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Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. Objective: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. Method: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. Results: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. Conclusion: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

Bacterial peptidoglycan hydrolases play an essential role in cell wall metabolism during bacterial growth, division, and elongation (autolysins) or in the elimination of closely related species from the same ecological niche (bacteriocins). Most studies concerning the peptidoglycan hydrolases present in Gram-positive bacteria have focused on clinically relevant Staphylococcus aureus or the model organism Bacillus subtilis, while knowledge relating to other species remains limited. Here, we report two new peptidoglycan hydrolases from the M23 family of metallopeptidases derived from the same staphylococcal species, Staphylococcus pettenkoferi. They share modular architecture, significant sequence identity (60%), catalytic and binding residue conservation, and similar modes of activation, but differ in gene distribution, putative biological role, and, strikingly, in their isoelectric points (pIs). One of the peptides has a high pI, similar to that reported for all M23 peptidases evaluated to date, whereas the other displays a low pI, a unique feature among M23 peptidases. Consequently, we named them SpM23_B (Staphylococcus pettenkoferi M23 “Basic”) and SpM23_A (Staphylococcus pettenkoferi M23 “Acidic”). Using genetic and biochemical approaches, we have characterized these two novel lytic enzymes, both in vitro and in their physiological context. Our study presents a detailed characterization of two novel and clearly distinct peptidoglycan hydrolases to understand their role in bacterial physiology.

Biobutanol is a valuable biochemical and one of the most promising biofuels. Clostridium saccharoperbutylacetonicum N1-4 is a hyperbutanol-producing strain. However, its strong autolytic behavior leads to poor cell stability, especially during continuous fermentation, thus limiting the applicability of the strain for longterm and industrial-scale processes. In this study, we aimed to evaluate the role of autolysin genes within the C. saccharoperbutylacetonicum genome related to cell autolysis and further develop more stable strains for enhanced butanol production. First, putative autolysin-encoding genes were identified in the strain based on comparison of amino acid sequence with homologous genes in other strains. Then, by overexpressing all these putative autolysin genes individually and characterizing the corresponding recombinant strains, four key genes were pinpointed to be responsible for significant cell autolysis activities. Further, these key genes were deleted using CRISPR-Cas9. Fermentation characterization demonstrated enhanced performance of the resultant mutants. Results from this study reveal valuable insights concerning the role of autolysins for cell stability and solvent production, and they provide an essential reference for developing robust strains for enhanced biofuel and biochemical production. El biobutanol es un bioquímico valioso y uno de los biocombustibles más prometedores. Clostridium saccharoperbutylacetonicum N1-4 es una cepa productora de hiperbutanol. Sin embargo, su fuerte comportamiento autolítico conduce a una mala estabilidad celular, especialmente durante la fermentación continua, lo que limita la aplicabilidad de la cepa para procesos a largo plazo y a escala industrial. En este estudio, nuestro objetivo fue evaluar el papel de los genes de autolisina dentro del genoma de C. saccharoperbutylacetonicum relacionado con la autólisis celular y desarrollar más cepas más estables para mejorar la producción de butanol. Primero, los genes que codifican la autolisina putativos se identificaron en la cepa basándose en la comparación de la secuencia de aminoácidos con genes homólogos en otras cepas. Luego, sobreexpresando todos estos genes de autolisina putativos individualmente y caracterizando las cepas recombinantes correspondientes, se identificaron cuatro genes clave como responsables de las actividades de autolisis celular significativas. Además, estos genes clave se eliminaron utilizando CRISPR-Cas9. La caracterización de la fermentación demostró un rendimiento mejorado de los mutantes resultantes. Los resultados de este estudio revelan información valiosa sobre el papel de las autolisinas para la estabilidad celular y la producción de solventes, y proporcionan una referencia esencial para desarrollar cepas robustas para mejorar la producción de biocombustibles y bioquímicos. Universidad Nacional, Costa Rica Universidad de Auburn, Estados Unidos. Centro de Investigaciones Biológicas del Noroeste, S.C., México. Instituto de Ciencias de Bashan, Estados Unidos. Universidad Oceánica de China Escuela de Química

