin B, echinocandins, and statins), produce a wide range of secondary metabolites with the potential to exhibit antibacterial activity. Moreover, natural products often possess complex chemical structures, which can target multiple bacterial pathways, enhancing their ability to inhibit or kill bacteria. Conclusions: This study contributes new knowledge and perspectives on combating antibiotic resistance by exploring alternative treatments and natural products, which represent an important area of research. It may offer new solutions to address the urgent need for effective antimicrobial treatments. Further research is necessary to isolate, identify, and characterize the active compounds, study their mechanisms of action, optimize their efficacy and safety, and evaluate their potential for clinical use.

A novel hydrophobic peptide FGMp11: Insights into antimicrobial properties, hydrophobic sites on Staphylococcus aureus and its application in infecting pasteurized milk

Hydrophobic peptides have received considerable attention in food preservation. The elucidation of the hydrophobic points of hydrophobic peptides is helpful in revealing the antimicrobial mechanism, and screening strategies for novel antimicrobials such as molecular docking and deep learning. A novel antimicrobial peptide (AMP) FGMp11 was identified and screened from fermented goat milk with strong hydrophobic properties, β-turn structure, and the amino acid sequence of TPVVVPPFLQP. The hydrophobic properties of FGMP11 could significantly damage the integrity of the cell membrane, resulting in changes in cell morphology. Hydrophobic interactions of FGMp11 with Mur enzymes (MurC, MurD, MurE, MurF, and MurG) are ideal targets for antimicrobial therapy, as these targets are involved in peptidoglycan synthesis via hydrogen bond interactions. The electrostatic binding sites of FGMp11 and the key amino acids GLN, LEU, PRO, and VAL were mainly -OH, -NH2, and -C=O groups, reducing cell activity. FGMp11 was able to significantly inhibit the growth of S. aureus during the storage of pasteurized milk. Overall, these findings provide a scientific basis for the antimicrobial activity of FGMp11 against food-borne pathogens and its application in food preservation.

Identification of potential inhibitors against Escherichia coli Mur D enzyme to combat rising drug resistance: an in-silico approach

Indiscriminate use of anti-microbial agents has resulted in the inception, frequency, and spread of antibiotic resistance among targeted bacterial pathogens and the commensal flora. Mur enzymes, playing a crucial role in cell-wall synthesis, are one of the most appropriate targets for developing novel inhibitors against antibiotic-resistant bacterial pathogens. In the present study, in-silico high-throughput virtual (HTVS) and Standard-Precision (SP) screening was carried out with 0.3 million compounds from several small-molecule libraries against the E. coli Mur D enzyme (PDB ID 2UUP). The docked complexes were further subjected to extra-precision (XP) docking calculations, and highest Glide-score compound was further subjected to molecular simulation studies. The top six virtual hits (S1–S6) displayed a glide score (G-score) within the range of −9.013 to −7.126 kcal/mol and compound S1 was found to have the highest stable interactions with the Mur D enzyme (2UUP) of E. coli. The stability of compound S1 with the Mur D (2UUP) complex was validated by a 100-ns molecular dynamics simulation. Binding free energy calculation by the MM-GBSA strategy of the S1-2UUP (Mur D) complex established van der Waals, hydrogen bonding, lipophilic, and Coulomb energy terms as significant favorable contributors for ligand binding. The final lead molecules were subjected to ADMET predictions to study their pharmacokinetic properties and displayed promising results, except for certain modifications required to improve QPlogHERG values. So, the compounds screened against the Mur D enzyme can be further studied as preparatory points for in-vivo studies to develop potential drugs. HIGHLIGHTS E.coli is a common cause of urinary tract infections. E.coli MurD enzyme is a suitable target for drug development. Novel inhibitors against E.coli MurD enzyme were identified. Molecular dynamics studies identified in-silico potential of identified compound. ADMET predictions and Lipinski’s rule of five studies showed promising results. Communicated by Ramaswamy H. Sarma

