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PubMed2005

GC/MS identification and quantification of constituents of bacterial lipids and glycoconjugates obtained after methanolysis as heptafluorobutyrate derivatives.

In previous articles [Anal. Biochem. 284 (2000) 201; J. Lipid Res. 43 (2002) 794], we reported that the GC/MS identification and quantification of nearly all constituents of glycolipids could be obtained on the same sample in a single GC/MS analysis as heptafluorobutyrate derivatives of the products liberated using acid-catalyzed methanolysis. The same type of data could be obtained on glycoproteins and proteoglycans [Biochemistry 42 (2003) 8342]. These experiments were performed on material from higher organisms, and there was no evidence that bacteria-specific constituents could also be identified and quantified. The current article reports that the GC/MS analysis of compounds liberated by acid-catalyzed methanolysis as heptafluorobutyrate derivatives allows the simultaneous qualitative and quantitative determinations of pentoses, deoxyhexoses, hexoses, hexosamines, uronic acids, Kdo, Mur, heptose, Kdn, and neuraminic acid as well as of most fatty acids (including hydroxylated fatty acids). This approach provides a way of obtaining fingerprints of bacterial constituents and quantification of the overall effect of gene inactivation or of culture conditions.

Bacteria↗
PubMed1982

Induction of coordinated movement of Myxococcus xanthus cells.

Rhythmically advancing waves of cells, called ripples, arise spontaneously during the aggregation of Myxococcus xanthus into fruiting bodies. Extracts prepared by washing rippling cells contain a substance that will induce quiescent cells to ripple. Three lines of evidence indicate that murein (peptidoglycan) is the ripple-inducing substance in the extracts. First, ripple-inducing activity is associated with the cell envelope of sonically disrupted M. xanthus cells. Second, whole cells, cell extracts, or purified murein from a variety of different bacteria are capable of inducing ripples. In contrast, extracts prepared from Methanobacterium spp. which contain pseudomurein instead of typical bacterial murein fail to induce ripples. Third, four components of M. xanthus murein, N-acetylglucosamine, N-acetylmuramic acid, diaminopimelate, and D-alanine, are able to induce ripples. Ripples produced by aggregating cells have a wavelength of 45 micrometers and a maximum velocity of 2 micrometers/min. Both of the multigene systems that control gliding motility appear to be required for rippling, and all known mutations at the spoC locus eliminate both rippling and sporulation.

Acetylglucosamine↗
PubMed1999

Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl- L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth.

The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the epsilon-amino group of L-lysine and the alpha-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.

Bacteriolysis↗
PubMed2005

Scission of the lactyl ether bond of N-acetylmuramic acid by Escherichia coli "etherase".

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

Carbon↗
PubMed1975

Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K 12. Purification and properties of the enzyme.

N-Acetylmuramyl-L-alanine amidase activity was detected in Escherichia coli K 12 by usine N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)-meso-[3H]diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N-acetylmuramyl acid and L-alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K 12. The enzyme was purified to near homogeneity. A molecular weight of 39 000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N-acetylmuramyl-L-alanine amidase activity was carried out by investigating several of its properties.

Alanine↗
PubMed1976

Correlation between the immunoadjuvant activities and pyrogenicities of synthetic N-acetylmuramyl-peptides or -amino acids.

A total of 14 different N-acetylmuramyl-peptides or -amino acids with or without configurations inherent to bacterial cell wall peptidoglycans were synthesized and their pyrogenicities on intravenous injection into rabbits were tested. N-Acetylmuramyl-peptides, and especially N-acetylmuramyl-L-alanyl-D-isoglutamine and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine, which were previously shown to be adjuvant-active in both induction of delayed-type hypersensitivity and stimulation of increased serum antibody levels to ovalbumin in guinea pigs, exhibited distinct pyrogenicity at as low dose as 16 mug per rabbit. However, none of the adjuvant-inactive analogues or diastereomers of the above N-acetylmuramyl-dipeptide or related compounds caused any significant febrile response in rabbits, even at a dose of 250 mug per animal.

