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.     

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.     

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.     

Muramic acid as a measure of microbial biomass in estuarine and marine samples.

Muramic acid, a component of the muramyl peptide found only in the cell walls of bacteria and blue-green algae, furnishes a measure of detrital or sedimentary procaryotic biomass. A reproducible assay involving acid hydrolysis, preparative thin-layer chromatographic purification, and colorimetric analysis of lactate released from muramic acid by alkaline hydrolysis is described. Comparison of semitropical estuarine detritus, estuarine muds, and sediments from anaerobic Black Sea cores showed muramic acid levels of 100 to 700 microng/g (dry weight), 34 microng/g, and 1.5 to 14.9 microng/g, respectively. Enzymatic assays of lactate from muramic acid gave results 10- to 20-fold higher. Radioactive pulse-labeling studies showed that [14C]acetate is rapidly incorporated into muramic acid by the detrital microflora. Subsequent loss of 14C, accompanied by nearly constant levels of total muramic acid, indicated active metabolism in procaryotic cell walls.     

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.     

Binding of Polysaccharide to Peptidoglycan in Cell Wall of Streptomyces roseochromogenes

The polysaccharide-peptidoglycan complex, which was prepared with lysozyme from Streptomyces roseochromogenes IAM53 cell walls, was hydrolyzed with lytic enzyme of Flavo-bacterium to separate polysaccharide. The enzymatically prepared polysaccharide (100 mg) contained 500 μmoles of hexoses, 40 μmoles of hexosamines and 31 μmoles of phosphate. Hexoses consisted of mannose and galactose in a molar ratio of 5 to 1. Hexosamines consisted of equimolar glucosamine and muramic acid, a half of which was identified as muramic acid 6-phosphate. The reducing end of the polysaccharide was muramic acid. The polysaccharide extracted with trichloroacetic acid contained no muramic acid-phosphate. So the polysaccharide moiety of S. roseochromogenes cell walls must be linked covalently to 6-position of muramic acid in peptidoglycan through phosphate,     

Improved method using muramic acid to estimate biomass of bacteria in sediments

Summary A method, which depends on the measurement of muramic acid content to estimate bacterial biomass, has been improved in sensitivity by two orders of magnitude. It is now applicable to any aquatic sediment, whereas previously it was mainly useful in the analysis of gut contents of deposit-feeding animals. Reduced NAD, a product of the oxidation of d-lactate derived from muramic acid, is assayed using bacterial luciferase. The amount of muramic acid in a number of terrestrial and marine bacteria was measured, and found to be lower than that obtained with the previous, less specific, assay procedure. The muramic acid content of a blue-green alga has been measured, thus allowing blue-green algae to be taken into account when estimating bacterial biomass. Experimental evidence is presented which shows that muramic acid in cell wall fragments of bacteria is rapidly degraded by microorganisms in a marine sediment.     

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.     

A simple method for the quantitative determination of muramic acid

A simple method for microdetermination of muramic acid is elaborated. The method is based on the degradation of muramic acid to lactic acid, followed by degradation of the latter to acetaldehyde which can be determined colorimetrically with p-hydroxydiphenyl (PHD). A linear relationship exists between the concentration of muramic acid (up to 20 μg), and absorbance at 560 nm. Substances usually present in the hydrolysates of bacterial cell wall peptidoglycan do not interfere in the determination.     

Capillary gas chromatography using electron capture or selected ion monitoring detection for the determination of muramic acid,diaminopimelic acid and the ratio of d/l-alanine in bacteria

Gas chromatographic analyses of muramic acid, diaminopimelic acid and D-alaline, which are specific components of the bacterial cell wall, have been performed using electron capture or selected ion monitoring detection. Intact cells or peptidogylycan preparations were hydrolyzed in HCl and DCl. After purification by cation exchange chromatography, followed by conversion to the N-heptafluobutyrliso-butyl esters, the components were separated on a 25 m fused silica column coated with SE-54 or on a chiral glass capillary column.The detection limits for muramic acid and diaminopimelic acid were about 10 pg using either detection method and the procedure has the potential sensitivity for detecting about 3 × 10⁵ bacterial cells, e.g., Escherichia coli.Mass spectrometric determination of the d/l ratio of alamine in intact cells of Group A streptococci, type M 15 and in peptidogylcan preparations thereof indicated the proportions 10.2% and 10.5% of D-alanine, respectively. The values uncorrected for racemization during acid hydrolysis were 10.3% and 10.7%, respectively.     

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.     

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.     

Peptidoglycan Differences in Strains of Bacillus cereus Constitutive and Inducible for Penicillinase Production

Strains of Bacillus cereus differing in penicillinase production were shown to possess cell walls of differing muramic acid contents.     

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.     

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.     

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.     

干湿交替条件下土壤氨基糖含量的动态变化

通过室内模拟培养试验,研究了恒湿和干湿交替条件下土壤中3种微生物来源氨基糖含量的动态变化,并且利用氨基葡萄糖和胞壁酸的比值分析了干湿交替条件下土壤真菌和细菌对土壤有机质转化的相对贡献.结果表明:恒湿条件下,细菌来源的胞壁酸在土壤中的分解速率大于真菌来源的氨基葡萄糖,氨基半乳糖在土壤中的分解速率较慢;干湿交替改变了土壤中3种氨基糖的分解特征,与恒湿处理相比,干湿交替培养前期以胞壁酸为代表的细菌残余物的分解速率高于以氨基葡萄糖为代表的真菌残余物,随着干湿交替频率的增大,以氨基葡萄糖为代表的真菌残余物分解速率高于以胞壁酸为代表的细菌残余物.可见,干湿交替条件改变了以氨基糖为代表的土壤氮素的微生物转化过程.     

Structure of muramic acid TMS derivative mass spectrum's base ion (m/z=185) used for quantification of bacterial peptidoglycan.

The use of trimethylsilyl (TMS)-derivatisation for determining muramic acid in environmental and clinical samples by gas chromatography-mass spectrometry provides high detection sensitivity; however, questions have been raised as concerns the chemical structure of the entity giving the strong signal of m/z 185. In the present communication we present evidence that this entity results from the formation of a lactam structure of muramic acid upon derivatisation.     

CHEMICAL CHARACTERIZATION OF THE ISOLATED CELL SURFACE OF AMOEBA

The cell surface has been isolated from uninucleate, freshwater, phagocytic amoebae by a new procedure. Several criteria were employed to demonstrate purity of the cell surface fraction. All morphological components of the tripartite surface were present in the isolated surface and the weight of the isolated surface was quantitatively accounted for by the components analyzed. Chemical analyses showed the presence of lipid, protein, and carbohydrate. Mannose was the predominant neutral sugar. Analyses for three different strains of Amoeba were similar. Phosphate was found to be the major anionic group in the cell surface material. Sulfate, uronic acid, sialic acid, muramic acid, and nonamidated glutamic acid and aspartic acid were absent. Evidence is presented suggesting that the phosphate is associated with an unidentified nonreducing polyol.     

Studies on the specificity of action of bacteriophage T4 lysozyme.

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endo-acetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide side-chains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted.