Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.     

A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis

Many cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection, yet remarkably little is known about the mechanism by which these non-sporulating bacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria. While Rpf proteins are clearly peptidoglycan glycosidases, the mechanism and role of Rpf in mediating reactivation remains unclear. Here we use a yeast two-hybrid screen to identify potential binding partners of RpfB and report the interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. The interacting domains map to the C-termini of both proteins, near predicted enzymatic domains. We show that RipA is a secreted, cell-associated protein, found in the same cellular compartment as RpfB. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. Finally, we demonstrate that RipA is capable of digesting cell wall material and is indeed a peptidoglycan hydrolase. The interaction between these two peptidoglycan hydrolases at the septum suggests a role for the complex in cell division, possibly during reactivation.     

A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.     

Interaction and Modulation of Two Antagonistic Cell Wall Enzymes of Mycobacteria

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions.     

Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation

The culturability of several actinobacteria is controlled by resuscitation-promoting factors (Rpfs). These are proteins containing a c. 70-residue domain that adopts a lysozyme-like fold. The invariant catalytic glutamate residue found in lysozyme and various bacterial lytic transglycosylases is also conserved in the Rpf proteins. Rpf from Micrococcus luteus, the founder member of this protein family, is indeed a muralytic enzyme, as revealed by its activity in zymograms containing M. luteus cell walls and its ability to (i) cause lysis of Escherichia coli when expressed and secreted into the periplasm; (ii) release fluorescent material from fluorescamine-labelled cell walls of M. luteus; and (iii) hydrolyse the artificial lysozyme substrate, 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside. Rpf activity was reduced but not completely abolished when the invariant glutamate residue was altered. Moreover, none of the other acidic residues in the Rpf domain was absolutely required for muralytic activity. Replacement of one or both of the cysteine residues that probably form a disulphide bridge within Rpf impaired but did not completely abolish muralytic activity. The muralytic activities of the Rpf mutants were correlated with their abilities to stimulate bacterial culturability and resuscitation, consistent with the view that the biological activity of Rpf results directly or indirectly from its ability to cleave bonds in bacterial peptidoglycan.     

The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.     

The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases

Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis. Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function. The family includes two different peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes. The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan. The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.     

Structural Changes and Cellular Localization of Resuscitation-Promoting Factor in Environmental Isolates of Micrococcus luteus

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.     

Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae

Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 A resolution. PBP3 folds into an NH(2)-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated beta-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel beta-sheet surrounded by alpha-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k(cat)/K(m) = 50,500 M(-1) x s(-1)), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.     

Molecular structure and peptidoglycan recognition of Mycobacterium tuberculosis ArfA (Rv0899)

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.     

Cutting edge: primary innate immune cells respond efficiently to polymeric peptidoglycan, but not to peptidoglycan monomers

The cell wall of bacteria induces proinflammatory cytokines in monocytes and neutrophils in human blood. The nature of the stimulating component of bacterial cell walls is not well understood. We have previously shown polymeric peptidoglycan (PGN) has this activity, and the cytokine response requires PGN internalization and trafficking to lysosomes. In this study, we demonstrate that peptidoglycan monomers such as muramyl dipeptide and soluble peptidoglycan fail to induce robust cytokine production in immune cells, although they activate the nucleotide-binding oligomerization domain proteins in transfected cell models. We further show that lysosomal extracts from immune cells degrade intact peptidoglycan into simpler products and that the lysosomal digestion products activate the nucleotide-binding oligomerization domain proteins. We conclude that naive innate immune cells recognize PGN in its polymeric form rather than monomers such as muramyl dipeptide and require PGN lysosomal hydrolysis to respond. These findings offer new opportunities in the treatment of sepsis, especially sepsis arising from Gram-positive organisms.     

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus

ABSTRACT

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.     

