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The peptidoglycan cell wall is a predominant defining structure of bacteria, determining cell shape and supporting survival in diverse conditions. As a single, macromolecular sacculus enveloping the bacterial cell during growth and division, peptidoglycan is necessarily a dynamic structure that requires highly regulated synthesis of new material, remodeling, and turnover, or autolysis, of old material. Despite ubiquitous clinical exploitation of peptidoglycan synthesis as an antibiotic target, much remains unknown about how bacteria modulate synthetic and autolytic processes. Here, we couple bacterial genetics in Vibrio cholerae with compositional analysis of soluble pools of peptidoglycan turnover products to uncover a critical role for a widely misunderstood class of autolytic enzymes, the lytic transglycosylases (LTGs). We demonstrate that LTG activity is specifically required for vegetative growth. The vast majority of LTGs, however, are dispensable for growth, and defects that are ultimately lethal accumulate due to generally inadequate LTG activity, rather than the absence of specific individual enzymes. Consistent with this, we found that a heterologously expressed E. coli LTG, MltE, is capable of sustaining V. cholerae growth in the absence of endogenous LTGs. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains accumulate in the WT, and, to a higher degree, in LTG mutants, and that LTG mutants are hyper-susceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that a key function of LTGs is to prevent toxic crowding of the periplasm with synthesis-derived PG fragments. Contrary to prevailing models, our data further suggest that this process can be temporally separate from peptidoglycan synthesis.

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.

The goal of this review is to address the complicated nature of autolysins found among oral bacteria and the roles they play within the oral biofilm. The various functions of autolysins are discussed, as well as the regulation of autolysins and overall contribution to biofilms and the oral environment. With their many functions, bacterial autolysins can be understood as contributing to both cell physiology and population dynamics within the oral environment. Cell division, peptidoglycan turnover/remodeling, fratricide, and programmed cell death are all influenced, if not controlled, by autolysins. The release of nutrients and DNA as functional byproducts of lysins can alter the biofilm by affecting the growth and overall genetic makeup of the diverse bacteria. Autolysins and their various functions contribute to biofilm formation, horizontal gene transfer, and the overall health of the oral cavity.

The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This "anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.

Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40&ndash, 45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

Neisseria gonorrhoeae (Gc, gonococcus) is a Gram-negative diplococcus and obligate human pathogen. Gc is the causative agent gonorrhea, a major global health problem. Gc colonization of mucosal surfaces leads to the abundant recruitment of neutrophils. Both the mucosa and neutrophils produce various defenses to neutralize microbes, yet Gc can survive in the presence of these defenses. One antimicrobial protein common to both the mucosa and neutrophils is lysozyme. In addition to targeted hydrolysis of bacterial cell wall peptidoglycan (PG), lysozyme can also kill bacteria through ionic interactions leading to membrane instability. Besides its antimicrobial activities, lysozyme hydrolysis and degradation of PG facilitates the release of immunomodulatory PG. Thus, lysozyme resistance in bacteria serves to limit both the antimicrobial and immunomodulatory activities of lysozyme. However, it is unclear how Gc resists lysozyme or how this resistance contributes to virulence. Our first observation for how Gc resists killing by lysozyme came from a serendipitous study investigating how two PG turnover proteins in Gc affect bacterial survival in the presence of neutrophils. During periods of cell wall remodeling, PG must be released from the intact cell wall in a process termed PG turnover. The lytic transglycosylases LtgA and LtgD are part of the PG turnover machinery in Gc, and both of these enzymes’ activities result in the extracellular release of immunomodulatory PG monomers. We found that ΔltgAΔltgD mutant Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type. Addition of the immunomodulatory PG monomers that are released by LtgA and LtgD failed to alter ΔltgAΔltgD mutant survival in the presence of neutrophils, pointing to a mechanism other than PG monomer release for LtgA- and LtgD-mediated survival. We found two reasons to explain decreased survival of the ΔltgAΔltgD mutant. First, ΔltgAΔltgD mutant Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD mutant Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude from this study that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. Notably, these observations are the first to implicate a possible link between bacterial degradation by lysozyme to increased neutrophil activation. Because some Gram-negative bacteria produce protein inhibitors that bind to and occlude the active site of lysozyme, we next investigated the possibility of Gc protein inhibitors of lysozyme. Here, we identified Ng_1063 as a new inhibitor of lysozyme in Gc, and defined its functions in light of a second, recently identified inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme’s active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea. Lysozyme-sensitive Gram-positive bacteria can activate macrophages, dependent on lysozyme and the PG-sensor NOD2. We hypothesize that the lysozyme-sensitive ΔltgAΔltgD mutant Gc may be similarly activating neutrophils. However, it is unknown how lysozyme or NOD2 functions to sense PG in neutrophils. Therefore, we began by testing what types of PG are immunomodulatory in neutrophils. Neutrophils exhibit increased markers of activation when exposed to insoluble, polymeric PG compared with soluble, monomeric PG. Activation by insoluble, polymeric PG was dependent on phagocytosis. Moreover, we found that pre-treatment of polymeric PG with lysozyme, which degrades PG into monomers, ablated neutrophil activation. We propose that soluble, monomeric PG has limited access to the neutrophil cytosol where NOD2 resides, while insoluble, polymeric PG, as occurs in an intact bacterium, is phagocytosed and processed intracellularly by lysozyme, preceding NOD2 activation. Future studies are aimed at testing the requirement for lysozyme and NOD2 in neutrophil responses to PG or lysozyme-sensitive Gc.

