Improved method of determining muramic acid from environmental samples

Muramic acid is an amino sugar that forms part of the peptidoglycan in prokaryotic cell walls. Since muramic acid is found only in prokaryotes it has been used as a measure of bacterial and cyanophyte biomass. Successful application of sensitive capillary gas‐liquid chromatographic (GLC) analysis required neutralization of the acid‐hydrolysate of a biomass sample followed by centrifugation to remove humic acids. After a further fractionation on a cation exchange column followed by derivatization and GLC analysis, recoveries of 98 ±9.5 (X± S.D.) % of authentic muramic acid from estuarine sediments with sensitivities of 10^‐13 mol were achieved. The structure of the GLC derivative was established by GLC infrared analysis and GLC mass spectrometry. The improvements in reproduci‐bility and sensitivity have allowed detection of ¹³C enrichments in muramic acid from the detrital microbiota incubated with relabeled precursors.     

Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid

The muramic acid assay is a powerful tool for detecting both intact bacteria and bacterial debris. Past use of aldononitrile acetate derivatization for determining muramic acid in complex samples by gas chromatography/mass spectrometry met detection needs in many instances; however, questions have been raised regarding the interpretation of the derivative structure and its electron ionization fragments. In this study, we applied different methods and proved that the aldononitrile acetate derivatized muramic acid yields a molecular weight of 398, associated with a lactam structure. We also presented evidence that the structure of aldononitrile acetate derivatized muramic acid is acetylated at four positions, 3 O-acetylations and 1N-acetylation. In practical manner, this communication provides a comprehensive reference to researchers using δ¹³C value or ion fragments of the muramic acid marker in biogeochemical studies.     

Quantification of Frankia Strains and Other Root-Associated Bacteria in Pure Cultures and in the Rhizosphere of Axenic Seedlings by High-Performance Liquid Chromatography-Based Muramic Acid Assay

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic Festuca rubra seedlings. Quantities of muramic acid from acid-hydrolyzed cells of Frankia strains, Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas sp., and Bacillus polymyxa were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera, Frankia and Streptomyces, than in the rest of the strains. Quantification of Frankia strains, S. griseoviridis, E. agglomerans, and Pseudomonas sp. was also attempted from the rhizosphere of F. rubra seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify Frankia cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the Festuca rhizosphere, 28 to 63% of the muramic acid in the Frankia inoculum was recovered.     

Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10⁻¹⁰ to 5.3 × 10⁻¹⁰ μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 10⁸ to 10¹¹ cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10⁻¹⁰ to 1.6 × 10⁻¹⁰ μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis.     

Muramic acid in the cell walls of Prochloron

Muramic acid has been detected in Prochloron with the aid of two different techniques. It was assayed by cleaving D-lactate from muramic acid and then reducing NAD with D-lactate dehydrogenase and measuring the NADH with bacterial luciferase. Gas-liquid chromatography of trimethylsilyl derivatives of cell extracts confirmed that muramic acid was present in about the quantity given by the D-lactate assay. The amount of muramic acid present was 1.7±0.2 g/mg dry weight or 1.3fg/m² of cell surface. This suggests that the thickness of the peptidoglycan layer in Prochloron is similar to that in blue-green algae.Abbreviations D-LDH d-lactate dehydrogenase - MA muramic acid - TMS trimethylsilyl - TLE thin layer electrophoresis - GLC gas-liquid chromatography     

A simple chromatographic route for the isolation of meso diaminopimelic acid

Meso diaminopimelic acid is an important noncoded amino acid found in Gram‐negative bacterial peptidoglycan. In spite of its importance, this stereoisomer is not available commercially. A simple, economical procedure was developed for the isolation of pure meso diaminopimelic acid via an high‐performance liquid chromatography separation. In our new approach, the underivatized three isomers of diaminopimelic acid were separated on a crown ether‐based chiral stationary phase. For the structure identification, ¹H NMR spectroscopy was applied. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Cell walls from Actinopolyspora halophila,an extremely halophilic actinomycete

Cell wall components were prepared from Actinopolyspora halophila (strain wt), an extremely halophilic actinomycete requiring a minimum 12% NaCl concentration for growth, and from an erythromycin-resistant strain of A. halophila (strain ER) that required only 6% NaCl for growth. Both cell wall preparations contained glutamic acid, alanine, and diaminopimelic acid in a 1:2:1 molar ratio. On the basis of muramic acid content, peptidoglycans from the wt and ER strains contained 255 and 245 disaccharide units per mg dry weight respectively. In addition, both cell wall preparations contained from 10 to 20% more glucosamine than muramic acid, and equimolar amounts of d-galactose and d-arabinose. Analysis of cell walls before and after digestion with Myxobacter AL-1 protease indicated that nearly all glycan disaccharide units were peptide-substituted and that peptide cross-bridging was facilitated by direct peptide linkages between N-diaminopimelic acid and C-terminal alanine. While the peptidoglycan of A. halophila wt was 50% peptide cross-linked, that from A. halophila ER was approximately 67% peptide cross-linked. Chemical modifications involving substitution of non-N-acetylated hexosamines of the cell walls greatly enhanced their sensitivity to lysozyme. Although differences in peptidoglycan structure between the two strains of A. halophila were observed, these probably do not account for the reduced salt requirement for growth of the erythromycin-resistant strain.Issued as NRCC 25165     