Structure of the OmpA-like domain of RmpM from Neisseria meningitidis

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

Peptidoglycan recognition by Pal, an outer membrane lipoprotein

Peptidoglycan-associated lipoprotein (Pal) is a potential vaccine candidate from Haemophilus influenzae that is highly conserved in Gram-negative bacteria and anchored to the outer membrane through an N-terminal lipid attachment. Pal stabilizes the outer membrane by providing a noncovalent link to the peptidoglycan (PG) layer through a periplasmic domain. Using NMR spectroscopy, we determined the three-dimensional structure of a complex between the periplasmic domain of Pal and a biosynthetic peptidoglycan precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-alpha-d-Glu-m-Dap-D-Ala-d-Ala (m-Dap is meso-diaminopimelate). Pal has a binding pocket lined with conserved surface residues that interacts exclusively with the peptide portion of the ligand. The m-Dap residue, which is mainly found in the cell walls of Gram-negative bacteria, is sequestered in this pocket and plays an important role by forming hydrogen bond and hydrophobic contacts to Pal. The structure provides insight into the mode of cell wall recognition for a broad class of Gram-negative membrane proteins, including OmpA and MotB, which have peptidoglycan-binding domains homologous to that of Pal.     

A close-up view of the VraSR two-component system. A mediator of Staphylococcus aureus response to cell wall damage

Staphylococcus aureus remains a clinical scourge. Recent studies have revealed that S. aureus is capable of mounting a response to antibiotics that target cell wall peptidoglycan biosynthesis, such as beta-lactams and vancomycin. A phosphotransfer-mediated signaling pathway composed of a histidine protein kinase, VraS, and a response regulator protein, VraR, has been linked to the coordination of this response. Herein, we report for the first time on the signal transduction mechanism of the VraSR system. We found that VraS is capable of undergoing autophosphorylation in vitro and its phosphoryl group is rapidly transferred to VraR. In addition, phosphorylated VraR undergoes rapid dephosphorylation by VraS. Evidence is presented that VraR has adopted a novel strategy in regulating the output response of the VraSR-mediated signaling pathway. The VraR effector domain inhibits formation of inactive VraR dimers and, in doing so, it holds the regulatory domain into an intermediate active state. We show that only phosphorylation induces formation of the biological active VraR-dimer species. Furthermore, we propose that damage inflicted to cell wall peptidoglycan could be the main source of the stimuli that VraR responds to due to the tight control that VraS has on the phosphorylation state of VraR. Our findings provide for the first time insights into the molecular basis for the proposed role of VraSR as a "sentinel" system capable of rapidly sensing cell wall peptidoglycan damage and coordinating a response that enhances the resistance phenotype in S. aureus.     

Substrate analogues to study cell-wall biosynthesis and its inhibition

It has been known for more than 30 years that Lipid II is an intermediate in peptidoglycan synthesis. Recently, it has become apparent that it is also an important target of numerous antibiotics, including the glycopeptides, the lantibiotics and ramoplanin. It is also utilized by sortases in the construction of Gram-positive cell walls. Recent progress has been made in the synthesis of peptidoglycan intermediates that can be used to study enzymes which make peptidoglycan. These intermediates also enable studies to probe the mechanism of action of a variety of substrate-binding antibiotics.     

Functional biosynthesis of cell wall peptidoglycan by polymorphic bifunctional polypeptides. Penicillin-binding protein 1Bs of Escherichia coli with activities of transglycosylase and transpeptidase

Dual enzyme activities for the biosynthesis of peptidoglycan of the cell wall are located in major higher molecular weight penicillin-binding proteins (PBP) of Escherichia coli. Each of these proteins catalyzes the two successive final reactions in the synthesis of cross-linked peptidoglycan from the precursor N-acetylglucosaminyl-N-acetylmuramyl peptide linked to undecaprenol diphosphate; namely, the transglycosylation that extends the glycan chain and the penicillin-sensitive DD-transpeptidation that cross-links the glycan chains through two peptide side chains. Both transglycosylation and transpeptidation catalyzed by PBP-1Bs represent de novo synthesis of cross-linked peptidoglycan. Under appropriate conditions, about 25% cross-linkage was observed during the reaction, the main reaction product supposedly being a regularly cross-linked network of peptidoglycan. The two domains for the transglycosylase and transpeptidase activities were found to be located on a 50-kDa portion of the PBP-1Bs, which are about 90 kDa. Gene recombination experiments indicated that the transglycosylase domain is located upstream, i.e. on the N-terminal side of the transpeptidase domain, suggesting that the gene for these bifunctional peptides may have been formed by fusion of the genes for transglycosylase and transpeptidase that were previously located separately on the chromosome in this order.