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Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens - their formidable cell wall - could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the present work, Principal coordinate analysis (PCoA) is introduced to develop a robust model to classify the chromatographic data sets of peptidoglycan sample. PcoA captures the heterogeneity present in the data sets by using the dissimilarity matrix as input. Thus, in principle, it can even capture the subtle differences in the bacterial peptidoglycan composition and can provide a more robust and fast approach for classifying the bacterial collection and identifying the novel cell wall targets for further biological and clinical studies. The utility of the proposed approach is successfully demonstrated by analysing the two different kind of bacterial collections. The first set comprised of peptidoglycan sample belonging to different subclasses of Alphaproteobacteria. Whereas, the second set that is relatively more intricate for the chemometric analysis consist of different wild type Vibrio Cholerae and its mutants having subtle differences in their peptidoglycan composition. The present work clearly proposes a useful approach that can classify the chromatographic data sets of chromatographic peptidoglycan samples having subtle differences. Furthermore, present work clearly suggest that PCoA can be a method of choice in any data analysis workflow., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An antibacterial Cp was extracted from the stem of Cissus quadrangularis and purified with a 5.39 fold increase in specific activity and 8.67% recovery. The molecular weight of the purified enzyme was estimated to be 39kDa by SDS-PAGE. The purified enzyme appeared as a single band on Native-PAGE. The optimum pH and temperature for protease activity were around 6.0 and 50°C respectively. The Cp showed pH stability from 3 to 10 and retained more than 90% of its relative protease activity. The addition of metal ions such as Mg 2+ and Ca 2+ also exhibited relative protease activity. Cp showed a potent antibacterial activity against pathogenic bacteria. About 4.74Uml -1 of Cp from C. quadrangularis was tested for antibacterial activity against Bacillus cereus and Bacillus megaterium which subsequently showed zone of inhibition of 21 and 20mm respectively. Cp from C. quadrangularis degraded the peptidoglycan layer of bacteria by Cp was confirmed by transmission electron microscopic analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25-100pg/ml of LPS and 25-100ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25μg/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3kDa to 100kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation. The released sulfonated derivative was water soluble which was suitable for detailed NMR analysis. The data exhibited the presence of two stoichiometric and one substoichiometric (relative abundance was about 0.5) acetylations, suggesting that the glycoconjugate is composed of two equimolar pentasaccharide-dipeptide repeating units each having either two or three acetyl groups. However, the position of substoichiometric acetylation could not be defined. To determine the position, the sheath was derivatized with a thiol selective fluorescent reagent followed by digestion with a specific polysaccharide lyase prepared from a sheath-degrading bacterium, Paenibacillus koleovorans. As expected, two fluorescent digests were recovered by reverse-phase HPLC and were subjected to NMR analysis. The data revealed that both digests are pentasaccharide-dipeptides which have unsaturated glucuronic acid and galactosamine residues at their reducing and non-reducing ends, respectively. It was also confirmed that one digest has 3-O-acetylated glucose residue while the other has non-derivatized glucose residue. The substoichiometric acetylation was thus identified with the 3-O-acetylation, and structural determination of the thiolic glycoconjugate was completed. By virtue of the clarification of the two digests' structures, the cleavage site was specified as (1→4)-α-galactosaminic bond to glucuronic acid. Based on the present and earlier findings, we propose a novel glycoconjugate category named thiopeptidoglycan and a novel polysaccharide lyase named thiopeptidoglycan lyase., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Quantification of solid cell material (cell debris) is necessary for the optimisation of the efficiency of bioseparations. Cell debris can be quantified by detection of a component present in the cell wall that can act as a marker for cell debris. Membrane-associated proteins have previously been used as a marker for cell debris. This marker was quantified by SDS-PAGE with densiometry. In this paper cell debris quantification methods are presented that are faster and more accurate, i.e. membrane-associated protein quantification with the Protein 50 Labchip of Agilent Technologies, or that make use of peptidoglycan as marker for cell debris, i.e. a spectrophotometric muramic acid assay.

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

A gas chromatographic-ion trap tandem mass spectrometric method was developed for the quantification of muramic acid, a marker of bacterial peptidoglycan, in environmental and clinical specimens. Samples (bacteria, house dust and urine) were heated in methanolic hydrochloric acid overnight and extracted with hexane for removal of hydrophobic compounds. The aqueous phase was evaporated and heated in acetic anhydride and pyridine after which the product, the acetate derivative, was washed with dilute hydrochloric acid and water. The described method is both rapid and simple to apply, and produces a stable derivative. It should become widely used for measuring peptidoglycan in chemically complex environments.

A high-performance anion-exchange chromatography method with pulsed-amperometric detection has been developed for the simultaneous analysis of both amino acids and amino sugars and applied to the compositional analysis of peptidoglycan hydrolysates. Chromatography of the acid hydrolysis products was performed on a CarboPac PA-1 anion-exchange column, with pulsed-amperometric detection. Complete resolution of the two amino sugars (glucosamine and muramic acid) and eight of the nine amino acids (Ala, diaminobutyric acid, diaminopimelic acid, Glu, Gly, homoserine, Lys, Orn, and Ser) known to occur in various peptidoglycans was achieved within 70 min. Only homoserine and glycine (retention times 26.8 and 26.9 min, respectively) were not resolved by this procedure, but the simultaneous occurrence of these two amino acids in peptidoglycan is extremely rare. Reproducibility of the separations was shown to be very high and detection limits exceeded 10 pmol for glucosamine. This convenient and simple analysis was applied to the quantitation of many crude peptidoglycan samples isolated from the species of the Proteeae (Proteus, Providencia, and Morganella) for the determination of the extent of peptidoglycan O-acetylation.