Exploring the in silico studies of the endophyte fungus Phoma herbarum against mur enzymes of Staphylococcus aureus – a computational approach

Exploring the in silico studies of the endophyte fungus Phoma herbarum against mur enzymes of Staphylococcus aureus – a computational approach

BCp12/PLA combination: A novel antibacterial agent targeting Mur family, DNA gyrase and DHFR

The combination of natural antimicrobial peptide BCp12/phenyllatic acid (BCp12/PLA) presents a more efficient antibacterial effect, but its antibacterial mechanism remains unclear. This study studied the synergistic antibacterial mechanism of BCp12 and PLA against S. aureus. The results demonstrated that the BCp12/PLA combination presented a synergistic antibacterial effect against S. aureus, with a fractional inhibitory concentration of 0.05. Furthermore, flow cytometry and scanning electron microscope analysis revealed that BCp12 and PLA synergistically promoted cell membrane disruption compared with the group treated only with one compound, inducing structural cell damage and cytoplasmic leakage. In addition, fluorescence spectroscopy analysis suggested that BCp12 and PLA synergistically influenced genomic DNA. BCp12 and PLA targeted enzymes related to peptidoglycan and DNA synthesis and interacted by hydrogen bonding and hydrophobic interactions with mur enzymes (murC, murD, murE, murF, and murG), dihydrofolate reductase, and DNA gyrase. Additionally, the combined treatment successfully inhibited microbial reproduction in the storage of pasteurized milk, indicating that the combination of BCp12 and PLA can be used as a new preservative strategy in food systems. Overall, this study could provide potential strategies for preventing and controlling foodborne pathogens.

Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

Anti-Tuberculosis Mur Inhibitors: Structural Insights and the Way Ahead for Development of Novel Agents.

Mur enzymes serve as critical molecular devices for the synthesis of UDP-MurNAc-pentapeptide, the main building block of bacterial peptidoglycan polymer. These enzymes have been extensively studied for bacterial pathogens such as Escherichia coli and Staphylococcus aureus. Various selective and mixed Mur inhibitors have been designed and synthesized in the past few years. However, this class of enzymes remains relatively unexplored for Mycobacterium tuberculosis (Mtb), and thus offers a promising approach for drug design to overcome the challenges of battling this global pandemic. This review aims to explore the potential of Mur enzymes of Mtb by systematically scrutinizing the structural aspects of various reported bacterial inhibitors and implications concerning their activity. Diverse chemical scaffolds such as thiazolidinones, pyrazole, thiazole, etc., as well as natural compounds and repurposed compounds, have been reviewed to understand their in silico interactions with the receptor or their enzyme inhibition potential. The structural diversity and wide array of substituents indicate the scope of the research into developing varied analogs and providing valuable information for the purpose of modifying reported inhibitors of other multidrug-resistant microorganisms. Therefore, this provides an opportunity to expand the arsenal against Mtb and overcome multidrug-resistant tuberculosis.

Insights from the molecular docking analysis of compounds from Vitex negundoi with targets from Klebsiella pneumoniai causing urinary tract infection.

Antimicrobial resistance among bacterial strains has emerged out to be a serious threat and contributes to the loss of effectiveness of the common antibiotics. New Delhi metallo-β-lactamase-1 (blaNDM-1) is an enzyme present in several pathogenic bacteria with a high incidence in Klebsiella pneumoniaie and plays a crucial role in the development of antibacterial resistance. Mur enzymes are also important alternative drug targets in addition to blaNDM-1 which are crucial for the survival of the bacteria. Vitex negundoi is an aromatic medicinaltree with proven antibacterial properties. Fifteen compounds from V. negundo were evaluated for their inhibitory effects on the target proteins blaNDM-1, Mur C, Mur E and Mur F of K. pneumoniae through molecular docking using the Glide (xp) module of Schrodinger. ADME toxicity was also predicted for all the fifteen compounds in the QikProp module. The docking results revealed that the compounds agnuside, negundoside and isoorientin showed promising inhibitory effects on all four targets blaNDM-1, Mur C, Mur E and Mur F of K. pneumoniae with docking scores greater than -7 kcal/mol and reasonable hydrogen bond interactions. The findings of this study provide a lead for developing novel drugs against potent multidrug-resistant K. pneumoniae.

