Murein type and polysaccharide composition of cell walls from Renibacterium salmoninarum

Abstract The primary structure of the murein of Renibacterium salmoninarum ATCC33209 was determined. It contained lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and the d -alanine of adjacent peptide subunits and a d -alanine-amide substitution at the α-carboxyl group of d -glutamic acid in position 2 of the peptide side chain.
Cell walls contained a considerable amount of polysaccharide with galactose as major sugar component. In addition N -acetyl-glucosamine, rhamnose and N -acetyl-fucosamine were detected.     

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.     

Covalent linkage of the type- and group-specific antigens to the peptide moiety of the peptidoglycan of serotype III group BStreptococcus

The attachment sites for the two major cell wall polysaccharides, the type-and group-specific antigens of a serotype III group B streptococcus (GBS) were investigated with [14C]lysine to label the peptide portion of the peptidoglycan and [3H]acetate to label both polysaccharide antigens as well as the glycan backbone of the peptidoglycan. Mutanolysin-treated cell walls were subjected to trypsin digestion, followed by exhaustive beta-elimination with 6N ammonium hydroxide at 37°C. The resulting products were purified by column chromatography prior to chemical, immunological, and high-voltage electrophoresis analyses. Data from these studies indicated that both cell wall polymers are covalently attached to the peptidoglycan via the peptide unit. Additionally, during synthesis and assembly both antigens attached only to nascent peptidoglycan.     

Presence of D-alanine in an endopeptidase from Streptococcus pyogenes

D-amino acids are commonly found in peptide antibiotics and the cell wall peptidoglycan of bacterial cell walls but have not been identified in proteins or enzymes. Here we report the presence of 6-7 A-alanine residues in an endopeptidase of Streptococcus pyogenes, a unique enzyme involved in surface protein attachment that we term LPXTGase. Using D-amino acid oxidase coupled with catalase for the deamination of D-alanine to pyruvic acid (a conversion unique to D-alanine), we were able to identify [14C]pyruvic acid in a [14C]alanine-labeled preparation of purified LPXTGase, which represents 27% of the amino acid composition. Because D-amino acids are not accommodated in ribosomal peptide synthesis, these results suggest that the same process used in assembling peptide antibiotics or a yet unidentified mechanism may synthesize the core protein of this endopeptidase.     

Structure of the peptide network of pneumococcal peptidoglycan

The peptide network of Streptococcus pneumoniae cell walls was solubilized using the pneumococcal autolytic amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28). The peptide material was fractionated into size classes by gel filtration followed by reverse-phase high-performance liquid chromatography which resolved the peptide population into over 40 fractions. About 40% of the lysines present participate in cross-links between stem peptides. The main components (3 monomers, 5 dimers, and 2 trimers), accounting for 77% of all the wall peptides, were purified. Their structures were determined using a combination of amino acid and end-group analysis, mass spectrometry, and gas-phase sequencing. Two different types of cross-links between stem peptides were found. In the most abundant type there is an alanylserine cross-bridge between the alanine in position 4 of the donor stem peptide and the lysine at position 3 of the acceptor peptide, as in type A3 peptidoglycan. In the second type of cross-link there is no intervening cross-bridge, as in the type A1 peptidoglycan of Gram-negative bacteria. The data indicate that pneumococcal peptidoglycan has a structural complexity comparable to that recently shown in some Gram-negative species.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Study of the composition of cell wall of group A Streptococcus after hydrolysis using muramidase from Streptomyces levoris

The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.     

Evidence for the synthesis of soluble peptidoglycan fragments by protoplasts of Streptococcus faecalis.