Tuberculosis is the leading killer among infectious diseases worldwide. Increasing multidrug resistance has prompted new approaches for tuberculosis drug development, including targeted inhibition of virulence determinants and of signaling cascades that control many downstream pathways. We used a multisystem approach to determine the effects of a potent small-molecule inhibitor of the essential Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB. We observed differential levels of phosphorylation of many proteins and extensive changes in levels of gene expression, protein abundance, cell wall lipids, and intracellular metabolites. The patterns of these changes indicate regulation by PknA and PknB of several pathways required for cell growth, including ATP synthesis, DNA synthesis, and translation. These data also highlight effects on pathways for remodeling of the mycobacterial cell envelope via control of peptidoglycan turnover, lipid content, a SigE-mediated envelope stress response, transmembrane transport systems, and protein secretion systems. Integrated analysis of phosphoproteins, transcripts, proteins, and lipids identified an unexpected pathway whereby threonine phosphorylation of the essential response regulator MtrA decreases its DNA binding activity. Inhibition of this phosphorylation is linked to decreased expression of genes for peptidoglycan turnover, and of genes for mycolyl transferases, with concomitant changes in mycolates and glycolipids in the cell envelope. These findings reveal novel roles for PknA and PknB in regulating multiple essential cell functions and confirm that these kinases are potentially valuable targets for new antituberculosis drugs. In addition, the data from these linked multisystems provide a valuable resource for future targeted investigations into the pathways regulated by these kinases in the M. tuberculosis cell., IMPORTANCE Tuberculosis is the leading killer among infectious diseases worldwide. Increasing drug resistance threatens efforts to control this epidemic; thus, new antitubercular drugs are urgently needed. We performed an integrated, multisystem analysis of Mycobacterium tuberculosis responses to inhibition of its two essential serine/threonine protein kinases. These kinases allow the bacterium to adapt to its environment by phosphorylating cellular proteins in response to extracellular signals. We identified differentially phosphorylated proteins, downstream changes in levels of specific mRNA and protein abundance, and alterations in the metabolite and lipid content of the cell. These results include changes previously linked to growth arrest and also reveal new roles for these kinases in regulating essential processes, including growth, stress responses, transport of proteins and other molecules, and the structure of the mycobacterial cell envelope. Our multisystem data identify PknA and PknB as promising targets for drug development and provide a valuable resource for future investigation of their functions.

Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.

Staphylococcus lugdunensis is a human skin commensal organism, but it is considered as a virulent Staphylococcus species. In a previous study, we described the first S. lugdunensis autolysin, AtlL. This enzyme displays two enzymatic domains and generates two peptidoglycan hydrolases, an N-acetylmuramoyl-l-alanine amidase and an N-acetylglucosaminidase. In this study, to further investigate the functions of this autolysin, a ΔatlL mutant was constructed. The microscopic examination of the mutant showed cell aggregates and revealed a rough outer cell surface demonstrating, respectively, the roles of AtlL in cell separation and peptidoglycan turnover. This ΔatlL mutant exhibited a lower susceptibility to Triton X-100-induced autolysis assays and appears to be more resistant to cell wall antibiotic-induced lysis and death compared with its parental strain. The atlL mutation affected the biofilm formation capacity of S. lugdunensis. Furthermore, the ΔatlL mutant showed trends toward reduced virulence using the Caenorhabditis elegans model. Overall, AtlL appears as a major cell wall autolysin of S. lugdunensis implicated in cell separation, in stress-induced autolysis and in bacterial pathogenesis.

Bacterial resistance to antibiotics is increasing at an alarming rate and many commonly used antibiotics are no longer effective. Thus, there is considerable interest in investigating novel antibacterial compounds, such as the plant-derived pentacyclic triterpenoids, including oleanolic acid (OA), ursolic acid (UA) and their derivatives. These compounds can be isolated from many medicinal and crop plants and their antibacterial, antiviral, antiulcer and anti-inflammatory effects are well documented. OA and UA are active against many bacterial species, particularly Gram-positive species, including mycobacteria. They inhibit bacterial growth and survival, and the spectrum of minimal inhibitory concentration (MIC) values is very broad. In addition, OA, UA and their derivatives display potent antimutagenic activity. Studies to identify the cellular targets and molecular mechanisms of OA and UA action were initiated a few years ago and it has already been demonstrated that both acids influence bacterial gene expression, the formation and maintenance of biofilms, cell autolysis and peptidoglycan turnover. Before these compounds can be used clinically as antimicrobial agents, further extensive studies are required to determine their cytotoxicity and the optimum mode of their application.