14 p.-9 fig.-4 tab., The ββ-solenoid structures are part of many proteins involved in the recognition of bacterial cell wall. They are elongated polypeptides consisting of repeated β-hairpins connected by linker sequences and disposed around a superhelical axis stabilised by short-range interactions. Among the most studied ββ-solenoids are those belonging to the family of choline-binding modules (CBMs) from the respiratory pathogen Streptococcus pneumoniae (pneumococcus) and its bacteriophages, and their properties have been employed to develop several biotechnological and biomedical tools. We have carried out a theoretical, spectroscopic and thermodynamic study of the ββ-solenoid structure of the CBM from the pneumococcal LytA autolysin using peptides of increasing length containing 1–3 repeats of this structure. Our results show that hints of native-like tertiary structure are only observed with a minimum of three β-hairpins, corresponding to one turn of the solenoid superhelix, and identify the linker sequences between hairpins as the major directors of the solenoid folding. This study paves the way for the rational structural engineering of ββ-solenoids aimed to find novel applications., Funding sources: grants BIO2016-79323-R, CTQ2017-84371P and PID2019-105126RB-I00 (Agencia Estatal de Investigacion AEI/FEDER-EU-10.13039/ 501100011033-, and Ministerio de Ciencia e Innovación, Spain) and Intramural incorporation project #201820I132 (CSIC, Spain).

Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall-binding (CWB) domains, to be involved in different physiological functions that require bacterial cell wall remodeling. We identified a novel autolysin, Acd24020, from Clostridioides (Clostridium) difficile (C. difficile), with an endopeptidase catalytic domain belonging to the NlpC/P60 family and three bacterial Src-homology 3 domains as CWB domains. The catalytic domain of Acd24020 (Acd24020-CD) exhibited C. difficile-specific lytic activity equivalent to Acd24020, indicating that Acd24020-CD has full-function as a lytic enzyme by itself. To elucidate the specific peptidoglycan-recognition and catalytic reaction mechanisms of Acd24020-CD, biochemical characterization, X-ray structure determination, a modeling study of the enzyme/substrate complex, and mutagenesis analysis were performed. Acd24020-CD has an hourglass-shaped substrate-binding groove across the molecule, which is responsible for recognizing the direct 3-4 cross-linking structure unique to C. difficile peptidoglycan. Based on the X-ray structure and modeling study, we propose a dynamic Cys/His catalyzing mechanism, in which the catalytic Cys299 and His354 residues dynamically change their conformations to complement each step of the enzyme catalytic reaction.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 μM. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the Δlytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

Advances in biochemical and molecular manipulation have led to increased biomass productivity and oil accumulation in the microalgae C. reinhardtii. However, scalable processes for the recovery of oil and other valuable biomolecules, such as protein, from C. reinhardtii are scarce. The use of aqueous enzymatic extraction, a non-solvent and environmentally friendly bioproduct recovery method, provides an opportunity to design an integrated process for oil and protein fractionation to reduce bioenergy and bioproducts costs. Based on the mechanistic understanding of biomolecule distribution and compartmentalization, an aqueous enzymatic treatment for the release of internally stored lipid bodies was designed. Application of a C. reinhardtii-produced protease, autolysin, for lysis of the microalgae cell wall was followed by a secondary treatment with trypsin for chloroplast disruption and lipid body release. Protein recovery after the primary treatment with autolysin indicated a 50.1 ± 4.2% release of total soluble protein and localization of lipid bodies still in the chloroplast. The development of a secondary enzyme treatment (trypsin) for chloroplast and lipid body lysis demonstrated a high percent of remaining lipids (73 ± 7%) released into the supernatant. The results indicate that the application of an enzymatic treatment scheme for protein and oil recovery is a promising alternative to traditional extraction processes.

Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 &plusmn, 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His50, His147, and Cys155, a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His51, Thr52, Tyr76, and Thr153, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies, the truncated version of LysC (LysC&Delta, 2&ndash, 23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC50 of 6 &mu, g/mL (1.5 &mu, M). This peptide was shown to have &alpha, helical conformation in solution in the presence of detergents which is a common feature of amphipathic &alpha, helical antimicrobial peptides.

International audience; Zymography is a widely used technique enabling visualization of in-gel peptidase/protease hydrolytic activities. This technique is used to study the activity of bacterial peptidoglycan (PG) hydrolytic enzymes named autolysins. Zymography is particularly suited for PG autolysin characterization as bulk PG is notorious to work with due to its highly insoluble nature. This recalcitrant property of PG therefore makes the setup of PG hydrolytic activity assay very challenging. In the course of studying the catalytic activity of the CwlS protein, a D,L NlpC/P60 endopeptidase possessing multiple LysM carbohydrate binding-domains from Bacillus subtilis, we observed a potential artifact of the zymography technique. The generation of CwlS truncated mutants impaired in their PG binding capacity presented lower apparent hydrolytic activities on zymograms. Furthermore, a catalytically dead version of CwlS, or a CwlS mutant that possesses only its LysM domains and no catalytic domain, maintained similar apparent PG hydrolytic properties as wild-type CwlS on zymograms. Additionally, a mutant harboring twelve mutations in the four LysM domains, previously demonstrated to be unable to bind PG but has a similar net positive charge as the wild-type protein presents also apparent activity on zymogram. We demonstrate in this study that zymography results, which are meant to be interpreted in favor of apparent PG hydrolytic activities, are instead reflecting impairment of gel staining probably due to the very high net positive charge of the protein. Opposed Reviewers: Abstract Zymography is a widely used technique enabling visualization of in-gel

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans­formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

Mycobacterium tuberculosis (Mtb) cell wall, built on a cross-linked sugar-peptide polymer called peptidoglycan, protects the bacterial cell from adverse environments. Peptidoglycan homeostasis is maintained by extracellular peptidoglycan synthases and hydrolases. Intricate coordination of their activities is required to maintain structural integrity of the cell wall during growth, division, and response to stress. The sensor protein kinase B (PknB) is likely to play a critical role in monitoring the state of the peptidoglycan outside the cell and inducing subsequent metabolic changes inside. In this study, computational, biochemical, and structural approaches were used to characterize the peptidoglycan hydrolases of Mtb and to investigate the molecular mechanism of peptidoglycan signaling through PknB.Peptidoglycan hydrolases are critical players in bacterial growth, division, cell shape determination, and peptidoglycan fragment-mediated communication. Computational analysis identified 22 mycobacterial peptidoglycan hydrolases based on homology to known enzymes from model organisms. The peptidoglycan degradation machinery of Mtb includes 4 N-acetylmuramoyl-L-alanine amidases, 8 lytic transglycosidases, and 10 peptidases of various specificities. Ten of these enzymes form a core set of mycobacterial peptidoglycan hydrolases, while four of them are essential for growth in Mtb. Comprehensive biochemical and structural investigation of the Mtb peptidoglycan hydrolases was initiated by cloning and heterologously expressing constructs representing all 22 Mtb peptidoglycan hydrolases in Escherichia coli. Robust expression was observed for all but one target protein. Twelve were successfully purified on large scale.The peptidoglycan amidases Rv3717 and Rv3915 share similar catalytic cores yet have non-redundant functions in peptidoglycan turnover. Hydrolase activity assays using polymerized peptidoglycan sacculi and soluble peptidoglycan fragments elucidated contributions of individual amino acid residues, metal binding, and disulfide bond formation to catalysis. The structure of product-bound Rv3717 suggested a mechanism that limits this enzyme's activity on polymerized sacculi.Peptidoglycan D,D-peptidases Rv2911, Rv3330 and Rv3627 are low molecular weight penicillin-binding proteins that participate in peptidoglycan maturation and degradation. Surprisingly, these three enzymes were inactive on peptidoglycan sacculi or peptidoglycan fragments, yet were active on beta-lactams meropenem and Bocillin. The structure of Rv3330 solved in complex with meropenem revealed a potential peptide-binging groove distant from the active site. The observed lack of activity of low molecular weight penicillin-binding proteins suggests a requirement for an activator. Discovery of such factors will significantly advance our understanding of Mtb peptidoglycan homeostasis.PknB is an essential sensor kinase that controls cell wall biosynthesis. Its homologs in Gram-positive bacteria have been implicated in binding peptidoglycan fragments and in mediating bacterial responses to cell wall stress. To investigate the mechanism of peptidoglycan recognition by PknB, the structure of its extracellular sensor domain was solved. It consists of four 70-amino acid PASTA repeat domains that adopt an extended conformation. The last repeat domain contains a hydrophobic pocket with a conserved tryptophan, a signature of a ligand-binding site. The structure of PknB extracellular domain suggests that ligand-dependent localization and oligomerization control kinase activity.