Adjuvants, Immunologic↗
PubMed1980

Release of soluble peptidoglycan from growing conococci: demonstration of anhydro-muramyl-containing fragments.

Previous analysis of soluble peptidoglycan (PG) fragments released by exponentially growing gonococci implicated the combined action of both hexosaminidase and amidase activities in PG turnover. Current studies further characterized PG fragments which were labeled in the glycan with D-glucosamine and in the peptide moiety with meso-diaminopimelic acid of L- and D-alanine. Labeled PG fragments were isolated by gel filtration and characterized on the bases of (i) KD values, (ii) free amino group analysis using fluorodinitrobenzene, (iii) borohydride reduction, (iv) alkali-catalyzed beta-elimination, (v) paper chromatography in various solvents, (vi) electrophoretic mobility at various pH values, (vii) digestibility by Charonia lampas glycosidases, and (viii) content of labeled D- and L-alanine. A set of well-characterized PG fragments was used as standards. The monomer fraction (the major extracellular product) was found to contain two components. Most (about 80%) appeared to be N-acetylglucosaminyl-beta-1 leads to 4-1,6-anhydro-N-acetylmuramyl-L-ala-D-glu-meso-diaminopimelic acid; the remainder was the corresponding disaccharide tetrapeptide containing a C-terminal D-alanine. An unusual feature of these products was the presence of the anhydro-muramyl (non-reducing) ends, reflecting the activity of a gonococcal transglycosylase, and the near absence of products containing detectable reducing ends. Otherwise, the structures of the monomer fragments were typical of those expected for a gram-negative bacterium (chemotype I). The corresponding peptide-cross-linked dimer and the free disaccharide also contained nonreducing ends, exclusively. Free peptides (products of amidase activity) consisted of both tripeptide and tetrapeptide. In summary, all gonococci examined appear to possess an unusual transglycosylase activity which contributes to the release of soluble PG fragments containing nonreducing, anhydro-muramyl ends. The release of these fragments in vivo might be a unique aspect of gonococci-host interactions.

Amino Acids↗
PubMed1973

Chemical analysis of cell walls and autolytic digests of Bacillus psychrophilus.

The amino acid composition of isolated cell walls of Bacillus psychrophilus has been determined before and after extraction of protein with ethylenediamine-tetraacetic acid at 45 C. This revealed that the peptidoglycan consists of Ala, Lys, and Glu in a molar ratio of 3:1:2. By using autolytic digests of log-phase cell walls, it was possible to detect 14 ninhydrin-positive degradation products. Chemical analyses of the seven major bands from these digests indicated that the amino acid sequence of the peptide subunit in the murein of this organism consists of muramyl-l-alanyl-gamma-d-glutamyl-l-lysyl- d-alanine, and the linkage between adjacent peptides is supplied by a second d-glutamic acid which is bound to the sigma-amino group of lysine and the carboxyl group of the d-alanine through its amino group. The nature of the solubilized wall fragments indicates that each of the peptide bonds in the murein is hydrolyzed by autolysins except the l-alanyl-gamma-d-glutamyl linkage.

Alanine↗
PubMed1989

Peptidoglycan synthesis during the cell cycle of Escherichia coli: composition and mode of insertion.

The composition and the mode of insertion of peptidoglycan synthesized during the cell cycle of Escherichia coli were determined. This was carried out on peptidoglycan that was periodically pulse-labeled in synchronously growing cultures. The chemical composition of the pulse-labeled (newly synthesized) peptidoglycan remained constant throughout the cell cycle, as judged from high-pressure liquid chromatography analysis of the muropeptide composition. The mode of insertion was deduced from the acceptor-donor radioactivity ratio in the bis-disaccharide tetratetra compound. The ratio was low in elongating cells and high in constricting cells. This indicates that during elongation, peptidoglycan was inserted as single strands, whereas during constriction, a multistranded (or sequential single-stranded) insertion occurred. Experiments with an ftsA division mutant suggested that the composition and mode of insertion of newly synthesized peptidoglycan remained the same throughout the constriction process. Our results imply that the changed mode of insertion rather than the chemical structure of the peptidoglycan might be responsible for the transition from cell elongation to polar cap formation.

