Structure of Bordetella pertussis peptidoglycan.

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing [3H]diaminopimelic acid and treated by a hot (96 degrees C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60%). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and less than 2% protein. Radiochemical analyses indicated that 3H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelic acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived peptidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of greater than 95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl++ +-alanine.     

Mycobacterial lipid II is composed of a complex mixture of modified muramyl and peptide moieties linked to decaprenyl phosphate

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.     

Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.     

Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, α-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan–teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala–d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the -amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.     

Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)]

Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.     

Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors

Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.     

Primary structure of the peptidoglycan from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714

A peptidoglycan fraction free of non-peptidoglycan components was isolated from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714. Hydrofluoric acid treatment (48%, 0 degrees C, 48 h) cleaved off from the peptidoglycan non-peptidoglycan glucosamine, mannosamine, and mannose. The purified peptidoglycan consists of N-acetyl muramic acid, N-acetyl glucosamine, L-alanine, D-alanine, D-glutamic acid, and meso-diaminopimelic acid in approximately equimolar amounts. At least partial amidation of carboxy groups in the peptide subunits is indicated. Peptide analyses and 2,4-dinitrophenyl studies of partial acid hydrolysates revealed the structure of the Synechocystis sp. strain PCC 6714 peptidoglycan to belong to the A1 gamma type (direct cross-linkage) of peptidoglycan classification. The degree of cross-linkage is about 56% and thus is in the range of that found in gram-positive bacteria. Some of the peptide units are present as tripeptides lacking the carboxy-terminal D-alanine.     

Myxospore Formation in Myxococcus xanthus: Chemical Changes in the Cell Wall During Cellular Morphogenesis

Vegetative cells of Myxococcus xanthus (strain FB) were induced to form myxospores by the glycerol induction technique. Several structural changes took place in the peptidoglycan during myxospore formation. The percent of the peptidoglycan comprised of monomer (disaccharide peptide) decreased from about 20% to approximately 7%. The proportion of the total diaminopimelic acid possessing a free amino group decreased about 11%. A carbohydrate containing only glucose was found to be bound, possibly covalently, to the vegetative cell and myxospore peptidoglycan. The amount of carbohydrate relative to peptidoglycan decreased by two-thirds during myxospore formation. None of the above changes in the peptidoglycan were observed in a mutant (strain GNI) of M. xanthus which was unable to convert to myxospores when incubated in the glycerol induction medium, or in the parental wild type (FB) when it was incubated in induction medium lacking the myxospore inducer, glycerol.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.     

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.     

Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity

Unlike numerous pore-forming amphiphilic peptide antibiotics, the lantibiotic nisin is active in nanomolar concentrations, which results from its ability to use the lipid-bound cell wall precursor lipid II as a docking molecule for subsequent pore formation. Here we use genetically engineered nisin variants to identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g. [S3T]nisin, led to reduced binding and increased the peptide concentration necessary for pore formation. The binding constant for the S3T mutant was 0.043 x 10(7) m(-1) compared with 2 x 10(7) m(-1) for the wild-type peptide, and the minimum concentration for pore formation increased from the 1 nm to the 50 nm range. In contrast, peptides mutated in the flexible hinge region, e.g. [DeltaN20/DeltaM21]nisin, were completely inactive in the pore formation assay, but were reduced to some extent in their in vivo activity. We found the remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit its incorporation into the peptidoglycan network. Therefore, through interaction with the membrane-bound cell wall precursor lipid II, nisin inhibits peptidoglycan synthesis and forms highly specific pores. The combination of two killing mechanisms in one molecule potentiates antibiotic activity and results in nanomolar MIC values, a strategy that may well be worth considering for the construction of novel antibiotics.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

Appearance of gamma-D-glutamyl-(L) meso-diaminopimealate peptidoglycan hydrolase during sporulation in Bacillus sphaericus

Particulate preparations from sporulating cells of Bacillus sphaericus 9602 contained an endopeptidase activity that hydrolyzed the gamma-d-glutamyl-(l)meso-diaminopimelic acid linkages found in the spore cortical peptidoglycan of this organism. Diaminopimelic acid did not occur in the vegetative cell wall peptidoglycan, and the gamma-d-glutamyl-l-lysine linkages found in this polymer were not hydrolyzed by the endopeptidase. The endopeptidase hydrolyzed (X)-l-alanyl-gamma-d-glutamyl-(l)meso-diaminopimelyl(l)-d-alanyl-d-alanine only after removal of the terminal d-alanine residue. The preparations contained an acyl-d-alanyl-d-alanine carboxypeptidase I activity which converted such pentapeptides into substrates for the endopeptidase and which was inhibited 50% by 4 x 10(-7) M benzylpenicillin. This activity also hydrolyzed the analogous pentapeptide substrates containing l-lysine. The preparations also contained an acyl-l-lysyl-d-alanine carboxypeptidase II activity that was not active on the meso-diaminopimelic acid-containing analogue. Neither this activity nor the endopeptidase was inhibited by 10(-3) M benzylpenicillin. The specificities of the carboxypeptidases were consistent with the exclusive presence of l-lysine C-termini in the vegetative peptidoglycan and of meso-diaminopimelyl-d-alanine C-termini in the spore cortical peptidoglycan of B. sphaericus 9602.     