Summary The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. It is formed by linear glycan chains composed of N-acetylmuramic acid-(β-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodeled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus. This article is protected by copyright. All rights reserved.

Microalgae has potential as a biofuel feedstock and as a source of valuable bioproducts for a variety of food, feed, nutraceutical, and pharmaceutical industries due to high yields of proteins, starch, and lipids. However, several challenges are associated with bioproduct extraction from microalgae. The complexity of microalgae cell walls necessitates use of energy intensive disruption methods, but current chemical or mechanical techniques can degrade economically valuable bioproducts. Therefore, disruption methods that target microalgae cell walls are essential, such as enzymatic biomass pretreatment for the release of specific biomolecules. Aqueous enzymatic pretreatment can preserve valuable bioproducts while permitting high levels of cell disruption. In this study, we optimized harvesting times that maximized protein yields in nitrogen depleted cultures and promoted lipid accumulation in the microalgae Chlamydomonas reinhardtii. Furthermore, an aqueous enzymatic assisted extraction (AEAE) treatment was developed. Four lytic enzymes were tested for their ability to permeate C. reinhardtii cell walls. Autolysin treatment was chosen as preferred cell disruption method. Treated cells were visualized by TEM imaging. TEM images and cell counts confirmed cell permeability (100%) and further cell lysis (50%) and product release when cells were treated with autolysin for 24 h. Biomass was also subjected to lipid and protein extraction after autolysin treatment and yields were compared to other mechanical and chemical extraction methods. Protein extractability was significantly enhanced by the autolysin pretreatment when compared to sonication pretreatment. Solvent extraction accompanied with autolysin biomass pretreatment significantly enhanced lipid extractable yields as compared to only solvent extraction and solvent plus sonication extraction.

Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.

β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall., Author summary The bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all β-lactam antibiotics has made treatment increasingly difficult. β-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of β-lactams. Without β-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to β-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that β-lactam antibiotics can prevent lysis of a mutant with deregulated activity of autolytic enzymes involved in daughter cell splitting, emphasizes the idea that β-lactams interfere with the coordination between cell division and daughter cell splitting, and do not kill S. aureus simply by weakening the cell wall.

Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β- N -acetylglucosaminidase encoded by the gene lba0176 ( acmB ), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus . Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the Δ acmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the Δ acmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro . These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

Summary The pneumococcal autolysin LytA is a key virulence factor involved in several important functions including DNA competence, immune evasion and biofilm formation. Here, we present the 1.05 A crystal structure of the catalytic domain of LytA in complex with a synthetic cell-wall-based peptidoglycan (PG) ligand that occupies the entire Y-shaped substrate-binding crevice. As many as twenty-one amino-acid residues are engaged in ligand interactions with a majority of these interactions directed towards the glycan strand. All saccharides are intimately bound through hydrogen bond, van der Waals and CH-π interactions. Importantly, the structure of LytA is not altered upon ligand binding, whereas the bound ligand assumes a different conformation compared to the unbound NMR-based solution structure of the same PG-fragment. Mutational study reveals that several non-catalytic glycan-interacting residues, structurally conserved in other amidases from Gram-positive Firmicutes, are pivotal for enzymatic activity. The three-dimensional structure of the LytA/PG complex provides a novel structural basis for ligand restriction by the pneumococcal autolysin, revealing for the first time an importance of the multivalent binding to PG saccharides.