Isolation of bacterial mutants hypersusceptible to antibiotics can reveal novel targets for antibiotic potentiators. However, identification of such mutants is a difficult task which normally requires laborious replica plating of thousands of colonies. The technique proposed here allows for the positive selection of genetic knockout mutants leading to hypersusceptibility. This technique, designated SDR (selection for DNA release), involves introduction of random insertions of a marker gene into the chromosome of a highly transformable bacterial species, followed by treatment of the obtained library with an antibiotic at subinhibitory concentrations. DNA released by lysing bacteria is collected and used to transform fresh bacteria, selecting for insertion of the marker gene. These selection cycles are repeated until variants with a hypersusceptibility phenotype caused by insertion of the marker begin to dominate in the library. This approach allowed for isolation of a number of mutants of the gram-negative opportunistic pathogen Acinetobacter sp. susceptible to 4- to 16-times-lower concentrations of ampicillin than wild-type bacteria. The mutations affected proteins involved in peptidoglycan turnover and, surprisingly, proteins involved in exopolysaccharide production. A further modification of the SDR technique is described which allows for selecting mutants hypersensitive to agents that affect bacterial physiology but do not cause cell lysis, e.g., inhibitors of translation. This application of SDR is illustrated here by identification of several mutants of Acinetobacter sp. with increased susceptibility (two- to fivefold decrease in the MIC) to erythromycin. The same technique can be used to identify prospective targets for potentiators of many other antibacterial agents.

Bacterial cell wall hydrolases are essential for peptidoglycan turnover and crucial to preserve cell shape. The d,d-carboxypeptidase DacA and l,d-carboxypeptidase DacB of Streptococcus pneumoniae function in a sequential manner. Here, we determined the structure of the surface-exposed lipoprotein DacB. The crystal structure of DacB, radically different to that of DacA, contains a mononuclear Zn(2+) catalytic centre located in the middle of a large and fully exposed groove. Two different conformations were found presenting a different arrangement of the active site topology. The critical residues for catalysis and substrate specificity were identified. Loss-of-function of DacA and DacB altered the cell shape and this was consistent with a modified peptidoglycan peptide composition in dac mutants. Contrary, an lgt mutant lacking lipoprotein diacylglyceryl transferase activity required for proper lipoprotein maturation retained l,d-carboxypeptidase activity and showed an intact murein sacculus. In addition we demonstrated pathophysiological effects of disabled DacA or DacB activities. Real-time bioimaging of intranasal infected mice indicated a substantial attenuation of ΔdacB and ΔdacAΔdacB pneumococci, while ΔdacA had no significant effect. In addition, uptake of these mutants by professional phagocytes was enhanced, while the adherence to lung epithelial cells was decreased. Thus, structural and functional studies suggest DacA and DacB as optimal drug targets.

Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

The cell wall is the essential exoskeleton of most bacteria that maintains cell shape and protects the cell against osmotic lysis. The major component of the bacterial cell wall is the peptidoglycan, a heteropolymer consisting of glycan strands cross-linked by short peptides. Despite its rigidity, the peptidoglycan is a highly dynamic structure, which is continuously undergoing turnover and remodeling during cell growth and division. In Gram-negative bacteria like Escherichia coli, peptidoglycan fragments released during this turnover process are reutilized (recycled) efficiently. However, it is still unknown whether recycling also proceeds in Gram-positive bacteria. Recently, a pathway for the recovery of peptidoglycan fragments, which involves the sequential processing of N-acetylglucosamine-N-acetylmuramic acid-peptides (muropeptides) by the secreted enzymes N-acetylglucosaminidase (NagZBs) and N-acetylmuramic acid-L-alanine amidase (AmiEBs), has been proposed for the Gram-positive bacterium Bacillus subtilis. Orthologs of muropeptide recycling enzymes of B. subtilis are also present in Clostridium acetobutylicum, an anerobic Gram-positive solvent producer that is closely related to Bacillus. We recognized that orthologs of NagZ (CA_C0182) and AmiE (CA_C0181) of C. acetobutylicum are apparently not secreted as in B. subtilis, indicating that catabolism of muropeptides may proceed different.This thesis presented here investigates the peptidoglycan recycling pathway of C. acetobutylicum and focuses on two enzymes showing a novel specificity for the amino sugars of the bacterial peptidoglycan, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). We identified a kinase of C. acetobutylicum, named MurK (formerly CA_C0183), which is encoded by a gene that is clustered with putative recycling genes. MurK catalyzes the phosphorylation of both GlcNAc and MurNAc at the 6-position, yielding GlcNAc-6-phosphate and MurNAc-6-phosphate, respectively. The biochemical characterization of MurK revealed a 1.5-fold higher catalytic activity for GlcNAc than for MurNAc. Applying phosphorylation assays using radioactive ATP indicated that structurally related cell wall sugars, i.e., epimers (N-acetylgalactosamine and N-acetylmannosamine), non-N-acetylated derivatives (glucosamine and muramic acid) and an anhydro form of MurNAc (1,6-anhydroMurNAc) are no substrates for this enzyme. MurK displays no amino acid sequence similarity with the amino sugar kinases AnmK and NagK of E. coli but with human GlcNAc kinase. We showed that MurK kinase can be used for a sensitive detection of the cell wall sugars at amounts as low as 5 fmol. Within the same gene cluster downstream of murK on the genome of C. acetobutylicum, we identified a second encoded enzyme, a novel acetyltransferase, named GlmA (formerly CA_C0184). It catalyzes the formation of GlcNAc by the transfer of an acetyl group from acetyl coenzyme A to glucosamine (GlcN) and terminal nonreducing GlcN residues, respectively. We determined a 3- to 4-fold higher catalytic efficiency of GlmA for linear di- and trisaccharides composed of β-1,4-glycosidically linked GlcN residues, compared to GlcN. However, the lactic acid derivative of GlcN, muramic acid, did not serve as an acetyl acceptor of GlmA. Acetylation assays with muropeptides derived from peptidoglycan of B. subtilis revealed that non-N-acetylated GlcN residues had to be N-acetylated by GlmA prior to the release of terminal GlcNAc by the N-acetylglucosaminidase NagZBs, whereas peptidoglycan of E. coli lacking these N-deacetylated modifications could not be acetylated by GlmA.In conclusion, the results presented in this thesis indicate that MurK, the GlcNAc/MurNAc kinase, and GlmA, the glucosamine/glucosaminide N-acetyltransferase, both are involved in a putative recycling pathway in C. acetobutylicum, which proceeds distinct to the known pathways of B. subtilis and E. coli. Our model of the C. acetobutylicum peptidoglycan recycling proposes that de-N-acetylated muropeptides liberated during cell wall turnover are transported into the cell by the putative ABC transporter AppABCDF. In the cytoplasm, recovery of GlcN residues requires N-acetylation at the nonreducing end by GlmA. The generated terminal GlcNAc can then be released from muropeptides by NagZ and MurNAc from the peptide moiety by AmiE. Both amino sugars can now be used for phosphorylation by the MurK kinase, yielding GlcNAc- and MurNac-6-phosphate, respectively. Further processing involves the conversion of MurNAc-6-phosphate to GlcNAc-6-phosphate by the MurQ etherase, which enters, after deacetylation, either glycolysis or the peptidoglycan biosynthesis pathway.

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

The mechanism of action of sceptrin, an antimicrobial agent isolated from the sponge Agelas mauritiana, was investigated. Sceptrin has been reported to exhibit antibacterial and antifungal activities. In our studies, sceptrin demonstrated a bacteriostatic rather than bactericidal effect on exponentially growing Escherichia coli cells at the MIC. Under these conditions, the culture produced chains of cells, and incorporation of radio-labelled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3H-uridine into RNA was slightly inhibited. At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabelled precursors, and induced the formation of unusual spheroplasts. Peptidoglycan turnover in E. coli appeared to be stimulated by sceptrin as demonstrated by a release of diaminopimelic acid-containing high molecular weight material. Subsequent studies of the release of potassium ions from E. coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells. It is proposed that spheroplasts formation may reflect a cell wall effect that occurs subsequent to membrane damage.

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.

Sporulation in low-G+C gram-positive bacteria (Firmicutes) is an important survival mechanism that involves up to 150 genes, acting in a highly regulated manner. Many sporulation genes have close homologs in non-sporulating bacteria, including cyanobacteria, proteobacteria and spirochaetes, indicating that their products play a wider biological role. Most of them have been characterized as regulatory proteins or enzymes of peptidoglycan turnover; functions of others remain unknown but they are likely to have a general role in cell division and/or development. We have compiled a list of such widely conserved sporulation and germination proteins with poorly characterized functions, ranked them by the width of their phylogenetic distribution, and performed detailed sequence analysis and, where possible, structural modeling aimed at estimating their potential functions. Here we report the results of sequence analysis of Bacillus subtilis spore germination protein GerM, suggesting that it is a widespread cell development protein, whose function might involve binding to peptidoglycan. GerM consists of two tandem copies of a new domain (designated the GERMN domain) that forms phylum-specific fusions with two other newly described domains, GERMN-associated domains 1 and 2 (GMAD1 and GMAD2). Fold recognition reveals a β-propeller fold for GMAD1, while ab initio modeling suggests that GMAD2 adopts a fibronectin type III fold. SpoVS is predicted to adopt the AlbA archaeal chromatin protein fold, which suggests that it is a DNA-binding protein, most likely a novel transcriptional regulator. Contact: drigden@liverpool.ac.uk Supplementary information: Supplementary data are available at ftp://ftp.ncbi.nih.gov/pub/galperin/Sporulation.html

Background The metabolism of bacterial peptidoglycan is a dynamic process, synthases and cleavage enzymes are functionally coordinated. Lytic Transglycosylase enzymes (LT) are part of multienzyme complexes which regulate bacterial division and elongation. LTs are also involved in peptidoglycan turnover and in macromolecular transport systems. Despite their central importance, no LTs have been identified in the human pathogen Streptococcus pneumoniae. We report the identification of the first putative LT enzyme in S. pneumoniae and discuss its role in pneumococcal peptidoglycan metabolism. Results Homology searches of the pneumococcal genome allowed the identification of a new domain putatively involved in peptidoglycan cleavage (PECACE, PEptidoglycan CArbohydrate Cleavage Enzyme). This sequence has been found exclusively in Gram-positive bacteria and gene clusters containing pecace are conserved among Streptococcal species. The PECACE domain is, in some instances, found in association with other domains known to catalyze peptidoglycan hydrolysis. Conclusions A new domain, PECACE, putatively involved in peptidoglycan hydrolysis has been identified in S. pneumoniae. The probable enzymatic activity deduced from the detailed analysis of the amino acid sequence suggests that the PECACE domain may proceed through a LT-type or goose lyzosyme-type cleavage mechanism. The PECACE function may differ largely from the other hydrolases already identified in the pneumococcus: LytA, LytB, LytC, CBPD and PcsB. The multimodular architecture of proteins containing the PECACE domain is another example of the many activities harbored by peptidoglycan hydrolases, which is probably required for the regulation of peptidoglycan metabolism. The release of new bacterial genomes sequences will probably add new members to the five groups identified so far in this work, and new groups could also emerge. Conversely, the functional characterization of the unknown domains mentioned in this work can now become easier, since bacterial peptidoglycan is proposed to be the substrate.

The immunodominant antigen A, IsaA, of Staphylococcus aureus was found to include a putative soluble lytic transglycosylase domain in its C-terminal region. Since the presence of this distinctive domain suggested that the protein might participate in peptidoglycan turnover, as indicated in Gram-negative bacteria, its cellular location was investigated. The protein was found not only in the culture supernatant but also in the cell wall fraction. To estimate its physiological role for the bacterium, its cell surface distribution was studied by immunoelectron microscopy. Protein A-gold particles binding to the immune complex were mainly located on the septal region of the bacterial cell surface. These data suggested that IsaA might be involved in bacterial cell separation through a preferential interaction with peptidoglycan chain.

Neisseria gonorrhoeae releases soluble fragments of peptidoglycan during growth. These molecules are implicated in the pathogenesis of various forms of gonococcal infection. A major peptidoglycan fragment released by gonococci is identical to the tracheal cytotoxin of Bordetella pertussis and has been shown to kill ciliated fallopian tube cells in organ culture. Previous studies indicated that a unique lytic peptidoglycan transglycosylase (AtlA) was responsible for some, but not all, of the peptidoglycan-derived cytotoxin (PGCT) production in certain gonococcal strains. To examine the role of other putative lytic transglycosylases in PGCT production, we made a deletion mutation in a gonococcal gene exhibiting similarity with genes encoding lytic transglycosylases from other bacterial species. The gonococcal mutant was viable and grew normally, but it was less autolytic than the wild-type strain in stationary-phase culture and under nongrowth conditions. The gonococcal mutant was reduced in peptidoglycan turnover, and the profile of the released products showed a reduction in monomeric peptidoglycan. Proportionally more multimeric fragments were released. These results suggest that this gonococcal gene ( ltgA ) encodes a lytic peptidoglycan transglycosylase and that it is responsible for a significant proportion of the PGCT released by N. gonorrhoeae .

Recently, Staphylococcus aureus strains with intermediate resistance to vancomycin, the antibiotic of last resort, have been described. Multiple changes in peptidoglycan turnover and structure contribute to the resistance phenotype. Here, we describe that structural changes of teichoic acids in the cell envelope have a considerable influence on the susceptibility to vancomycin and other glycopeptides. S. aureus cells lacking d -alanine esters in teichoic acids exhibited an at least threefold-increased sensitivity to glycopeptide antibiotics. Furthermore, the autolytic activity of the d -alanine mutant was reduced compared to the wild-type, and the mutant was more susceptible to the staphylolytic enzyme lysostaphin. Vancomycin inhibited autolysis at very high concentrations but neither in the wild-type nor in the mutant was the autolytic activity influenced in the range of the MIC. Mutant cells had a considerably higher capacity to bind vancomycin.