Determination of Cu environments in the cyanobacterium Anabaena flos-aquae by X-ray absorption spectroscopy

Whole cells and peptidoglycan isolated from cell walls of the cyanobacterium Anabaena flos-aquae were lyophilized and used at pH 2 and pH 5 in Cu(II) binding studies. X-ray absorption spectra measured at the Cu K-edge were used to determine the oxidation states and chemical environments of Cu species in the whole-cell and peptidoglycan samples. In the whole-cell samples, most of the Cu retained at both pH values was coordinated by phosphate ligands. The whole-cell fractions contained significant concentrations of Cu(I) as well as Cu(II). An X-ray absorption near-edge spectrum analysis suggested that Cu(I) was coordinated by amine and thiol ligands. An analysis of the peptidoglycan fractions found that more Cu was adsorbed by the peptidoglycan fraction prepared at pH 5, due to increased chelation by amine and carboxyl ligands. The peptidoglycan fractions, also referred to as the cell wall fractions, contained little or no Cu(I). The Cu loading level was 30 times higher in the cell wall sample prepared at pH 5 than in the sample prepared at pH 2. Amine and bidentate carboxyl ligands had similar relative levels of importance in cell wall peptidoglycan samples prepared at both pH values, but phosphate coordination was insignificant.     

Isolation and characterization of a cDNA clone for the amino-terminal portion of the pro-alpha 1(II) chain of cartilage collagen

We have isolated a cDNA clone (pRcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(II) chain mRNA. A synthetic oligonucleotide was used both as a primer for cDNA synthesis and as a probe for screening a cDNA library. The probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(II) chain. This primer was chosen so that the resulting cDNA would contain the sequence of the 5' end of the mRNA. The nucleotide sequences of the cDNA were determined and compared with that of three other interstitial procollagen chain mRNAs (pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) chain mRNA). pRcol 2 contains a 521-base pair (bp) insert, including 153 bp of the 5' untranslated region plus 368 bp coding for the signal peptide, the amino-terminal propeptide, and a part of the telopeptide. The signal peptide of the type II collagen chain is composed of about 20 amino acids. There is little homology between the amino acid sequence of the signal peptide in the pro-alpha 1(II) chain and that of three other interstitial procollagen chains. The NH2-terminal propeptide is deduced to contain short nonhelical sequences at its amino and carboxyl ends and an internal helical collagenous domain comprising 25 repeats of Gly-X-Y with one interruption. There is a strong conservation of the amino acid sequence of the carboxyl-terminal part of the NH2-terminal propeptide in the pro-alpha 1(II), pro-alpha 1(I), and pro-alpha 2(I) chains. Type II collagen mRNA does not contain a sequence corresponding to a uniquely conserved nucleotide sequence around the translation initiation site which occurs in mRNA for other procollagen chains.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.     

Characteristics of the active and inactive variants of Actinomyces sp. 26-115, a producer of actinomycin C

Two natural variants, i.e. No. 1 and No. 2, not producing actinomycin were isolated from cultures of the actinomycin C-producing organism Actinomyces sp. 26-115. Variant No. 1 differed from the active variant by the growth dynamics and colony morphology. Variant No. 2 was close to the active variant by the growth dynamics. It was shown with electron microscopy that the cells of variant No. 1 differed from those of the active variant in the number and form of the mycelial septa, more even and compact structure of the cell walls and higher sensitivity to actinomycin. Still, they were more stable to the effect of lysozyme and ultrasound. The cell walls of the inactive variant No. 1 gradually lost teichoic acid during development, while the loss of peptidoglycan was observed only on transfer to the stationary phase. The cell walls of the active variant lost teichoic acid and peptidoglycan at the same time on transfer to the stationary phase. Peptidoglycans of both variants contained diaminopimelic acid (the configuration of which was not determined) and glycine (1:1) as differentiating amino acids. The two adjacent tetrapeptides were joined with one glycine radical. The peptidoglycan peptide chains of both variants contained muramic, glutamic and diaminopimelic acids and alanine (1:1:1:2). The peptidoglycans of the inactive variant No. 1 contained in addition valine and isoleucine. However, it is hardly probable that they are contained by the peptidoglycan peptide chains.     

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.     

The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis.

Physiological properties of the murG gene product of Escherichia coli were investigated. The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors. The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I. In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway. Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed. In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.