Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.     

Cadaverine Covalently Linked to Peptidoglycan Is Required for Interaction between the Peptidoglycan and the Periplasm-Exposed S-Layer-Homologous Domain of Major Outer Membrane Protein Mep45 in Selenomonas ruminantium

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal β-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal β-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded β-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.Polyamines, the ubiquitous polycationic compounds composed of a hydrocarbon backbone with multiple amino groups, exist in all living cells and participate in a wide variety of biological reactions, including DNA, RNA, and protein synthesis (34). However, it has been revealed that some strictly anaerobic eubacteria belonging to the Veillonellaceae family, such as Selenomonas ruminantium, Veillonella alcalescens, Veillonella parvula, and Anaerovibrio lipolyticus, possess polyamines covalently linked to their peptidoglycan (PG) as an essential constituent (8, 16, 17). S. ruminantium possesses a peptidoglycan associated with cadaverine. Cadaverine binds covalently to the α-carboxyl group of the d-glutamic acid residue of peptidoglycan by one of its two amino groups, and the other amino group remains as a free cation (15). In this bacterium, cadaverine is synthesized constitutively from lysine by lysine/ornithine decarboxylase (LDC/ODC [EC 4.1.1.18]), a bifunctional enzyme that decarboxylates both l-lysine and l-ornithine at similar K_m and V_max values (35, 36) and is transferred to a d-glutamic acid residue by a particulate enzyme designated as lipid intermediate:diamine transferase (20). The cadaverine synthesis by LDC/ODC is completely inhibited by dl-α-difluoromethyllysine (DFML) or dl-α-difluoromethylornithine (DFMO), which inhibits the decarboxylating activity toward both l-lysine and l-ornithine (35), and the prevention of the cadaverine synthesis in S. ruminantium was shown to lead to the significant decrease of the amount of the cadaverine covalently linked to peptidoglycan (PG-cadaverine) and result in the growth inhibition (17). Since this inhibitory effect accompanies a drastic morphological change of the cells resulting in an aberrant cell surface structure, PG-cadaverine has been assumed to play a significant role in maintaining the integrity of the cell surface (17).The cell surface structure of S. ruminantium has a typical Gram-negative three-layer organization, comprising a cytoplasmic membrane, peptidoglycan layer, and outer membrane (18). However, it contains neither the free nor bound form of murein-lipoprotein (19), which plays an important role in the structural linkage between the outer membrane and peptidoglycan, thereby maintaining the structural integrity of the cell surface structures of Gram-negative bacteria (5, 33). The Escherichia coli lpo mutant that lacks murein-lipoprotein becomes hypersensitive to EDTA, resulting in rapid cell lysis upon exposure to EDTA. In contrast, S. ruminantium shows no cell lysis, even in the presence of high concentrations of EDTA, despite the absence of murein-lipoprotein (19). One possible interpretation for these findings was the assumption that PG-cadaverine associates with the structural connection between the outer membrane and peptidoglycan, thereby replacing the function of murein-lipoprotein with an outer membrane component or components. Nevertheless, the factors in the outer membrane interacting with PG-cadaverine have not been identified.The outer membrane of S. ruminantium contains a 45-kDa major protein (Mep45), which has been proposed to be a peptidoglycan-associating protein (18, 19). Kalmokoff et al. reported that the major outer membrane protein of S. ruminantium OB268, which is similar to Mep45 in size, contains an N-terminal surface-layer homology (SLH) domain (13), a putative functional domain that interacts with cell wall components (27). These findings prompted us to investigate the Mep45 major outer membrane protein of S. ruminantium as a putative outer membrane component interacting with PG-cadaverine. In this report, we characterize the Mep45 protein and its interactions with PG-cadaverine and prove their involvement in the structural linkage between the outer membrane and peptidoglycan.     

Structural specificity of diamines covalently linked to peptidoglycan for cell growth of Veillonella alcalescens and Selenomonas ruminantium.

Putrescine and cadaverine are essential constituents of the peptidoglycan of Veillonella alcalescens, Veillonella parvula, and Selenomonas ruminantium and are necessary for the growth of these organisms (Y. Kamio and K. Nakamura, J. Bacteriol. 169:2881-2884, 1987, and Y. Kamio, H. P?s?, Y. Terawaki, and L. Paulin, J. Biol. Chem. 261:6585-6589, 1986). In this study, the structural specificity of the diamine requirement for normal cell growth of these bacteria was examined by using a series of diamines with a general structure of NH3+ X (CH2)n X NH3+. Diaminohexane (n = 6) which was incorporated into the peptidoglycan was as effective as putrescine (n = 4) and cadaverine (n = 5) for normal cell growth. However, diaminopropane (n = 3) and diaminoheptane (n = 7) were less effective for growth than diaminohexane, although they were incorporated into the peptidoglycan to the same extent.     

ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan‐binding domain (Pfam number PF01471) conserved in many peptidoglycan‐related proteins is present in the periplasmic region of ExeA (P‐ExeA). In this study, co‐sedimentation analysis revealed that P‐ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross‐linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30°C and above. These results provide evidence that the putative peptidoglycan‐binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB‐dependent assembly of the secretin multimer in the outer membrane.     

Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli

A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.     

Cadaverine covalently linked to a peptidoglycan is an essential constituent of the peptidoglycan necessary for the normal growth in Selenomonas ruminantium

Cadaverine links covalently to the D-glutamic acid residue of the peptidoglycan in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium (Kamio, Y., Itoh, Y., and Terawaki, Y. (1981) J. Bacteriol. 146, 49-53). This report clarifies a physiological function of cadaverine in this organism by using DL-alpha-difluoromethyllysine, which had previously been shown to be a selective irreversible inhibitor of lysine decarboxylase of Mycoplasma dispar (P?s?, H., MaCann, P.P., Tanskanen, R., Bey, P., and Sjoerdsma, A. (1984) Biochem. Biophys. Res. Commun. 125, 205-210). DL-alpha-Difluoromethyllysine is now shown to be a potent and irreversible inhibitor of lysine decarboxylase of S. ruminantium in vitro; however, it did not inhibit the transfer of cadaverine to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan. DL-alpha-Difluoromethyllysine at 5 mM markedly inhibited the growth of the bacterium and caused rapid cell lysis. Immediately before the cell lysis, almost all cells became swollen, and such cells showed a loosened envelope structure when studied by electron microscopy. The peptidoglycan prepared from the DL-alpha-difluoromethyllysine-treated cells did not have covalently linked cadaverine. The growth inhibition by DL-alpha-difluoromethyllysine was completely reversed by adding cadaverine (1 mM) to the medium. Furthermore, the exogenous cadaverine was exclusively incorporated into the peptidoglycan in the presence of DL-alpha-difluoromethyllysine (5 mM), and a normal peptidoglycan was synthesized. The cell lysis and the formation of an abnormal cell structure were completely prevented by cadaverine added to the medium. We conclude that cadaverine covalently linked to the peptidoglycan in S. ruminantium is an essential constituent of the peptidoglycan and is required for cell surface integrity and the normal growth of S. ruminantium.     

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer.     

Molecular assembly of the lipoprotein trimer on the peptidoglycan layer of Escherichia coli

The molecular assembly of the major outer membrane lipoprotein on the peptidoglycan layer was studied using two hybrid genes coding for different OmpF-lipoprotein hybrid proteins. One gene codes for a "lipoprotein" in which the diacylglyceryl cysteine residue is replaced with the Ala-Glu residue of the NH2 terminus of the OmpF protein (hybrid protein I). The other gene codes for the lipid-free "lipoprotein" from which the COOH-terminal lysine residue was further deleted (hybrid protein II). Hybrid protein I existed as a trimer. A significant portion of it was found to be composed of only the free form, which was noncovalently associated with the peptidoglycan layer. The purified hybrid protein I trimer was dissociated into the subunit in the presence of guanidine-HCl and reassociated on dialysis. Both the native and reassociated trimers were bound to the lipoprotein-free peptidoglycan layer. No enhancement of the binding was observed when the reassociation reaction was carried out simultaneously. Hybrid protein II, on the other hand, did not exhibit association with peptidoglycan in both the cellular fractionation and in vitro binding experiments, although it existed as a trimer. It is concluded that 1) the protein domain of the lipoprotein exists as a trimer which is noncovalently as well as covalently associated with the peptidoglycan layer and 2) although the deletion of the COOH terminal lysine residue did not interfere with the trimerization, it interfered with the noncovalent interaction with the peptidoglycan layer.     

Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology

The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall Kd of 7.7 x 10(-7) M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 10(-11) M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.     

