Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.     

Quantitative Chemical Analyses and Antigenic Properties of Peptidoglycans from Clostridium botulinum and Other Clostridia

The cell wall peptidoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The peptidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histolyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the Clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other Clostridia as well as various types of C. botulinum.     

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.     

Structure of rigid-layer of Rhizobium cell wall. I. Purification on the peptidoglycan from the cellulose microfibrils

The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion. Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose. Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils. The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80%. Methods of peptidoglycan purification from the cellulose microfibrils are described.     

Extent of peptidoglycan O acetylation in the tribe Proteeae.

The degree of peptidoglycan O acetylation in 18 strains of the different genera of the tribe Proteeae (Proteus, Providencia, and Morganella) has been determined by high-performance liquid chromatography-based organic acid analysis of mild-base-released acetic acid and quantitation of peptidoglycan concentrations by simultaneous amino sugar-amino acid analysis using high-performance anion-exchange chromatography with pulsed amperometric detection. The N,O-diacetylmuramyl content of all isolated and purified peptidoglycans was greater than 29% and ranged up to 57% relative to total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by hen egg white lysozyme.     

Carbohydrate Metabolism in Spirochaeta stenostrepta

The pathways of carbohydrate metabolism in Spirochaeta stenostrepta, a free-living, strictly anaerobic spirochete, were studied. The organism fermented glucose to ethyl alcohol, acetate, lactate, CO(2), and H(2). Assays of enzymatic activities in cell extracts, and determinations of radioactivity distribution in products formed from (14)C-labeled glucose indicated that S. stenostrepta degraded glucose via the Embden-Meyerhof pathway. The spirochete utilized a clostridial-type clastic reaction to metabolize pyruvate to acetyl-coenzyme A, CO(2), and H(2), without production of formate. Acetyl-coenzyme A was converted to ethyl alcohol by nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenase activities. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-coenzyme A. Hydrogenase and lactate dehydrogenase activities were detected in cell extracts. A rubredoxin was isolated from cell extracts of S. stenostrepta. Preparations of this rubredoxin stimulated acetyl phosphate formation from pyruvate by diethylaminoethyl cellulose-treated extracts of S. stenostrepta, an indication that rubredoxin may participate in pyruvate cleavage by this spirochete. Nutritional studies showed that S. stenostrepta fermented a variety of carbohydrates, but did not ferment amino acids or other organic acids. An unidentified growth factor present in yeast extract was required by the organism. Exogenous supplements of biotin, riboflavin, and vitamin B(12) were either stimulatory or required for growth.     

Lysis of modified walls from Lactobacillus fermentum.

The N and O substitution in wall peptidoglycan from Lactobacillus fermentum was studied in relation to growth phase, as well as the lytic activities and the effect of trypsin on them. The N-nonsubstituted sites were determined by dinitrophenylation techniques. The results indicate that an extensive substitution at the O groups takes place as cells go into the stationary growth phase, concomitant with a decrease in their lysozyme sensitivity. N-nonsubstituted residues, mainly glucosamine, occurred in both exponential-phase and stationary-phase walls but not in the corresponding peptidoglycans. Small amounts of N-nonsubstituted muramic acid were detected in walls and peptidoglycan from cells in the stationary growth phase only. N acetylation of isolated walls did not increase their lysozyme sensitivity but rather decreased it. Autolysis of walls was completely inhibited by the chemical modifications used. Trypsin stimulates the lysozyme sensitivity of native walls but has no effect on walls that had been O deacetylated and N acetylated. It is suggested that the effect of trypsin is due to its action as an esterase removing the O acetylation in lysozyme-resistant walls.     

Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation

The degree of peptidoglycan O-acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild-base-released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation-exchange amino acid analysis. The beta-D-N,6-O-diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20-52.8%, relative to the total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by both human and hen egg-white lysozymes and for hen egg-white lysozyme, the extent of this resistance was dependent upon the degree of O-acetylation. The steady-state parameters, Km and V, for the hen-egg-white-lysozyme-catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25 degrees C. Values of Km for the different peptidoglycan samples were found to increase with increasing O-acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [delta(delta G#)], a consequence of increasing O-acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.     

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.     

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.     

Quantitative characteristics of human antibodies to the peptidoglycans of different Staphylococcus species

The content of antibodies to peptidoglycans of 13 staphylococcal species was studied in the ELISA with the IgG fraction isolated from commercial human immunoglobulin. The content of antibodies to S. aureus peptidoglycan (calculated per mg of protein) was 3.0 micrograms and that of antibodies to peptidoglycans of other species amounted to 1.8-4.1 micrograms. The maximal value was obtained for S. xylosus and the minimal value, for S. lentus. No relationship between the content of antibodies to peptidoglycans and the degree of their antigenic affinity to S. aureus peptidoglycan was noted: the scatter of the values in species with relatively low and even minimal affinity did not exceed the range of variance in the group with a high degree of homology (2.2-2.4 micrograms). The production of antibodies to staphylococcal peptidoglycans was supposed to result from the action of a complex of antigenic determinants, both common and specific for each staphylococcal species.     