Advances in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Covalent Inhibition.

Peptidoglycan is a cross-linked polymer responsible for maintaining the bacterial cell wall integrity and morphology in Gram-negative and Gram-positive bacteria. The peptidoglycan pathway consists of the enzymatic reactions held in three steps: cytoplasmic, membrane-associated, and periplasmic. The Mur enzymes (MurA-MurF) are involved in a cytoplasmic stage. The UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme is responsible for transferring the enolpyruvate group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG) to form UDP-N-acetylglucosamine enolpyruvate (EP-UNAG). Fosfomycin is a natural product analogous to PEP that acts on the MurA target enzyme via binding covalently to the key cysteine residue in the active site. Similar to fosfomycin, other MurA covalent inhibitors have been described with a warhead in their structure that forms a covalent bond with the molecular target. In MurA, the nucleophilic thiolate of Cys115 is pointed as the main group involved in the warhead binding. Thus, in this minireview, we briefly describe the main recent advances in the design of MurA covalent inhibitors.

Discovery of multi-target mur enzymes inhibitors with anti-mycobacterial activity through a Scaffold approach

In the present study, we generated a ligand-based scaffold model from a known bioactive datasets of mur enzymes of other species to identify multi-targeting inhibitors as antitubercular agents. Compounds in the ChEMBL database were first filtered to screen for substructure molecules ofMtb’s multi-target enzymes. 5′-O-(5-Amino-5-deoxy-β-D-ribofuranosyl)uridine has been identified as scaffold to develop compounds targeting Mtb’s mur enzymes. A library of Murcko scaffolds was extracted and evaluated for their in-silico antitubercular activity against Mtb’s mur enzymes. The screened compounds were subjected to molecular docking, molecular dynamics simulations, MM/PBSA calculation with Mtb’s mur enzymes to evaluate the mechanism of interaction to assess inhibitory activity against the target protein. The results revealed that 15 compounds have higher docking scores and good interactions with multiple mur enzymes of Mtb. From the docking analysis, compound HPT had the best score and binding affinity with the all mur enzymes. Further, protein-ligand interactions were evaluated by molecular dynamics simulations to assess their stability throughout 100 ns period. From the MD trajectory, we calculated RMSD, RMSF, Rg, PCA, DCCM, FEL, hydrogen bonding, and vector motion. Furthermore, the binding free energies of the all nine mur enzymes with compound HPT exhibited good binding affinity might show the anti-mycobacterial activity. The compound HPT revealed from this computational study could act as potent anti-mycobacterial inhibitors and further serve as lead scaffolds to develop more potent pharmaceutical molecules targeting multiple mur enzymes of Mtb based on 5′-O-(5-Amino-5-deoxy-β-D-ribofuranosyl)uridine in the future. Communicated by Ramaswamy H. Sarma

Exploring the interaction mechanism between potential inhibitor and multi-target Mur enzymes of mycobacterium tuberculosis using molecular docking, molecular dynamics simulation, principal component analysis, free energy landscape, dynamic cross-correlation matrices, vector movements, and binding free energy calculation