Autolysin E (AtlE) is a bacteriolytic enzyme which plays an important role in division and growth of bacterial cells and therefore represents a promising potential drug target. Its 3D structure has been recently elucidated. We used in silico prediction tools to study substrate or ligand (inhibitor) binding regions of AtlE. We applied several freely available tools and a commercial tool for binding site identification and compared results of the prediction. Calculation time, number of predictions and output data provided by specific software vary according to the different approaches utilized by specific method categories. Despite different approaches, binding sites in similar locations on the protein were predicted. Specific amino acid residues that form these binding sites were predicted as binding residues. The predicted residues, especially those with predicted highest conservation score, could theoretically have catalytic and binding properties. According to our results, we assume that E138, which has the highest conservation score, is the catalytic residue and F161, G162 and Y224, which are also highly conserved, are responsible for substrate binding. Ligands developed with binding specificity towards these residues could inhibit the catalysis and binding of the substrate of AtlE. The molecules with inhibitory potency could therefore represent potential new antibacterial agents.

A structure-based approach is applied for the development of inhibitors of bacterial N-acetyglucosaminidase (autolysin). Autolysins are enzymes involved in the degradation of peptidoglycan and therefore participate in bacterial cell growth and different lysis phenomena. Several studies indicate that by the inhibition of autolysins, and consequently of bacterial cell division, antibacterial activity can be obtained, thus paving the road to a novel group of therapeutics against human pathogens. As crystal structures of the autolysin E (AtlE)-ligand complexes were obtained in our laboratories, fragment-based virtual screening was the method of choice for the initial studies. Fragment libraries from various databases were merged to increase the number of compounds for the virtual screening. Twenty-four commercially available virtual hits were selected and subjected to quantitative analysis of binding interactions using the surface plasmon resonance technique. Twelve fragments showed fragment-AtlE interactions. For F1, the top hit of the virtual screening, a KD of 228 µM was determined, while other fragments displayed non-stoichiometric binding. Blind docking of potential binders uncovers three possible allosteric sites. Ligands of N-acetyglucosaminidase identified in our study represent valuable information for the further development of AtlE inhibitors, which could in future represent antibacterial agents acting by a novel mode of action.

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.

The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity.

N-acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors as both tools for studying the chemical biology of autolysins and also serving as anti-bacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (5) and fgka (21), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 µM respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrate a propensity for cells to form linked chains, suggesting impaired cell growth and division.

Human thrombospondin 1 (hTSP-1) is a matricellular glycoprotein facilitating bacterial adherence to and invasion into eukaryotic cells. However, the bacterial adhesin(s) remain elusive. In this study, we show a dose-dependent binding of soluble hTSP-1 to Gram-positive but not Gram-negative bacteria. Diminished binding of soluble hTSP-1 to proteolytically pretreated staphylococci suggested a proteinaceous nature of potential bacterial adhesin(s) for hTSP-1. A combination of separation of staphylococcal surface proteins by two-dimensional gel electrophoresis with a ligand overlay assay with hTSP-1 and identification of the target protein by mass spectrometry revealed the major staphylococcal autolysin Atl as a bacterial binding protein for hTSP-1. Binding experiments with heterologously expressed repeats of the AtlE amidase from Staphylococcus epidermidis suggest that the repeating sequences (R1ab-R2ab) of the N-acetyl-muramoyl-L-alanine amidase of Atl are essential for binding of hTSP-1. Atl has also been identified previously as a staphylococcal vitronectin (Vn)-binding protein. Similar to the interaction with hTSP-1, the R1ab-R2ab repeats of Atl are shown here to be crucial for the interaction of Atl with the complement inhibition and matrix protein Vn. Competition assays with hTSP-1 and Vn revealed the R1ab-R2ab repeats of AtlE as the common binding domain for both host proteins. Furthermore, Vn competes with hTSP-1 for binding to Atl repeats and vice versa. In conclusion, this study identifies the Atl repeats as bacterial adhesive structures interacting with the human glycoproteins hTSP-1 and Vn. Finally, this study provides insight into the molecular interplay between hTSP-1 and Vn, respectively, and a bacterial autolysin.

