How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.     

Peptidoglycan turnover and recycling in Gram-positive bacteria

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.     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.     

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.     

Structure and Function of a Novel ld-Carboxypeptidase A Involved in Peptidoglycan Recycling

Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.     

Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The Listeria monocytogenes Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of Listeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using L. monocytogenes serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant tagO homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either tagO1 (EGDe lmo0959) or tagO2 (EGDe lmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient Listeria cells. Substitution of tagO from an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other Listeria phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.     

Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.     

Cell shape determination in Escherichia coli

The rigid cell wall peptidoglycan (murein) is a single giant macromolecule whose shape determines the shape of the bacterial cell. Insight into morphogenetic mechanism(s) responsible for determining the shape of the murein sacculus itself has begun to emerge only in recent years. The discovery that MfreB and Mbl are cytoskeletal actin homologues that form helical structures extending from pole to pole in rod-shaped cells has opened an exciting new field of microbial cell biology. MreB (in Gram-negative rods) and Mbl (in Gram-positive species) are essential for murein synthesis along the lateral wall and hence, the rod shape of the cell. Known members of the morphogenetic system include MreB (or Mbl), MreC, MreD and PBP2, but Rod A and murein biosynthetic enzymes involved in peptidoglycan precursor synthesis and assembly are likely to be recruited to the same multimolecular apparatus. However, the actual role of MreB in assembly of the morphogenetic complex is still not clear and little is known about regulatory mechanisms controlling the switch from lateral murein elongation to septa1 murein synthesis at the time of cell division.     

Identification of Two Binding Domains, One for Peptidoglycan and Another for a Secondary Cell Wall Polymer, on the N-Terminal Part of the S-Layer Protein SbsB from Bacillus stearothermophilus PV72/p2

First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation,Not the Acquisition,of Cell Wall-Modifying Enzymes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Bacteriocin Protein BacL1 of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL₁ and BacA. We previously reported that BacL₁ protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL₁ remains unknown. In this study, we investigated the targeting specificity of BacL₁. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL₁ specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL₁. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL₁ and BacA. The static growth state also abolished the binding and targeting of BacL₁ to the cell division-associated site. Furthermore, the targeting of BacL₁ was detectable among Gram-positive bacteria with an l-Ala-l-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL₁ specifically targets the l-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.     

Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytB_CAT), excluding the choline binding domain. LytB_CAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.     

LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.     

Specialized Peptidoglycan Hydrolases Sculpt the Intra-bacterial Niche of Predatory Bdellovibrio and Increase Population Fitness

Bdellovibrio are predatory bacteria that have evolved to invade virtually all Gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound β-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that “regular” PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.     

Cell Wall Hydrolases Affect Germination,Vegetative Growth,and Sporulation in Streptomyces coelicolor