Multi-targeting enzyme approaches are considered to be the most significant in suppressing pathogen growth and disease control for MDR and XDR-resistant Mycobacterium tuberculosis. The multiple Mur enzymes involved in peptidoglycan biosynthesis play a key role in a cell’s growth. Firstly, homology modeling was employed to construct the 3 D structure of the Mur enzymes. The computational approaches, including molecular docking and molecular dynamics simulations and MM-PBSA methods, were performed to explore the detailed interaction mechanism to evaluate the inhibitory activity against targeted proteins. The computational calculations revealed that the best-docked phytochemical compound (gallomyricitrin) inhibits the selected targets: Mur enzymes by forming stable hydrogen bonds. The analysis of RMSD, RMSF, Rg, PCA, DCCM, cross-correlation network, FEL, H-bond, and vector movement reveal that the docked complex of MurA, MurI, MurG, MurC, and MurE is more stable compared to MurB, MurF, MurD, and MurX docked complexes during MD simulations. Moreover, FEL exposed that gallomyricitrin stabilized to the minimum global energy of Mur Enzymes. The PCA, DCCM, and vector movements and binding free energy results provided further evidence for the stability of gallomyricitrin’s interactions inside the binding sites by forming hydrogen bonds. The cross-correlation analysis reveals that Mur enzymes exhibit a positive and negative correlated motion between residues in different protein domains. The computational results contribute in several ways to our understanding of inhibition activity and provide a basic insight into the binding activity of gallomyricitrin as a multi-target drug for tuberculosis. Communicated by Ramaswamy H. Sarma

The Mur Enzymes Chink in the Armour of Mycobacterium tuberculosis cell wall.

TUBERCULOSIS: (TB) transmitted by Mycobacterium tuberculosis (Mtb) is one of the top 10 causes of death globally. Currently, the widespread occurrence of resistance toward Mtb strains is becoming a significant concern to public health. This scenario exaggerated the need for the discovery of novel targets and their inhibitors. Targeting the "Mtb cell wall peptidoglycan synthesis" is an attractive strategy to overcome drug resistance. Mur enzymes (MurA-MurF) play essential roles in the peptidoglycan synthesis by catalyzing the ligation of key amino acid residues to the stem peptide. These enzymes are unique and confined to the eubacteria and are absent in humans, representing potential targets for anti-tubercular drug discovery. Mtb Mur ligases with the same catalytic mechanism share conserved amino acid regions and structural features that can conceivably exploit for the designing of the inhibitors, which can simultaneously target more than one isoforms (MurC-MurF) of the enzyme. In light of these findings in the current review, we have discussed the recent advances in medicinal chemistry of Mtb Mur enzymes (MurA-MurF) and their inhibitors, offering attractive multi-targeted strategies to combat the problem of drug-resistant in M.tuberculosis.

Identification of novel multitarget antitubercular inhibitors against mycobacterial peptidoglycan biosynthetic Mur enzymes by structure-based virtual screening

Current therapeutic strategies for several diseases, including Mycobacterium tuberculosis infection, have evolved from an initial single-target treatment to a multitarget one. A multitarget antitubercular drugs targeting different mycobacterial proteins are more effective at suppressing bacterial growth. In this study, a high throughput virtual screening was performed to identify hits to the potential antitubercular multitarget: murA, murB, murC, murD, murE, murF, murG and murI from M. tuberculosis that is involved in peptidoglycan biosynthesis. In the virtual screening, we were docked 56,400 compounds of the ChEMBL antimycobacterial library and re-scored and identified the top 10 ranked compounds as antitubercular drug candidates. Further, the best common docked complex CHEMBL446262 was subjected to molecular dynamics simulation to understand the molecule's stability in the presence of an active site environment. After that, we have calculated binding free energy the top-ranked docked complexes using the MM/PBSA method. These ligands exhibited the highest binding affinity; find out novel drug-likeness might show the M. tuberculosis effect's inhibitor by interacting with multitarget Mur enzymes. New antitubercular therapies that include multitarget drugs may have higher efficacy than single-target medicines and provide a more straightforward antitubercular therapy regimen. Communicated by Ramaswamy H. Sarma

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases, MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection

Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin. Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent CDI.

Identification of a Potential Inhibitor Targeting MurC Ligase of the Drug Resistant Pseudomonas aeruginosa Strain through Structure-Based Virtual Screening Approach and In Vitro Assay.