Carbohydrate Sequence↗
PubMed1976

Escherichia coli mutants altered in murein lipoprotein.

Mutants with alterations in the structure, biosynthesis, or assembly of murein lipoprotein were selected by a procedure based on radiation suicide of wild-type organisms by [3H]arginine under conditions where the radioactive arginine was preferentially incorporated into lipoprotein. Further screening for the potential mutants among the survivors of [3H]arginine suicide was carried out by using a sensitive immunodiffusion test, followed by radioactive double-labeling experiments. Three mutants were obtained and partially characterized.

Alanine↗
PubMed1975

N-acetylmuramyl-L-alanine amidase of Vi phage III.

1. A lytic enzyme was isolated from Vi phage III-induced lysate of Salmonella typhi, and purified about 200-fold by chromatography on IRC-50, CM-cellulose, and Sephadex G-75 columns. 2. Both E. coli B murein and muropeptide C6 were digested on incubation with the lytic enzyme. The main product of murein and muropeptide C6 digestion is identical with tetrapeptide Ala-Glu-DAP-Ala. The release of amino groups during digestion was not accompanied by the appearance of either reducing groups or hexosamines. 3. It is concluded that Vi phage III-induced lytic enzyme is N-acetylmuramyl-L-alanine amidase, which cleaves the amide bond between N-acetylmuramic acid and L-alanine.

Amidohydrolases↗
PubMed1997

Structural characterization of peptidoglycan muropeptides by matrix-assisted laser desorption ionization mass spectrometry and postsource decay analysis.

In this study we report the development of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)-based methods for the structural characterization of muropeptides derived from peptidoglycan. Prior to analysis, peptidoglycan samples were subjected to enzymatic digestion with muramidase and the resulting muropeptides were purified by HPLC. A new matrix, 5-chloro-2-mercaptobenzothiazole, was employed for the MALDI-MS analysis. The results have demonstrated that sub-picomole to femtomole detection can be achieved in both positive mode and negative mode, allowing unambiguous determination of the molecular masses of monomeric and oligomeric muropeptides. Structural information from monomeric muropeptides was obtained by further postsource decay (PSD) analysis. Fragmentation patterns in positive mode and negative mode PSD were complementary for the elucidation of the peptide chain sequence. Lysostaphin digestion was also incorporated with MALDI mass mapping analysis for determination of peptide chain cross-linking patterns of muropeptide oligomers from Staphylococcus aureus strains.

Amino Acid Sequence↗
PubMed1975

Alterations in the composition and bacteriophage-binding properties of walls of Staphylococcus aureus H grown in continuous culture in simplified defined media.

A nutritional mutant of Staphylococcus aureus H has been isolated and grown in media in which the only amino acids are arginine, cysteine, glutamic acid and proline. Walls of the bacteria grown in such media in continuous culture under potassium limitation differ in composition from walls of the bacteria grown in batch culture in rich nutrient broth in that they contain less glycine, the peptidoglycan component is less highly cross-linked and the teichoic acid component contains a reduced proportion of N-acetylglucosaminyl substituents. Walls of the potassium-limited bacteria retain the ability to bind bacteriophage 52a but are more susceptible to the action of lytic peptidases than are wall samples in which the peptidoglycan is more highly cross-linked. Teichoic acid was present in walls of the bacteria grown under phosphate limitation in the defined medium and these walls were also able to absorb bacteriophage 52a.

Amino Acids↗