Enzymatic activities for interconversion of purines in spirochetes.

Enzymatic activities that catalyze the interconversion of purines and purine derivatives were detected in cell extracts of Spirochaeta aurantia, Spirochaeta stenostrepta, Treponema succinifaciens, and Treponema denticola. Phosphoribosyltransferase activities present in cell extracts of each of the four spirochete species functioned in the conversion of adenine, hypoxanthine, and guanine to AMP, IMP, and GMP, respectively. Nucleotidase activities in the extracts mediated the formation of nucleosides from nucleotides. The conversion of adenosine, inosine, and guanosine to the respective purine bases was catalyzed by nucleoside phosphorylase and, in some instances, by nucleoside hydrolase activities. Guanine deaminase activity was found in both S. aurantia and S. stenostrepta, whereas adenosine deaminase activity was detected only in S. aurantia. Adenine deaminase activity in T. succinifaciens extracts was sensitive to O2 and was relatively resistant to heating. Our results indicate that the four species of spirochetes studied possess a broad spectrum of purine interconversion enzymes. It is suggested that these enzymes may function in metabolic processes important for the survival of spirochetes in nutrient-poor natural environments.     

The nature of the peptidoglycan immunodeterminants of a Group A streptococcus strain demonstrable by the immunoferritin technique

Analogs (di- and trialanine, tetra- and pentapeptide) to the peptide sequence in Group A streptococcus peptidoglycan were synthetized and were used to inhibit the antipeptide portion of peptidoglycan antibodies. The reactions between these peptidoglycan antibodies and peptidoglycan immunodeterminants on whole cells, isolated cell walls, and peptidoglycans were studied by the immunoferritin technique. Of the peptides used, pentapeptide exhibited the highest inhibiting capacity. The nature and distribution of ferritin-labeled immunodeterminants were identical on isolated peptidoglycans and cell walls as well as on both surfaces of either of these materials. A very low capacity of the M-protein amino acid sequence to inhibit the immunoferritin reaction indicated that the ferritin-labeled structures on whole-cell surfaces were the pentapeptide of peptidoglycan and not the M-protein residues.     

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Chemical compositions of cell walls and polysaccharide fractions of spirochetes

Cellular polysaccharide fractions of various representative members of genera of the family Spirochaetaceae were obtained by the ammonium hydroxide extraction method. The sugar composition of the polysaccharide preparations was complex and many kinds of sugars such as rhamnose, fucose, ribose, xylose, mannose, galactose, and glucose were detected in all of the spirochetes tested. Of particular interest was the presence of 4-O-methylmannose as a constituent polysaccharide in members of the genus Leptospira. This sugar was not detected in the polysaccharides of Spirochaeta, Borrelia, and Treponema. The chemical compositions of cell wall fractions were also examined. 4-O-Methylmannose was detected in the cell wall polysaccharides of the genus Leptospira but not in cell walls prepared from the Spirochaeta, Borrelia, and Treponema. The diaminopimelic acid present in cell wall peptidoglycans of the genus Leptospira was meso-diaminopimelic acid (A2pm). The molar ratios of alanine, glutamic acid, A2pm, glycine, muramic acid, and glucosamine in leptospiral cell walls were found to be approximately 2:1:1:1:1:1. In contrast to the Leptospira, the peptidoglycans of genera Spirochaeta, Borrelia, and Treponema contained ornithine (Orn) but not A2pm. Since 4-O-methylmannose and A2pm were found in the cell wall fractions of genus Leptospira but not in Spirochaeta, Borrelia, or Treponema, it was suggested that the chemical compositions of the cell wall might become an important criterion for the chemotaxonomy of Spirochaetales.     

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.     

Fatty Acid Composition of Spirochaeta stenostrepta

The fatty acid composition of Spirochaeta stenostrepta consists primarily of saturated, branch-chained fatty acids. Iso-C(15), anteiso-C(15), iso-C(17), and anteiso-C(17) represent 66% of the total fatty acids.     

Structures and biological activities of peptidoglycans of Listeria monocytogenes and Propionibacterium acnes

The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.     

Peptidoglycan recognition protein LF: a negative regulator of Drosophila immunity

Peptidoglycan recognition proteins (PGRPs) play important roles in the innate immune defence. Each PGRP detects a distinct subset of peptidoglycans and initiate immune signalling or enzymatic degradation of peptidoglycans. Here we characterize one of the 13 Drosophila PGRPs, PGRP-LF. PGRP-LF is membrane bound and has its two PGRP domains, z and w, localized outside the cell. Our data demonstrate that the z-and w-domain differ in their affinities to peptidoglycan. The z-domain has affinity to several groups of peptidoglycans while the w-domain only recognizes peptidoglycan from Escherichia coli. In addition, we observed that overexpression of PGRP-LF in Drosophila melanogaster Schneider 2 cells (S2 cells) promotes aggregation of cells. Furthermore, following immune stimulation of S2 cells overexpressing PGRP-LF, we noticed a reduced up-regulation of expression of antimicrobial peptide genes, in consonance with an immune suppressive role for PGRP-LF.