Pseudomonas aeruginosa shows resistance to a large number of antibiotics, including carbapenems and third generation cephalosporin. According to the World Health Organization global report published in February 2017, Pseudomonas aeruginosa is on the priority list among resistant bacteria, for which new antibiotics are urgently needed. Peptidoglycan serves as a good target for the discovery of novel antimicrobial drugs. Biosynthesis of peptidoglycan is a multi-step process involving four mur enzymes. Among these enzymes, UDP-N-acetylmuramate-L-alanine ligase (MurC) is considered to be an excellent target for the design of new classes of antimicrobial inhibitors in gram-negative bacteria. In this study, a homology model of Pseudomonas aeruginosa MurC ligase was generated and used for virtual screening of chemical compounds from the ZINC Database. The best screened inhibitor i.e. N, N-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazole-5-sulfonamide was then validated experimentally through inhibition assay. The presented results based on combined computational and in vitro analysis open up new horizons for the development of novel antimicrobials against this pathogen.

Screening of Antitubercular Compound Library Identifies Inhibitors of Mur Enzymes in Mycobacterium tuberculosis.

The rapid rise in the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) mandates the discovery of novel tuberculosis (TB) drugs. Mur enzymes, which are identified as essential proteins in Mtb and catalyze the cytoplasmic steps in the peptidoglycan biosynthetic pathway, are considered potential drug targets. However, none of the clinical drugs have yet been developed against these enzymes. Hence, the aim of this study was to identify novel inhibitors of Mur enzymes in Mycobacterium tuberculosis. We screened an antitubercular compound library of 684 compounds, using MurB and MurE enzymes of the Mtb Mur pathway as drug targets. For experimental validation, the top hits obtained on in silico screening were screened in vitro, using Mtb Mur enzyme-specific assays. In all, seven compounds were found to show greater than 50% inhibition, with the highest inhibition observed at 77%, and the IC50 for these compounds was found to be in the range of 28-50 μM. Compound 5175112 showed the lowest IC50 (28.69 ± 1.17 μM), and on the basis of (1) the binding affinity, (2) the stability of interaction noted on molecular dynamics simulation, and (3) an in vitro assay, MurE appeared to be its target enzyme. We believe that the overall strategy followed in this study and the results obtained are a good starting point for developing Mur enzyme-specific Mtb inhibitors.

Complex Formation between Mur Enzymes from Streptococcus pneumoniae.

Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE–MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.

Single Cell and Single-Molecule Assays Reveal Bacteria Regulate their Rate of Growth by Actively Reading Out the Level of Cell Wall Precursors

How cells regulate their growth rate in response to nutrient conditions remains an outstanding question in biology. Bacteria are surrounded by a peptidoglycan cell wall that holds their shape. This structure is built by MreB filaments that regulate the enzymes that synthesize the cell wall. Collectively termed the Rod Complex, these machines moving around the cell as they insert new material. To understand how cells grow, we used Structured Illumination Microscopy (SIM) and single-molecule tracking to quantitate the number of Rod complexes in Bacillus subtilis growing in different media. First, we found that cells regulate the filament density but not the speed in response to nutrient availability. The directional motion of enzymes is regulated by MreB filaments and their motions transitioning into a diffusive state when MreB filaments are disrupted. Second, we demonstrated that the number of MreB filaments is regulated by the cell wall precursor lipid II, as increased expression of key Mur enzymes that build lipid II increases the number of MreB filaments and the rate of growth. Furthermore, we showed that lipid II is sensed by the serine/theonine kinase PrkC, which phosphorylates RodZ, a MreB filament nucleator. Surprisingly we found that strains containing a phosphomimetic RodZ mutation or overexpression of PrkC causes cells to grow faster than they would in carbon limited media. Together these demonstrate that bacteria regulate their rate of growth by measuring the flux of peptidoglycan precursors through cell wall synthesis.