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis , the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N -acetylmuramoyl- l -alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis . We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

It has recently been shown that the major autolysin Acm2 from Lactobacillus plantarum WCFS1 undergoes intracellular O-GlcNAcylation [Fredriksen L, Mathiesen G, Moen A, Bron PA, Kleerebezem M, Eijsink VG, Egge-Jacobsen W. 2012. The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation. J Bacteriol. 194(2):325-333]. To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsY and the DNA translocase FtsK1. Among the other, glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for N-acetylhexosamine (HexNAc) were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK).

Background: Esp, a secreted protease of Staphylococcus epidermidis, blocks biofilm formation of Staphylococcus aureus and its ability to colonize human nares. Results: Esp cleaves autolysin, thereby preventing the release of staphylococcal DNA as biofilm matrix. Conclusion: Secreted proteases control S. aureus biofilm development and host colonization. Significance: Methods that promote autolysin degradation may also prevent S. aureus colonization of humans., Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan, thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion.

Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes was insertionally inactivated with an erythromycin resistance cassette, and the mutants were analyzed for autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This analysis revealed a single TDOR, SdbA, which exhibited a pleiotropic mutant phenotype. Using an in silico analysis approach, we identified the major autolysin AtlS as a natural substrate of SdbA and showed that SdbA is critical to the formation of a disulfide bond that is required for autolytic activity. Analysis by BLAST search revealed homologs to SdbA in other Gram-positive species. This study provides the first in vivo evidence of an oxidoreductase, SdbA, that affects multiple phenotypes in a Gram-positive bacterium. SdbA shows low sequence homology to previously identified oxidoreductases, suggesting that it may belong to a different class of enzymes. Our results demonstrate that SdbA is required for disulfide bond formation in S. gordonii and indicate that this enzyme may represent a novel type of oxidoreductase in Gram-positive bacteria.

Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

Unlike other members of coagulase negative staphylococci (CNS), strain warneri has proMCD operon, a homologue of sspABC proteinase operon of S. aureus. The proM and proC encode serine glutamyl endopeptidase and cysteine protease respectively, whereas proD directs homologue of SspC, putative cytoplasmic inhibitor which protects the host bacterium from premature activation of SspB. We determined whole nucleotide sequence of proMCD operon of S. warneri M, succeeded in expression of these genes, and investigated their functions by gene inactivation and complementation experiments. In gelatin zymography of the culture supernatant, a 20-kDa band corresponding to PROC cysteine protease was detected. By Western blotting, PROD was also confirmed in the cytoplasmic protein fraction. PROC and PROD showed significant similarity to SspB and SspC of S. aureus (73% and 58%, respectively). Inactivation mutants of proMCD, proCD and proD genes were established, separately. In the proMCD mutant, degradation/processing of extracellular proteins was drastically reduced, suggesting that PROM was responsible for the cleavage of extracellular proteins. By the proD mutation, the growth profile was not affected, and secretion of PROC was retained. Extracellular protein profiles of the proCD and proD mutants were not so different each other, but autolysin profiles were slightly dissimilar, around 39-48 kDa and 20kDa bands in zymogram. Experiments in buffer systems showed that autolysis was significantly diminished in proMCD mutant, and was promoted by addition of purified PROM. The proC gene was cloned into a multicopy plasmid, and introduced into the proMCD mutant. Compared with the wild type, autolysis of the proC-complemented strain was definitely enhanced by addition of purified PROM. These results suggested that PROM and PROC affected the coccal autolysis, through processing of the autolysin.

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three alpha-helices, one 3(10)-helix, and eight beta-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between beta 4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the beta 4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the beta 4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes., Article, JOURNAL OF BIOLOGICAL CHEMISTRY. 287(53):44736-44748 (2012)

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract.

