Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum.

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.     

A method for the enzymatic synthesis and HPLC purification of the peptidoglycan precursor UDP-N-acetylmuramic acid

UDP-N-acetylmuramic acid (UDP-MurNAc) is a precursor for peptidoglycan biosynthesis in bacteria. A major difficulty in the study of this pathway is that UDP-MurNAc is not commercially available. We have developed an enzymatic synthesis scheme for UDP-MurNAc using two easily purified Escherichia coli polyhistidine tagged peptidoglycan biosynthesis enzymes, MurZ and MurB, followed by a single-step purification of UDP-MurNAc by high-performance liquid chromatography. The identity of the UDP-MurNAc synthesized by our method was confirmed by electrospray ionization mass spectrometry. Furthermore, we show that the UDP-MurNAc can support a UDP-MurNAc-L-alanine ligase reaction.     

Sec-secretion and sortase-mediated anchoring of proteins in Gram-positive bacteria

Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Selectivity for D-lactate incorporation into the peptidoglycan precursors of Lactobacillus plantarum: role of Aad, a VanX-like D-alanyl-D-alanine dipeptidase

Lactobacillus plantarum produces peptidoglycan precursors ending in D-lactate instead of D-alanine, making the bacterium intrinsically resistant to vancomycin. The ligase Ddl of L. plantarum plays a central role in this specificity by synthesizing D-alanyl-D-lactate depsipeptides that are added to the precursor peptide chain by the enzyme MurF. Here we show that L. plantarum also encodes a D-Ala-D-Ala dipeptidase, Aad, which eliminates D-alanyl-D-alanine dipeptides that are produced by the Ddl ligase, thereby preventing their incorporation into the precursors. Although D-alanine-ended precursors can be incorporated into the cell wall, inactivation of Aad failed to suppress growth defects of L. plantarum mutants deficient in d-lactate-ended precursor synthesis.     

The biosynthesis and functionality of the cell-wall of lactic acid bacteria

The cell wall of lactic acid bacteria has the typical Gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attribut es of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.     

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.     

In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.     

The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan

Abstract The prevalence of aac(2')-Ia , a gene coding for gentamicin 2'-JV-acetyltransferase in Providenda stuartii , among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O -acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providenda rettgeri . Other bacteria that O -acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava . These data suggest that AAC(2')-Ia may contribute to the rO -acetylation of peptidoglycan in P. stuartii , but a more specific enzyme must also be produced for this function.     

PGRP-SB1: an N-acetylmuramoyl L-alanine amidase with antibacterial activity

The peptidoglycan recognition protein (PGRP) family is conserved from insects to mammals and is involved in immune regulation and bacterial clearance. They form at least three functional classes; receptors required for immune gene expression; amidases that degrade peptidoglycan and scavenge the tissues from immune-stimulating peptidoglycan; and as proteins with antibacterial activity. We here report that PGRP-SB1 is an N-acetylmuramoyl l-alanine amidase, which (in contrast to the previously described PGRP-amidases) shows antibacterial activity. PGRP-SB1 is highly active against peptidoglycans that have a diaminopimelic acid (DAP) residue in the cross-linking peptide, but lack activity to most lysine-containing peptidoglycans. The antibacterial activity is pronounced against Bacillus megaterium with an LD(50) of 1.5microg ml(-1). The bactericidal effect of PGRP-SB1 is dependent on its enzymatic activity, as the zinc co-factor is essential. The bactericidal mode of action is thus different from non-enzymatic vertebrate PGRPs that have been reported to be antibacterial.     

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor a from human monocytes

Abstract Elevated plasma concentrations of the cytokine tumor necrosis factor α (TNFα) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans , serovar copenhageni , was able to induce the release of TNFa from peripheral blood mononuclear cells. TNFa induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNFa release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNFa production.     