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor, highlighting both the lack of redundancy in hydrolase activity and the importance of cell wall remodeling in the S. coelicolor life cycle.Peptidoglycan (PG) is a primary constituent of the gram-positive bacterial cell wall and, despite its rigid structure, is a remarkably dynamic macromolecule. It functions in maintaining cell shape and cytoplasmic turgor pressure and serves as the scaffolding to which cell wall-associated components, such as proteins and teichoic acids, are anchored (16). PG comprises alternating N-acetylglucosamine and N-acetylmuramic acid residues, which make up the glycan backbone, and peptide side chains that link the glycan strands together (49). PG biosynthesis is a complex process involving the concerted efforts of many enzymes, beginning with precursor synthesis in the cytoplasm and concluding with polymerization outside the cytoplasmic membrane (3, 8, 48). During bacterial growth, PG is actively remodeled to allow incorporation of new PG and to accommodate changes in cell shape. The enzymes responsible for this remodeling are collectively termed cell wall hydrolases, and they act by cleaving covalent bonds within either the glycan strands or the peptide side chains. The essential nature of PG requires that synthesis and cleavage be tightly regulated, with the activities of biosynthetic and hydrolytic enzymes coordinated in both space and time.Cell wall hydrolases are diverse enzymes that are typically grouped on the basis of substrate specificity and the resulting cleavage products. The major groups include the lysozymes and lytic transglycosylases, which hydrolyze the β-(1,4)-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine; the endopeptidases, which cleave the peptide bonds in the amino acid side chains connecting the parallel glycan strands; the carboxypeptidases, which cleave the C-terminal amino acids of peptide chains; and the amidases, which cleave between N-acetylmuramic acid and the first residue (l-Ala) of the peptide side chain (55).In addition to remodeling the PG, cell wall hydrolases also contribute to a multitude of specialized cellular processes, from the assembly of secretion systems, flagella, and pili (55) to the resuscitation of dormant cells by a recently discovered class of hydrolases known as the resuscitation-promoting factors (Rpfs) (37). The Rpfs are secreted proteins that are structurally related to lysozymes (12, 13) and are found in a subset of the actinomycetes, including Micrococcus, Mycobacterium, Corynebacterium, and Streptomyces (44). The sole Micrococcus luteus Rpf is essential for viability (36), while in Mycobacterium tuberculosis, which encodes five Rpf proteins, the enzymes are required for virulence and resumption of active growth during emergence from a latent state (26). The sporulating actinomycete Streptomyces coelicolor is predicted to encode seven Rpf proteins, along with a plethora of other cell wall hydrolases. Surprisingly little is known about cell wall remodeling in the streptomycetes, despite the fact that significant remodeling must accompany the filamentous growth and morphological changes associated with the different stages of the Streptomyces life cycle. The S. coelicolor life cycle initiates with spore germination; this process likely depends on cell wall hydrolase activity, as spore germination in Bacillus subtilis requires the activity of at least two hydrolases (50). Following spore germination in S. coelicolor, germ tubes elongate and branch in a filamentous manner, forming a network of cells termed the vegetative mycelium. A second type of filamentous (but nonbranching) cells, the aerial hyphae, then emerge from the vegetative mycelium, and it is within these cells that chains of spores develop. Cell wall hydrolase activity and the associated cell wall remodeling are thought to be essential for vegetative hyphal branch formation, vegetative and aerial hyphal tip extension, spore chain formation, and spore dispersal. In this work, we describe the first investigation of cell wall hydrolase activity and function in Streptomyces. We identify a subset of hydrolases whose genes share a conserved 5′ untranslated region (UTR), demonstrate enzymatic activity for four of these proteins, and reveal that these enzymes function at multiple stages in the S. coelicolor life cycle.     

Reduction of the Peptidoglycan Crosslinking Causes a Decrease in Stiffness of the Staphylococcus aureus Cell Envelope

We have used atomic-force microscopy (AFM) to probe the effect of peptidoglycan crosslinking reduction on the elasticity of the Staphylococcus aureus cell wall, which is of particular interest as a target for antimicrobial chemotherapy. Penicillin-binding protein 4 (PBP4) is a nonessential transpeptidase, required for the high levels of peptidoglycan crosslinking characteristic of S. aureus. Importantly, this protein is essential for β-lactam resistance in community-acquired, methicillin-resistant S. aureus (MRSA) strains but not in hospital-acquired MRSA strains. Using AFM in a new mode for recording force/distance curves, we observed that the absence of PBP4, and the concomitant reduction of the peptidoglycan crosslinking, resulted in a reduction in stiffness of the S. aureus cell wall. Importantly, the reduction in cell wall stiffness in the absence of PBP4 was observed both in community-acquired and hospital-acquired MRSA strains, indicating that high levels of peptidoglycan crosslinking modulate the overall structure and mechanical properties of the S. aureus cell envelope in both types of clinically relevant strains. Additionally, we were able to show that the applied method enables the separation of cell wall properties and turgor pressure.     

Preparation and Characterization of Intact Globular Cell Wall Peptidoglycan from Micrococcus lysodeikticus

The intact globular cell wall peptidoglycan was prepared from Micrococcus lysodeikticus without any mechanical disruption. The purification procedure consists mainly of proteolytic digestion and extractions with hot 5% TCA and with 0.01 N NaOH. The purified preparation showed an amphoteric, heteroporous three-dimensional network structure of the peptidoglycan, which amounted to 77% of the dry weight of the preparation, retaining the chemical and morphological integrity as judged by an electron microscopic observation and chemical analyses.