Growing protoplasts of Streptococcus faecalis 9790 were found to synthesize and excrete soluble peptidoglycan fragments. The presence of soluble peptidoglycan derivatives in culture supernatants was determined by (i) incorporation of three different radioactively labeled precursors (L-lysine, D-alanine, and acetate) into products which, after hen egg-white lysozyme hydrolysis, had the same KD values on gel filtration as muramidase hydrolysis products of isolated walls; (ii) inhibition of net synthesis of these products by cycloserine and vancomycin; and (iii) identification of disaccharide-peptide monomer using the beta-elimination reaction, gel filtration, and high-voltage paper electrophoresis. Under the conditions of these experiments the presence of newly synthesized, acid-precipitable (macromolecular) peptidoglycan was not detected. The predominance of monomer (70 to 80%) in lysozyme digests of peptidoglycan synthesized by protoplasts was in sharp contrast to digest of walls from intact streptococci which contain mostly peptide cross-linked products. Biosynthesis and release of relatively uncross-linked, soluble peptidoglycan fragments by protoplasts was related to the absence of suitable, preexisting acceptor wall.     

A model for the three-dimensional structure of peptidoglycan in staphylococci

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.     

Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria.

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups.     

Chemical Composition of the Cell Walls of Bacillus stearothermophilus

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.     

Induction of delayed type hypersensitivity-like skin reaction by peptidoglycans of bacterial cell walls

Water-soluble glycopeptides isolated from Lactobacillus plantarum and Staphylococcus epidermidis cell walls elicited a delayed type hypersensitivity (DTH)-like skin reaction in rats previously immunized with Mycobacterium tuberculosis cell walls, but not in unimmunized rats. Histological examination of the skin reaction sites in immunized animals revealed a close similarity of this skin reaction to a typical DTH reacton with respect to the time course of development and the types of cells that infiltrated into the skin reaction sites, which were characterized by a predominant infiltration of mononuclear cells at 48 hr. This DTH-like reaction was also demonstrated by immunizing the rats with the cell wall peptidoglycans of L. plantarum or S. epidermidis and skin testing them with homologous as well as heterologous peptidoglycans. The DTH-like reaction appeared to be caused by peptidoglycans that exist in common in the cell walls of phylogenetically distant bacterial species. Furthermore, it was also suggested that the putative antigenic determinants(s) might include both the glycan chain and part of the peptide moieties of the cell wall peptidoglycan rather than either of the single moieties.     

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.     

Sphere-Rod Morphogenesis in Arthrobacter crystallopoietes I. Cell Wall Composition and Polysaccharides of the Peptidoglycan

Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone- and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.     

Isolation and Chemical Structure of the Peptidoglycan of Spirillum serpens Cell Walls

The peptidoglycan layer of Spirillum serpens cell walls was isolated from intact cells after treatment with sodium dodecylsulfate and digestion with Pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid, and meso-diaminopimelic acid in the approximate molar ratio of 1:1:2:1:1. Aspartic acid and glycine were the only other amino acids found in significant quantities. N-terminal amino acid analyses of the tetrapeptide amino acids in the peptidoglycan revealed that 54% of the diaminopimelic acid molecules are involved in cross-linkage between tetrapeptides. This amount of cross-linkage is greater than that found in the peptidoglycan of previously studied cell walls of gram-negative bacteria. The polysaccharide backbone was isolated, after myxobacter AL-1 enzyme digestion of the peptidoglycan, by fractionation with ECTEOLA-cellulose and Sephadex G-100. An average length of 99 hexosamines for the polysaccharide chains was found (ratio of total hexosamines to reducing end groups).     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

Peptidoglycan-polysaccharide complex in the cell wall of the filamentous prochlorophyte Prochlorothrix hollandica.

A peptidoglycan-polysaccharide complex composed of N-acetylglucosamine, N-acetylmuramic acid, muramic acid 6-phosphate, L-alanine, D-alanine, D-glutamic acid, meso-diaminopimelic acid, N-acetylmannosamine, mannose, galactose, glucose, and phosphate was isolated from cell walls of the filamentous prochlorophyte Prochlorothrix hollandica; this complex was similar in chemical composition and structure to that found in cyanobacteria. Peptide patterns of partial acid hydrolysates of the isolated peptidoglycan revealed an A1 gamma structure with direct cross-linkage (m-diaminopimelic acid-D-alanine) of the peptide side chains. The degree of cross-linkage (63%) was found to be in the range of values obtained for gram-positive bacteria and cyanobacteria.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.