Occurrence of diaminopimelic epimerase in maize

Extracts of maize leaves catalyzed the interconversion of meso-diaminopimelic acid its L-isomer. Three observations support the existence of this epimerase activity: (i) detection of the reversible interconversion of L-diaminopimelic acid and meso-diaminopimelic acid by paper chromatography after incubation of either isomer with extract; (ii) formation of [¹⁴C]CO₂ from L-[¹⁴C]diaminopimelic acid in an incubation mix containing meso-diaminopimelic acid decarboxylase; and (iii) inhibition of [¹⁴C]CO₂ evolution from L-diaminopimelic acid by unlabeled meso-diaminopimelic acid. The demonstration of the diaminopimelic acid epimerase lends support to the occurrence in plants of the complete diaminopimelic acid pathway for biosynthesis of lysine as it occurs in Escherichia coli and most bacteria.     

Murein (peptidoglycan) structure of Vibrio fetus comparison of a venereal and an intestinal strain

The murein of a venereal and an intestinal strain of Vibrio fetus was isolated by extraction with hot 4% sodium dodecyl sulfate, indicating the absence of covalently bound protein. Murein was composed of muramic acid, glucosamine, alanine, glutamic acid and diaminopimelic acid in molar ratios of 1:1:2:1:1. Approx. 30% of Dpm molecules were involved in peptide cross linkages and analyses of lysozyme split products indicated a structure similar to that of other Gram-negative genera. Evidence was obtained for the occurrence of chromatographically distinct fractions of the disaccharide tetrapeptide (GlcNAcMurNAclAladGlumesoDpmdAla). Digestion products also included variable concentrations of free murein peptides and glucosamine, whose origin is unexplained. In no instance were differences observed between mureins of intestinal and venereal strains of V. fetus.     

Evidence for the incorporation of molecular oxygen,a pathway in biosynthesis of N-glycolylmuramic acid in Mycobacterium phlei

The hypothesis that the biosynthesis of the glycolyl group of muramic acid in the peptidoglycan of Myobacterium phlei is catalyzed by an N-acetyl hydroxylase is strongly supported by the experiments reported in this paper. ¹⁸O is incorporated into the N-substituent of muramic acid isolated from the peptidoglycan of M. phlei grown under pure oxygen enriched with the ¹⁸O isotope.     

Analysis of muramic acid in holocene microbial environments by gas chromatography,electron impact,and fast atom bombardment mass spectrometry

Analytical procedures have been modified to determine the abundance of muramic acid in four different Holocene sediment samples. Muramic acid is specific to the peptidoglycan moiety of the cell walls of most eubacterial pro‐karyotic organisms. The following procedure seemed to be the most appropriate for the detection of muramic acid and amino acids, including diaminopimelic acid. Hydrolysis of the samples (in 6 N HCl, 4.5 h, at 100°C) was followed by separation and purification of amino sugars and amino acids using Amberlite XAD‐2 and then Bio‐Rad AG 50W‐X8 resins. The N,O‐heptafluorobutyryl‐n‐butyl ester derivatives were prepared by esterification in acidified (3 N HCl) n‐butanol for 3 h at 100°C, followed by acylation by refluxing with heptafluorobutyric anhydride in acetonitrile (2:1 v/v) for 12 min at 150°C. The derivatives were analyzed by gas chromatography (GC) and gas chromatography‐mass spectrometry. Fast atom bombardment (FAB) ionization was used for the muramic acid derivative to determine its molecular weight and structure, d‐and l‐amino acids were separated by GC and a capillary chiral column. By using this technique a stable N,O‐heptafluo‐robutyryl‐n‐butyl ester derivative of muramic acid was identified at picogram levels in Holocene sedimentary microbial communities. It has been reported previously that microorganisms in sediments rapidly degrade muramic acid from cell walls of dead prokaryotes. Kinetic experiments revealed that muramic acid was relatively stable in intact cell walls but decomposed rapidly in the free form. These investigations noted above showed that the concentration of muramic acid may be used as an indicator of the presence of the intact cell walls of cyanobacteria and most other bacteria in Holocene microbial communities, and of microbial contamination in samples older than the Holocene.     

Envelope structure in three different L-forms ofProteus mirabilis

One A-type, stable and two different B-type, unstable L-forms were obtained from a strain ofProteus mirabilis and studied by electron microscopy and by chemical analysis for the presence of peptidoglycan. The wall of the parent bacterium is characterized by a profile of three superimposed dense lines and a content of 11.07 nmoles of muramic acid (MUR) and of 7.85 nmoles of diaminopimelic acid (DAP) per mg of dry weight. The stable, A-type L-form has completely lost the cell wall of the bacterium and is enveloped only by the plasma membrane to which very small quantities of peptidoglycan components are associated (MUR: 0.041 nmoles/mg; DAP: 0.075 nmoles/mg). The two B-type, unstable L-forms have the same wall structure in only two dense lines, but they differ in their peptidoglycan content. The first one does not contain more peptidoglycan components than the A-type, L-form (MUR: 0.022 nmoles/mg; DAP: 0.016 nmoles/mg), whereas the peptidoglycan content of the second one (MUR: 2.6 nmoles/mg; DAP: 1.65 nmoles/mg) is about one fifth of the content of muramic acid and diaminopimelic acid of the bacterial cell wall.     

The effect of cell wall components on glycine-enhanced sterol side chain degradation to androstene derivatives by mycobacteria

β-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of β-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria. Received: 12 January 1999 / Received revision: 21 June 1999 / Accepted: 27 June 1999     

Die Bildung und die Reversion von Sphäroplasten einer Diaminopimelinsäure-auxotrophen Mutante von Escherichia coli

1. The formation and reversion of spheroplasts of the diaminopimelic acid-auxotrophic mutant Escherichia coli K 12, 335, dap ⁻, R⁺TEM in a medium lacking diaminopimelic acid have been investigated by microphotography: During their development from rod form cells to spheroplasts cells on slide-surface-agar preparations underwent two successive cell divisions in the course of which the cells retained their rod form. The cells formed by these divisions partitioned into a varying number of spheroplasts of different size. The reversion of spheroplasts to rod form cells, started by the addition of diaminopimelic acid showed two characteristic steps: Each spheroplast partitioned again into several spheroplast-like cell bodies; most of them reverted directly to rod form cells.
2. The release of the R-factor mediated periplasmic TEM-β-lactamase, E. C. 3.4.2.6., into the growth medium during the development of spheroplasts attained more than 50% of the entire TEM-β-lactamase activity.

The spheroplasts showed a multiple enhancement of TEM-β-lactamase activity per mg cell protein compared with rod form cells.     

Isolation, Characterization, and Ultrastructure of the Peptidoglycan Layer of a Marine Pseudomonad

The peptidoglycan layer of a marine pseudomonad was observed by electron microscopy in thin sections of plasmolyzed intact cells and mureinoplasts but not in untreated intact cells. Only fragments of this layer could be isolated by sodium lauryl sulfate (SLS) treatment of mureinoplast envelopes. Sacculus-like peptidoglycan structures were obtained from growing cells by immediate heat inactivation of cellular autolytic enzymes and subsequent SLS, trypsin, and nuclease treatments. Recently, similar peptidoglycan sacculus-like structures have been obtained by adding SLS to the growing culture and treating the isolated particulate material with nucleases. Thin-sectioned and negatively stained preparations of whole cell peptidoglycan showed compressed profiles of cell-shaped sacculi. Peptidoglycan prepared by SLS treatment of mureinoplast envelopes had a similar composition to that prepared from whole cells. The major amino sugars and amino acids in the peptidoglycan component were glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in the molar ratios 1.18:1.24:1.77:1.00:0.79. Forty-five per cent of the epsilon-amino groups of diaminopimelic acid were cross-linked. The peptidoglycan was estimated to account for about 1% of the cell dry weight.     

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.     

Determination of muramic acid in organic dust by gas chromatography-mass spectrometry

A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography-mass spectrometry. The muramic acid derivative gave two peaks upon injection into the gas chromatograph-mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5–1500 ng of muramic acid. In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Metabolism of abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgaris

When (±)-abscisic acid-[2-¹⁴C] or (±)-abscisic acid-[4′-¹⁸O] was fed to bean (Phaseolus vulgaris) shoots, phaseic acid (PA) and dihydrophaseic acid (DPA) were the major metabolites, while epi-dihydrophaseic acid (epi-DPA) appeared as a minor metabolite. In the acidic fraction the amount of epi-DPA ranged from 18 to 42% of the DPA content, in the conjugated form from 50 to 200%. The content of endogenous epi-DPA amounted to only 1–2% of that of the DPA. These data indicate that the applied abscisic acid is not metabolised in a manner identical with that of the endogenous material. DPA and epi-DPA were shown to be formed separately from PA and could not be inter-converted either by the extraction conditions employed or when fed to bean shoots during short term experiments.