The effects of mecillinam, ampicillin and cephalexin on peptidoglycan synthesis in Salmonella typhimurium 2616 have been studied at equivalent concentrations or "isoactivities". Using antibiotics at isoactivities allows a direct comparison of the biochemical effects of different antibiotics. When mecillinam was added at different times during the division cycle at a concentration that produced 50% inhibition of peptidoglycan synthesis in an exponential culture over a short period of time, the inhibition of synthesis was greatest in the newborn cells and least in the dividing cells. Antibiotic competition experiments showed that mecillinam preferentially bound to penicillin-binding protein 2 in S. typhimurium 2616. High performance liquid chromatography analysis of the residual peptidoglycan synthesized in the presence of mecillinam showed an unexpected increase in pentapeptides and a significant increase in cross-linking. Other antibiotics added at equivalent activities did not show an increase in cross-linking.

The preparation and characterization of the essential components to be used in the radioimmunoassay of peptidoglycan monomer (PGM) is described. In order to raise the anti-peptidoglycan monomer antibodies 14C-labelled peptidoglycan monomer-bovine serum albumin conjugate was prepared by the coupling of 14C-peptidoglycan monomer to bovine serum albumin in the presence of glutaraldehyde in 0.1 M NaHCO3 at pH 8.3. The prepared conjugate elicited anti-PGM response in rabbits. A synthetic analog of peptidoglycan monomer, Boc-L-tyrosyl-peptidoglycan monomer was prepared by condensation of unprotected peptidoglycan monomer and N-hydroxysuccinimidester of Boc-L-tyrosine in the presence of triethylamine and the obtained disaccharide-hexapeptide was labelled with Na125I. This compound exhibited the ability of binding to anti-peptidoglycan monomer antibodies. The prepared compounds, namely anti-PGM antibodies and 125I-labelled Boc-L-tyrosyl-peptidoglycan monomer, were used as essential components in competitive radioimmunoassay for peptidoglycan monomer determination in mammalian and human sera and plasma, respectively.

Separation of amino compounds, including galactosamine and glucosamine, found in lipopolysaccharide and peptidoglycan, can be accomplished by two-dimensional thin-layer chromatography on cellulose layers. The solvent systems used are: 2-propanol-90% formic acid-water (80:4:20,v/v) in the first dimension and lutidine-water (65:35, v/v) in the second dimension. Compounds were detected using a chromogenic ninhydrin spray.

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.

We describe a method for determination of lysostaphin activity using Remazol Brilliant Blue R (RBB)-dyed staphylococcal cells or RBB-dyed staphylococcal peptidoglycan as substrate. The dyed substrates are easy to prepare and are stable for at least 6 months. Soluble hydrolytic products released by lysostaphin are measured spectrophotometrically at 595 nm after the insoluble substrate is removed by filtration or centrifugation. The dye release assay is more sensitive and more accurate than the previously described turbidimetric assay.

This review deals with the chemistry of marker substances used in microbial classification and identification, their isolation and purification and their biomedical application. A critical evaluation of current methods is also included. The information presented is partly based on personal experience, partly derived from more than 300 publications in this rapidly expanding field of science. Much has been done to improve the recognition of microorganisms by GC, but there are also a series of other techniques available that can assist in bacterial classification and identification. Some of these techniques have been made available to the clinical microbiologist through commercial systems, e.g., assessment of bacterial fatty acids. A fingerprint library has been developed by Hewlett-Packard for the analysis of fatty acids from approximately 6000 different bacteria. Other chemotaxonomic methods require great personal expertise and advanced equipment. Efforts should therefore be made to adapt and simplify such methods for application in the routine clinical laboratory. Chemical markers will probably have a great impact on future microbial taxonomy, particularly in cases where conventional methods fail to give satisfactory classifications. In order to make taxonomy more objective, there seems to be a need for screening of chemical markers in bacterial species and for compiling chemotaxonomic fingerprints in clinical manuals.

The transition from rod-shaped to spheroidal cells was studied in a temperature-sensitive strain (SP45) of Escherichia coli K12, carrying a mutation (pbpA) in the gene coding for penicillin-binding protein 2 (PBP-2). This transition imposed by the restrictive temperature was associated with reduction of peptidoglycan/surface area and of cellular osmotic stability. Addition of nalidixic acid (20 micrograms/ml) at the temperature shift from 30 to 42 degrees C resulted in lysis of some cells and appearance of spheroidal bulges along the cylinders in other cells, consistent with the hypothesis of envelope weakening due to inactivation of PBP-2.

A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.

D-Alanine and diaminopimelic acid originating from bacterial peptidoglycans and hydroxy fatty acids from lipopolysaccharides (endotoxins) were analysed by gas chromatography using a chiral column (Chirasil-Val as stationary phase) and selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry. The amino acids were analysed as N-heptafluorobutyryl isobutyl esters after rapid hydrolysis of peptidoglycan followed by isolation of the amino acids with disposable ion-exchange columns. Racemization of amino acid enantiomers was controlled by using deuterium chloride in the hydrolysis. The hydroxy acids were analysed as O-pentafluorobenzoyl methyl esters. Most of the bacteria present in airborne dust from a poultry confinement building were found to be Gram-positive according to the analytical chemical method whereas the Limulus amoebocyte lysate test suggested the presence of appreciable amounts of lipopolysaccharides of Gram-negative bacteria. Further studies are required to compare the utility of these two methods for determining endotoxins in complex environments.