The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using Escherichia coli and Pseudomonas aeruginosa as a baseline for gramnegative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network’s properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium. Bearing in mind that a thorough knowledge of structure can lead to an understanding of function, it is important to elucidate the detailed molecular structures of bacterial surfaces. There have been few recent studies of the physical properties of murein sacculi (12, 18, 20), and yet this cell wall component is of paramount importance to the integrity of the cell. It must have high tensile strength and at the same time be highly permeable to nutrients, waste products, and secreted proteins. Even large macromolecules such as DNA (in genetic exchange, such as transformation) and S-layer proteins can pass through (1, 17). The murein of a gram-negative bacterium is composed of one to three layers of peptidoglycan (12, 19) and is bonded via lipoprotein moieties to the outer membrane; these can be either covalently or electrostatically attached to the peptidoglycan (1). Our recent work on the elasticity and thickness of Escherichia coli and Pseudomonas aeruginosa murein sacculi has helped define several physical properties of this intriguing cell wall fabric (20), but the work did not elucidate the network’s porosity. Since 35 to 50% of the peptide stems of peptidoglycan are cross-linked at a given time in the cell’s growth cycle (5, 7), this bonding between neighboring glycan strands should be important not only for the fabric’s ability to withstand physical strain but also for its porosity. Indeed, it could even be possible for this limited cross-linking in combination with the finite length of each glycan strand to form peptidoglycan arrangements such that holes of various kinds occur within the network. Because of the dynamic nature of cell growth and peptidoglycan turnover, these holes could be transient and difficult to find, especially since they would be at nanoscale dimensions. Yet, the question of holes in these networks is of fundamental importance, and here we resort to computer modeling to predict their existence and distribution. A knowledge of the shape and size distributions of such holes, as well as the variances in these distributions, can be relevant for understanding the passage of molecules through the peptidoglycan layer, the elasticity of the layer, and, possibly, aspects of the mechanics of cell division. In this note, we show the expected distribution of holes deduced from the distribution of glycan strand lengths (5, 7, 9) and discuss their possible consequences for the viability of the bacterium. Peptidoglycan chemical structure and initial discussion of holes in a monomolecular thick peptidoglycan network. There can be two types of cross-linkages occurring between adjacent glycan strands in a peptidoglycan network: cross-links that oc

Staphylococcus aureus is a major pathogen in human infectious diseases. It appears to differ considerably from the coagulase-negative staphylococci (CoNS), such as Staphylococcus epidermidis and Staphylococcus saprophyticus, with respect to its pathogenicity in that it elaborates a large number of extracellular virulence factors and other proteins, while the CoNS, which are usually more indolent pathogens, generally produce few if any of these factors. CoNS cause infections associated with implanted foreign bodies such as intravascular catheters. Previous reports of adherence by staphylococci have concentrated primarily on the CoNS (8, 24, 33). However, an essential step in the establishment of any staphylococcal infection is attachment to the host tissues themselves or to foreign bodies within host tissues. Environmental stimuli have been shown to play an important role in expression of virulence genes during pathogenesis. Because S. aureus has the ability to invade different tissues, it responds to environmental stimuli in order to survive and express virulence genes during the infection. To survive, the cells must monitor external conditions and adjust their structure, physiology, and behavior accordingly. Thus, bacteria have developed sophisticated signaling systems for eliciting adaptive responses to their environment. The molecular mechanisms responsible for stimulus-response coupling often involve two types of components encoded by an operon, a transmembrane sensor (histidine protein kinase) and its associated response regulator (50). In general, the transmembrane protein binds a specific ligand, the signal, and binding triggers autophosphorylation at a conserved histidine residue. The phosphorylated sensor then transfers the phosphate to an aspartic residue in the response regulator (39). The response regulator can in turn enhance or repress transcription of target genes, as occurs with PhoB in the phosphate regulon (28) and OmpR in the porin regulon (27, 55) of Escherichia coli. A two-component regulatory system, AgrC-AgrA, involved in the production of virulence factors was previously described for S. aureus (21, 37, 43). This locus has also been shown to affect autolysin expression (16). Peptidoglycan hydrolases, enzymes that hydrolyze either the glycan or the peptide moieties of peptidoglycan, participate in a number of important biological processes occurring during cell growth and division, including cell wall synthesis, daughter cell separation, and peptidoglycan turnover and recycling (1). As these hydrolases are also involved in lysis of bacteria, some have been called autolysins. Thus, the same two-component regulatory system can be involved in pathogenesis as well as in cellular physiology. In order to find loci involved in the regulation of the multidrug efflux pump NorA of S. aureus, we used a library of Tn917LTV1 insertions in the chromosome of strain MT23142 with selection for higher and lower levels of resistance to tetraphenylphosphonium bromide (TPP), a substrate of NorA, than are found in MT23142. Overproduction of norA leads to resistance to drugs such as quinolones, ethidium bromide, and TPP (14, 35). MT23142 carries the flqB mutation. flqB, a cis-acting mutant of norA, is localized downstream of the initiation site of norA and overexpresses norA (35). One transposition mutant, BF15, obtained from the library showed a slight increase of resistance to TPP and modifies norA expression (14). In addition to effects on norA expression, the mutant unexpectedly showed alterations in autolysis. In order to characterize this mutant, we analyzed the sequence of chromosomal DNA flanking the insertional site of Tn917LTV1. We found a new two-component regulatory system, ArlS-ArlR. Insertion of Tn917 in the arlS gene resulted in changes in adhesive properties, the rate of autolysis, and extracellular proteolytic activity. All these properties were complemented by the cloned arlR and arlS genes, indicating that the disruption of arlS was responsible for the mutant phenotype.

It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16 h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation., Author Summary Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillin. Among these bacteria some of them possess a beta-lactamase that is only produced at a high level when a beta-lactam antibiotic is present outside the cell. This mechanism of regulation, named beta-lactamase induction, has been described both in Gram-negative and Gram-positive bacteria. In Staphylococcus aureus and Bacillus licheniformis, two Gram-positive bacteria, their beta-lactamase is induced by homologous proteins including a membrane-bound penicillin receptor, BlaR1, and a cytoplasmic DNA-binding protein, BlaI, acting as repressor. The first step in the induction mechanism is the acylation of the extracytoplasmic domain of BlaR receptor by the beta-lactam antibiotic concomitant with the activation of the peptidase activity of its cytoplasmic domain. The activated receptor launched a cytoplasmic signal leading to the inactivation of BlaI repressor. The nature of this cytoplasmic signal is not well understood. In our study, we identified that a dipeptide issued from the bacterial cell wall is able to inactivate the BlaI repressor. It is the first time that a cell wall fragment is shown to reenter in the cytoplasm of Gram-positive bacteria to regulate gene expression.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.

The composition of the peptidoglycan of Bordetella pertussis and the nature of its turnover products was determined by a new combination of analytical techniques: high performance liquid chromatography of an enzymatic peptidoglycan hydrolysate and fast atom bombardment mass spectrometry and fast atom bombardment collision-activated dissociation tandem mass spectrometry. Sixteen major components of the peptidoglycan were purified, and assignment of complete or partial chemical structures was achieved for nine and seven species, respectively. At this level of resolution, a previously unrecognized heterogeneity of monomeric (five new species; nine total) and dimeric species (five new species; five total) was detected. No species containing diaminopimelyl-diaminopimelic acid cross-links or lysyl-arginine substitutions were found. Previous estimates of total cross-linkage and average chain length were revised downward to 32% and 21 disaccharide residues, respectively. Detection of a chemically novel species, a disaccharide octapeptide monomer, in both the peptidoglycan hydrolysate and culture supernatant fluid, suggests that an N-acetyl-muramyl-L-alanine amidase acts on the intact peptidoglycan of Bordetella and participates in cell wall turnover. Five peptidoglycan turnover products were identified in the supernatant fluid of late logarithmic phase cultures, including the 1,6-anhydro monomeric species known as tracheal cytotoxin. Peptidoglycan turnover was detected at a low rate of approximately 10%/generation, a value sufficient to account for the generation of all tracheal cytotoxin found in culture supernatant fluids.

The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases.

The study of diaminopimelic acid (DAP) incorporation and turnover during growth recovery in chloramphenicol-treated (CMP-treated) Bacillus megaterium cells showed that two kinds of turnover occurred. A low acid-soluble turnover appeared as soon as growth resumed in bacteria labeled before the CMP treatment and at the middle of the first generation in those labeled during the treatment. The acid-insoluble turnover appeared only at the beginning of the second generation of growth resumption in bacteria labeled before CMP addition and at the beginning of the third generation in those labeled during the CMP treatment. The acid-soluble release observed during the period of cell wall thinning is too low to account for the decrease of the wall thickness and the acid-insoluble loss appears after this period.When bacteria were transferred into partially spent medium instead of fresh culture medium the acid-insoluble release started to appear half a generation sooner. Electron microscopic observations showed that in this case, large scales detached from the cell wall. This activity of wall degradation was not observed when the partially spent medium was previously heated for 10 min at 100 °C.The persistence of a thick wall on cell ends during the first generation does not reflect an absence of growth sites because their labeling on autoradiographs is high. Rather, it seems to be due to a low lytic activity at the poles.

Bacillus subtilis, Neisseria subflava , and LYT coccus were found to undergo massive lysis after growth in media containing 0.01 to 10 mM NaF. When cells of these organisms were transferred from late-exponential-phase cultures to 0.02 M sodium phosphate buffer plus 0.1 M KCl, they underwent spontaneous autolysis. Cells grown in media with fluoride were more liable to autolysis, and walls isolated from them also showed enhanced autolytic sensitivity, even though added fluoride did not directly stimulate autolysins. Sporadic or partial lysis occurred in populations of Streptococcus sanguis and Streptococcus mutans BHT or LM-7 after growth in fluoridated media. Most bacteria that were tested did not undergo fluoride-induced lysis. However, cells of all test bacteria were found to have reduced amounts of peptidoglycan per unit of cell weight when grown in the presence of fluoride. Incorporation of labeled lysine or glucosamine into peptidoglycan (Park-Hancock residue) was stimulated, instead of inhibited, by fluoride. However, fluoride also stimulated the loss of radioactivity from Park-Hancock residues of cells that had previously incorporated labeled lysine or glucosamine. Thus, fluoride appeared to enhance peptidoglycan turnover, and this turnover reduced the peptidoglycan contents of all bacteria tested, but induced lysis in only those bacteria that normally have highly active autolytic systems.

Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium.

The cell wall peptidoglycan of Lactobacillus acidophilus strain 63 AM Gasser has been shown to turn over rapidly during balanced exponential growth and recovery from amino acid deprivation. In contrast, turnover of either pulse-labeled or extensively labeled peptidoglycan of Streptococcus faecalis ATCC 9790 was not detected by the same method and experimental conditions. In S. faecalis, peptidoglycan turnover could not be induced either by brief exposure of the cells to a low concentration of lysozyme or by growth in the presence of a low concentration of penicillin. The peptidoglycan turnover rate of rapidly growing cultures of L. acidophilus was equivalent to a loss of about one third of their peptidoglycan per generation. This was not due to cellular lysis since turnover of protein was below detectable levels. Comparably rapid peptidoglycan turnover rates per generation were observed in slowly growing cultures (30% lost per generation) and in a mutant which autolyzed at about one fifth the rate of the wild type (25% lost per generation). In cultures labeled for 6 or more generations, turnover was preceded by a lag equivalent to 0.8 to 2 generation times. Turnover of pulses of less than 0.2 generations was not observed for periods exceeding 2 generations. These last two results cannot be interpreted as indicating that newly made wall is immune to turnover since in both cases nonsynchronized cultures were used, and the short pulses would eventually age as the culture grew. The addition of chloramphenicol or deprivation of valine completely prevented peptidoglycan turnover. The absence of detectable peptidoglycan turnover in S. faecalis (and other species) suggests that turnover is not an essential feature of cell wall growth. On the other hand, in L. acidophilus, peptidoglycan turnover appears to be closely associated with that portion of wall synthesis related to wall (and surface) enlargement and not to the portion related to wall thickening.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.

Bdellovibrio peptidoglycan is of typical gram-negative composition. The molar ratios of alanine:glutamic acid:diaminopimelic acid:muramic acid:glucosamine were about 2:1:1:1:1. Nascent, nongrowing Bdellovibrio bacteriovorus 109J were converted from highly motile vibrios to highly motile spheres when shaken in dilute buffer plus penicillin, cephalothin, bacitracin, or D-cycloserine. The spherical forms contained essentially no sedimentable peptidoglycan; i.e., they were spheroplasts. Spheroplasts induced by penicillin, D-cycloserine, and lysozyme were stable in dilute buffer and did not lyse when subjected to osmotic shock. Normal Bdellovibrio suspended in buffer turned over their peptidoglycan at a rate of approximately 30% h during the initial 120 min of starvation. Chloramphenicol and sodium azide strongly inhibited Bdellovibrio peptidoglycan turnover and the induction of spheroplasts by penicillin. The data indicate that nongrowing B. bacteriovorus are sensitive to penicillin and other antibiotics affecting cell walls because of their high rate of peptidoglycan turnover. It is also concluded that an intact peptidoglycan layer is required for maintaining cell shape, but is not required for osmotic stability of B. bacteriovorus.

Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation. The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures. Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B. subtilis. Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures. In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls. This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.

Background The efficacy of antibiotics against bacterial infections is decreasing due to the development of resistance in bacteria, and thus, there is a need to search for potential alternatives to antibiotics. In this scenario, peptidoglycan hydrolases can be used as alternate antibacterial agents due to their unique property of cleaving peptidoglycan cell wall present in both gram-positive and gram-negative bacteria. Along with a role in maintaining overall peptidoglycan turnover in a cell and in daughter cell separation, peptidoglycan hydrolases also play crucial role in bacterial pathophysiology requiring development of a computational tool for the identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data. Results In this study, the known peptidoglycan hydrolases were divided into multiple classes based on their site of action and were used for the development of a computational tool ‘HyPe’ for identification and classification of novel peptidoglycan hydrolases from genomic and metagenomic data. Various classification models were developed using amino acid and dipeptide composition features by training and optimization of Random Forest and Support Vector Machines. Random Forest multiclass model was selected for the development of HyPe tool as it showed up to 71.12 % sensitivity, 99.98 % specificity, 99.55 % accuracy and 0.80 MCC in four different classes of peptidoglycan hydrolases. The tool was validated on 24 independent genomic datasets and showed up to 100 % sensitivity and 0.94 MCC. The ability of HyPe to identify novel peptidoglycan hydrolases was also demonstrated on 24 metagenomic datasets. Conclusions The present tool helps in the identification and classification of novel peptidoglycan hydrolases from complete genomic or metagenomic ORFs. To our knowledge, this is the only tool available for the prediction of peptidoglycan hydrolases from genomic and metagenomic data. Availability: http://metagenomics.iiserb.ac.in/hype/ and http://metabiosys.iiserb.ac.in/hype/. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2753-8) contains supplementary material, which is available to authorized users.

Background: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now. Results: We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance. Conclusions: LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to β-lactam antibiotics in this bacterial model., Spanish Ministerio de Ciencia e Innovación, DIVINOCELL FP7 HEALTH-F3-2009-223431 from the European Commission and DIUFRO08-0060 from the Dirección de Investigación of the Universidad de La Frontera of Chile

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-β-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-β-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.