The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain,a new peptidoglycan binding domain

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram‐negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N‐acetylmuramyl‐L‐alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β‐sandwich of two symmetrical four‐stranded β‐sheets exposing highly conserved motifs on the two outer faces. We show that this N‐terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan‐binding domain. The C‐terminal catalytic domain shows an auto‐inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity.     

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.     

Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12.

The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.     

DipM,a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus

In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C‐terminal lysostaphin‐like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.     

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.     

Suppression of the Temperature-Sensitive Mutation of the bamD Gene Required for the Assembly of Outer Membrane Proteins by Multicopy of the yiaD Gene in Escherichia coli

We isolated temperature-sensitive mutants of the Escherichia coli bamD gene, which is essential for the assembly of β-barrel outer membrane proteins. As their multicopy suppressor, we identified a novel yiaD gene encoding a putative lipoprotein, YiaD. Mutations of its OmpA domain, which is required for interaction with peptidoglycan, affected suppression, suggesting that interaction with peptidoglycan is important to YiaD function.     

Chemical structure of peptidoglycan in Selenomonas ruminantium: cadaverine links covalently to the D-glutamic acid residue of peptidoglycan

The peptidoglycan of Selenomonas ruminantium, a strictly anaerobic bacterium, contains cadaverine (Y. Kamio, Y. Itoh, Y. Terawaki, and T. Kusano, J. Bacteriol. 145:122-128, 1981). This report describes the chemical structure of the peptidoglycan of this bacterium. The [14C]cadaverine-labeled peptidoglycan was degraded with the lytic enzymes prepared from Streptomyces albus G into three small fragments including a major fragment (band A compound). Bank A compound was composed of L-alanine, D-glutamic acid, meso-diaminopimelic acid, D-alanine, and cadaverine in the molar ratio 0.98:1.0:1.0:0.98:0.97. Diaminopimelic acid, L-alanine, and cadaverine were N-terminal residues in band A compound. When the [14C]cadaverine-labeled band A compound was subjected to partial acid hydrolysis, two peptide fragments were obtained. One of them consisted of diaminopimelic acid and D-alanine; diaminopimelic acid was the N-terminal amino acid, and the other fragment was composed of L-alanine, D-glutamic acid, and cadaverine, of which L-alanine and cadaverine were N-terminal. These results lead us to conclude that the primary peptide structure of band A compound is L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine and that cadaverine links covalently to the D-glutamic acid residue.     

Assembly of the type II secretion system: identification of ExeA residues critical for peptidoglycan binding and secretin multimerization

Aeromonas hydrophila secretes a number of protein toxins across the outer membrane via the type II secretion system (T2SS). Assembly of the secretion channel ExeD secretin into the outer membrane is dependent on the peptidoglycan binding domain of ExeA. In this study, the peptidoglycan binding domain PF01471 family members were divided into a prokaryotic group and a eukaryotic group. By comparison of their sequence conservation profiles and their representative crystal structures, we found the prokaryotic members to have a highly conserved pocket(s) that is not present in the eukaryotic members. Substitution mutations of nine amino acids of the pocket were constructed in ExeA. Five of the substitution derivatives showed greatly decreased lipase secretion, accompanied by defects in secretin assembly. In addition, using in vivo cross-linking and in vitro cosedimentation assays, we showed that these mutations decreased ExeA-peptidoglycan interactions. These results suggest that the highly conserved pocket in ExeA is the binding site for its peptidoglycan ligand and identify residues critical for this binding.     

Putrescine and cadaverine are constituents of peptidoglycan in Veillonella alcalescens and Veillonella parvula.

Veillonella alcalescens ATCC 17745, a strictly anaerobic, gram-negative small coccus, requires putrescine or cadaverine for growth (M. B. Ritchey, and E. A. Delwiche, J. Bacteriol. 124:1213-1219, 1975). Both putrescine and cadaverine were demonstrated to be incorporated exclusively into the peptidoglycan layer of V. alcalescens ATCC 17745. V. parvula GAI 0574 also proved to contain putrescine as a component of peptidoglycan. The primary chemical structure of the peptidoglycan common to the two Veillonella species is N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid gamma-meso-diaminopimelic acid-D-alanine. Putrescine or cadaverine links covalently to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal cell growth. In V. alcalescens ATCC 17745, above 40% saturation at cadaverine linked to the alpha-carboxyl group of the D-glutamic acid residue of the peptidoglycan is necessary for normal growth.     

The biosynthesis of peptidoglycan lipid-linked intermediates

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.