To combat the limitations of commercializing microalgal-derived biofuels, alternative lipid extraction procedures must be employed. The use of enzymes for cell wall and organelle disruption and lysis are advantageous over traditional extraction techniques such as the energy-intensive process of drying and pressing. The use of autolytic enzymes provides an opportunity for establishing a pretreatment method as part of an integrated aqueous enzymatic lipid extraction process from microalgae. A study was conducted to evaluate the effect of autolysin, an autolytic metalloproteinase, on Chlamydomonas reinhardtii cell lysis and enhanced lipid extraction in comparison to traditional disruption techniques. Increased temperature and controlled mixing were utilized as augmenting factors to improve the pretreatment of microalgae cells. The extent of cell lysis was measured by direct cell count and visualized using electron microscopy. Furthermore, recoverable lipids before and after autolysin treatment were determined by subsequent solvent extraction. Autolysin treatment at room temperature and constant mixing was capable of lysing over 50% of cells which could be increased to over 80% by increasing the incubation temperature. Pretreatment with autolysin significantly increased lipid yield as quantified by solvent extraction. Results indicate that autolysin combined with controlled mixing and incubation temperatures can enhance cell lysis and facilitate improved lipid extraction with subsequent secondary treatments. The pretreatment established represents a novel application of autolysin in catalyzing whole cell lysis beyond previous cell transformation applications of this enzyme.

International audience; In Staphylococcus aureus, peptidoglycan metabolism plays a role in the host inflammatory response and pathogenesis. Transcription of the peptidoglycan hydrolases is activated by the essential 2-component system WalKR at low cell density. During stationary growth phase, WalKR is not active and transcription of the peptidoglycan hydrolase genes is repressed. In this work, we studied regulation of expression of the glycylglycine endopeptidase LytM. We show that, in addition to the transcriptional regulation mediated by WalKR, the synthesis of LytM is negatively controlled by a unique mechanism at the stationary growth phase. We have identified 2 different mRNAs encoding lytM, which vary in the length of their 5' untranslated (5'UTR) regions. LytM is predominantly produced from the WalKR-regulated mRNA transcript carrying a short 5'UTR. The lytM mRNA is also transcribed as part of a polycistronic operon with the upstream SA0264 gene and is constitutively expressed. Although SA0264 protein can be synthesized from the longer operon transcript, lytM cannot be translated because its ribosome-binding site is sequestered into a translationally inactive secondary structure. In addition, the effector of the agr system, RNAIII, can inhibit translation of lytM present on the operon without altering the transcript level but does not have an effect on the translation of the upstream gene. We propose that this dual regulation of lytM expression, at the transcriptional and post-transcriptional levels, contributes to prevent cell wall damage during the stationary phase of growth.

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many--and quite possibly all--bacteria, the peptidoglycan fragments are recovered and recycled. Although cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall-targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall-recycling inhibitors.

Listeria monocytogenes is a foodborne pathogen capable of surviving under challenging conditions both outside and inside the host. During the transition from exponential to stationary phase it experiences a series of environmental changes that require an appropriate response to maintain cell viability. In this study the autolytic behaviour of a L. monocytogenes strain was investigated by two-dimensional electrophoresis. The study was done at the permissive autolysis temperature, 30℃ and at 20℃, an autolysis non-permissive temperature. An autolytic strain proteome was also compared to a non-autolytic strain at the permissive autolysis temperature. The autolytic strain proteome at 30℃ in comparison to 20℃ evidenced increased synthesis of the P60 autolysin, glycolytic enzymes and proteins related with environmental stress responses. The over-production of P45 autolysin, was observed when the autolytic strain proteome was compared with the non-autolytic strain. The proteomes at the non-permissive temperature and the proteome of the non-autolytic strain were characterized by a diminished synthesis of several stress related proteins. The lack of autolysis seems to be associated to the over-production of proteins linked to fatty acid and amino acid synthesis, transcription regulation and cell morphogenesis as evidenced by the proteome at the non-permissive temperature and the non-autolytic strain. Autolysis proteome evidenced the over-production of P60 autolysins, glycolysis and stress proteins whereas the proteome obtained in conditions of absence of autolysis reveal a completely different group of proteins. Possible targets to activate listerial autolysis were identified.

Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division., The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.

A bacterium hydrolysing various organic materials including cellulose, protein, starch and lipid was isolated. The isolate was identified as Bacillus subtilis, and named Bacillus subtilis CK-2 in this paper. This bacterium showed optimal growth at 40~45℃, pH 6~9, and 0~3% of NaCl. B. subtilis CK-2 seemed to synthesis highly active autolysin. The hydrolytic enzymes produced by B. subtilis CK-2 were primary enzymes because extracellular enzyme activities varied similarly to the growth curve. The hydrolytic enzymes seemed to be stable at basic pH conditions. From these results, B. subtilis CK-2 was found to bea useful bacterial agent for composting, or for use in feed-production waste in agriculture, fishery, forest materials, livestock farming, and food.

Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the D-Ala-D-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues exploit noncanonical target sites. In order to address all vancomycin targets in clinically relevant Staphylococcus aureus and Enterococcus faecalis strains we developed a series of small-molecule photoaffinity probes based on vancomycin. Proteomic profiling revealed the specific labeling of two previously unknown vancomycin targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of variants with improved activity.

Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-L-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins.

Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as “nonclassical protein secretion.” Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.

Abh is a transition state regulator of Bacillus subtilis that controls biofilm formation and the production of several diverse antimicrobial compounds. Using a high-throughput non-biased technique, we show for the first time that Abh influences the sensitivity of B. subtilis to diverse antimicrobial compounds. Following up on these findings with a combination of classical genetics and antibiotic susceptibility assays, we demonstrate that Abh influences cellular processes such as the remodelling of the cell wall. We present data demonstrating that the extracytoplasmic function sigma factor σ(X) controls resistance to β-lactam antibiotics by activating abh transcription. Downstream from Abh, activation of slrR expression by Abh is responsible for controlling the sensitivity of B. subtilis to such antibiotics due to the role that SlrR plays in regulating autolysin biosynthesis. The abh mutant additionally exhibits increased resistance to aminoglycoside antimicrobials. We confirm that aminoglycoside killing of B. subtilis is likely to be caused by oxidative damage but rule out the possibility that the increased resistance of the abh mutant to aminoglycosides is due to a general increase in resistance to oxidative stress.

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons., Author summary Staphylococcus aureus is a major human pathogen, and has become a significant worldwide health concern due to the rapid emergence of antibiotic resistant strains. Like most bacteria, S. aureus adapts to its environment by adjusting its genetic expression through sensing and regulatory systems. We show here that the SpdC membrane protein is a novel virulence factor of S. aureus, controlling biofilm formation and pathogenesis. We show that SpdC interacts with the WalK histidine kinase to inhibit activity of the WalKR two-component system. SpdC also interacts with nine other histidine kinases of S. aureus, suggesting it acts as a pleiotropic global regulator.

Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance-surface protein SACOL0050 (pls) and penicillin-binding protein 2' (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.

4 pages, 3 figures, 1 table.-- PMID: 20383019 [PubMed], LytC, one of the major autolysins from the human pathogen Streptococcus pneumoniae, has been crystallized as needles by the hanging-drop technique using 10%(w/v) PEG 3350 as precipitant and 10 mM HEPES pH 7.5. LytC crystals were quickly soaked in mother liquor containing 2 mM of the complex Gd-HPDO3A to produce derivatized crystals (LytC(Gd-HPDO3A)). Both native LytC and isomorphous LytC(Gd-HPDO3A) crystals were flash-cooled in a nitrogen flow at 120 K prior to X-ray data collection using an in-house Enraf-Nonius rotating-anode generator (lambda = 1.5418 A) and a MAR345 imaging-plate detector. In both cases, good-quality diffraction patterns were obtained at high resolution. LytC(Gd-HPDO3A) crystals allowed the collection of a SAD X-ray data set to 2.6 A resolution indexed in terms of a P2(1) monoclinic unit cell with parameters a = 59.37, b = 67.16, c = 78.85 A, beta = 105.69 degrees . The anomalous Patterson map allowed the identification of one heavy-atom binding site, which was sufficient for the calculation of an interpretable anomalous map at 2.6 A resolution., This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711, SAF2006-00390 and the Factoría de Cristalización from the CONSOLIDER-INGENIO 2010 program), EU-CP223111 (CAREPNEUMO, European Union). CIBER de Enfermedades Respiratorias is an initiative of Spanish Instituto de Salud Carlos III. IP-D and SR were granted I3P-CSIC and FPI fellowships, respectively.

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (alpha/beta)(5)(beta)(3)-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4A resolution. We show that the active center is extremely similar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this 'super-family' of enzymes.