In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

利用文本提取工具构建乳杆菌肽聚糖特异性免疫调节网络

目的探索乳杆菌肽聚糖免疫调节作用的机制。方法BALB／c小鼠腹腔注射乳杆菌肽聚糖,从腹腔巨噬细胞和脾淋巴细胞提取RNA,基因芯片分析基因表达情况,利用Medscan从pubmed文献摘要提取肽聚糖相关基因网络,映射芯片数据获得乳杆菌肽聚糖特异基因网络。结果乳杆菌肽聚糖主要通过TLR2-NF-κB信号通路激活炎性细胞因子的表达,但是PGRP-L可能通过降解肽聚糖对此通路有负调节作用,NF-κB的激活可能诱导NOD2表达,对此通路进行负调节。结论乳杆菌肽聚糖通过与多种受体作用诱导独特的免疫反应,维持机体免疫稳态。     

Protein content of peptidoglycan of liquid-grown cells differs from that of surface-grown,gliding Cytophaga johnsonae

The peptidoglycan sacculi of surface-grown Cytophaga johnsonae had associated with them a large amoutn of protein (the major species is 50 kDa) whereas sacculi from liquid-grown cells had little or no attached protein. The 50 kDa protein was localized in the outer membrane of liquid-grown cells. A portion of this membrane-derived 50 kDa protein was attached to the peptidoglycan only when the cells made contact with the substratum. Protein synthesis did not appear to be required for attachment as the process was not inhibited by chloramphenicol. Association of the 50 kDa protein with the peptidoglycan in response to cell contact with the substratum is suggested.     

Helical growth and the curved shape of Vibrio cholerae

The curved, comma, or bent shape of Vibrio cholerae is attributed to, and explained by, the normal helical growth of the cell. The comma-like shape of V. cholerae is not due to an asymmetrical positioning of peptidoglycan such that some chains of peptidoglycan are placed so they are more spread out on one side of the cell and squeezed together on the other side.     

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain

The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.     

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.     

A three dimensional model of the digestion of peptidoglycan by lysozyme

The digestion of single peptidoglycan chains of the recently proposed conformation (Formanek et al., 1974) can be described with the same enzymatic mechanism as proposed by Phillips for a hexasaccharide consisting of alternating N-acetylglucosamine, N-acetylmuramic acid residues (Phillips, 1966). It is shown by model building, that in a peptidoglycan lysozyme complex the peptide chains do not exhibit any sterical hindrance.The digestion of the peptidoglycan sacculus by lysozyme may occur at lattice defects of its paracrystalline structure. A slit of about 30 å lenght and 10–15 å width between peptidoglycan micells may be sufficient for the attachment of lysozyme.     

Chlorobiphenyl-desleucyl-vancomycin inhibits the transglycosylation process required for peptidoglycan synthesis in bacteria in the absence of dipeptide binding

Novel glycopeptide analogs are known that have activity on vancomycin resistant enterococci despite the fact that the primary site for drug interaction, D-ala-D-ala, is replaced with D-ala-D-lactate. The mechanism of action of these compounds may involve dimerization and/or membrane binding, thus enhancing interaction with D-ala-D-lactate, or a direct interaction with the transglycosylase enzymes involved in peptidoglycan polymerization. We evaluated the ability of vancomycin (V), desleucyl-vancomycin (desleucyl-V), chlorobiphenyl-vancomycin (CBP-V), and chlorobiphenyl-desleucyl-vancomycin (CBP-desleucyl-V) to inhibit (a) peptidoglycan synthesis in vitro using UDP-muramyl-pentapeptide and UDP-muramyl-tetrapeptide substrates and (b) growth and peptidoglycan synthesis in vancomycin resistant enterococci. Compared to V or CBP-V, CBP-desleucyl-V retained equivalent potency in these assays, whereas desleucyl-V was inactive. In addition, CBP-desleucyl-V caused accumulation of N-acetylglucosamine-beta-1, 4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II). These data show that CBP-desleucyl-V inhibits peptidoglycan synthesis at the transglycosylation stage in the absence of binding to dipeptide.     

Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.