Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Factors affecting sensitivity of group B streptococci to an exogenous murein hydrolase

Group B streptococci treated with cell wall synthesis inhibitors (penicillin or vancomycin) or by a variety of membrane-acting agents are sensitized to the lytic action of exogenous M1 muramidase. Muramidase without a sensitizing agent caused rupture of bacterial chains only, accompanied by the release of a small amount of cell wall peptidoglycan label and an increase of the number of colony-forming units. In combination with sensitizing agents the exogenous muramidase appeared to initiate hydrolysis of biosynthetically new peptidoglycan. Treatment of the cells with chloramphenicol or starvation for nutritionally required amino acids suppressed the rate of cell lysis and peptidoglycan hydrolysis during subsequent sensitization and muramidase treatment of the bacteria. Purified cell walls prepared from the amino acid starved cells were also hydrolyzed with a slower rate by muramidase. It is suggested that agents sensitizing the bacteria to the exogenous muramidase act by perturbing or removing some nonmurein components of the cell envelope which protect the peptidoglycan from the activity of exogenous enzyme. Agents increasing resistance against exogenous muramidase may also cause some alteration in peptidoglycan structure.     

The effect of the structural components of Staphylococcus aureus on the interaction of T- and B-lymphocytes]

The results of studies on the influence of staphylococcal peptidoglycan and cell walls on the cooperative interaction of T- and B-lymphocytes in the process of immune response to thymus-dependent antigen are presented. Peptidoglycan has been found to produce, depending on its dose, a suppressive and stimulating effect on the interaction of T- and B-cells. Cell walls exhibit only stimulating action under the same experimental conditions. The suppressive action of peptidoglycan is mediated by T-lymphocytes.     

Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan

Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross-wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide β-N-acetylmuramic acid, (1→4)-β-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid, a ribitol-phosphate polymer tethered to murein linkage units, prevents the LysM domain from binding to peptidoglycan. The localization of LytN and Sle1 to the cross-wall is abolished in staphylococcal tagO mutants, which are defective for wall teichoic acid synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross-wall and separating bacterial cells.     

An electron microscopic study of the location of peptidoglycan in group A and C streptococcal cell walls.

The morphological appearance of deproteinized Group A and C streptococcal walls after treatment by different procedures extracting teichoic acids and polysaccharides (formamide, hydrochloric acid, nitrous acid, trichloroacetic acid, sulphuric acid, sodium hydroxide and sodium deoxycholate) was compared with the content of teichoic acids and polysaccharides remaining in the treated walls. All procedures extracted teichoic acids almost completely, but polysaccharides were extracted to various degrees. The ultrastructural appearance of walls after these extractions still exhibited the triple-layered wall profile; only a reduction of thickness of the wall and of electron density of the layers occurred. There was no direct correlation between the reduction of rhamnose content and thickness of walls. The ultrastructural localization of peptidoglycan in the streptococcal walls was explored by means of the indirect immunoferritin technique using anti-peptidoglycan antibodies isolated from anti-Group A-variant antisera. Ferritin particles were bound predominantly to filamentous structures which protruded from both surfaces of peptidoglycan fragments and isolated walls. Peptidoglycan was also detected on the filamentous protrusions of whole cocci. These results contradict models of the streptococcal wall in which peptidoglycan forms the innermost layer and support a mosaic structure in which peptidoglycan forms a network of the peptidoglycan-polysaccharide complex.     

Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-β-N-acetylglucosaminidase

The bacteriolytic enzyme with an isoelectric point of 9.5 that is produced by all strains of Staphylococcus aureus investigated was purified from strain M18 (Wadström & Hisatsune, 1970). This enzyme released reducing groups from cell walls of Micrococcus lysodeikticus and was thus shown to be a bacteriolytic hexosaminidase. Although dinitrophenylation and acid hydrolysis of cell walls hydrolysed by a partially purified enzyme gave DNP-alanine and DNP-glycine from staphylococcal peptidoglycan, which indicated the presence of a peptidase and probably also an N-acetylmuramyl-l-alanine amidase, hydrolysis of cell walls by the extensively purified enzyme did not give any DNP-amino acids. The enzyme digest was purified by Amberlite CG-120 and Sephadex G-10 chromatography. Reduction by sodium borohydride of the disaccharide obtained was followed by acid hydrolysis and paper chromatography. Glucosamine completely disappeared after this treatment and a new spot identical with glucosaminitol appeared. The muramic acid spot remained unchanged. The purified enzyme was found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of a bacteriolytic endo-β-N-acetylglucosaminidase. It is also proposed that this enzyme is probably identical with the staphylococcal lysozyme. The mode of action of this has not previously been investigated.     

Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges

ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 angstroms crystal structure of the targeting domain shows an all-beta fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.     

A rapid fluorimetric test for lysozyme with purified fluorescamine-labelled peptidoglycan

A sensitive and rapid fluorometric lysozyme assay is described. It is based on the hydrolysis of fluorescamine-labelled peptidoglycan from Micrococcus luteus cell walls. Lysozyme levels as low as 0.1 microgram can be detected.     

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.     

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).     

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.     

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.     

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.     

溶葡球菌酶的比色测定及某些性质的研究

溶葡球菌酶是一种专一地溶解葡萄球菌的溶菌酶。和蛋清溶菌酶一样,通常采用比浊法进行测定,底物或为葡萄球菌、或为该菌的细胞壁、或为该菌细胞壁的肽聚糖。本文报道一种简便灵敏的溶葡球菌酶比色测定法,以偶联了KNR艳蓝染料的葡萄球菌(死)细胞或偶联了KNR艳蓝染料的该菌细胞壁肽聚糖为色源底物,根据酶作用后释放出的可溶性KNR生成物计算酶活性。本文采用该比色测定法检定了溶葡球菌酶的某些动力学性质。     

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.     

Oleanolic acid and ursolic acid affect peptidoglycan metabolism in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.     

Structural study on teichoic acids of Listeria monocytogenes types 4a and 4d

The chemical compositions of the cell walls obtained from 10 strains (serotypes 1a, 3a, 4a, 4b, 4c, 4d, 4e, 4e, 4f, 6, and 7) of Listeria monocytogenes were analyzed. These cell walls were shown to be mainly composed of peptidoglycan and ribitol teichoic acids. Considerable variations in the composition of neutral sugars were observed among these cell walls. Chemical and NMR analyses indicated that the teichoic acids from L. monocytogenes serotypes 4a and 4d are composed of the following repeating units: Formula: See Text.     

Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.     

Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that the TCA-wall consisted of mostly peptidoglycan. By digesting the TCA-wall with lysozyme, the circular structures were greatly disturbed, and they disappeared after 60 min of treatment. From these observations it can be expected that the peptidoglycan is arranged in a concentric circular manner in the newly generated cell wall of Staphylococcus.     

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.