Insoluble Peptidoglycan Research Articles

Evidence for the presence of sulfated polysaccharides in the cell walls of an Arthrobacter sp.

The chemical composition of the cell walls of Arthrobacter sp. AT-25 was investigated. The purified cell walls were composed of D-galactose, D-glucose, sulfate, phosphorus, muramic acid, glucosamine, alanine, glutamic acid, glycine, and LL-diaminopimelic acid in a molar ratio of 4.1:0.88:5.7:0.44:0.65:0.85:2.0:1.0:1.1:1.1. On mild treatment with acid, the cell walls readily released a special structure, the non-peptidoglycan portion, and gave an insoluble peptidoglycan, soluble sulfated polysaccharides, and a phosphorus-rich fraction in respective yields of 41, 40 and 6%. The peptidoglycan contained two phosphate esters, muramic acid 6-phosphate and glucosamine 6-phosphate, while the special structure (sulfated polysaccharides plus a phosphorus-rich fraction) contained three phosphate esters, glucose 6-phosphate, and glycerol 1- and 2-phosphate. These results indicate that the cell walls of the bacteria contain highly sulfated polysaccharides with low degrees of phosphorylation. This paper reports the presence of a novel special structure in the cell walls of the grampositive bacteria.

Major protein components in the cell envelope of Clostridium Tyrobutyricum

The overall composition of the Clostridium tyrobutyricum cell envelope did not vary significantly during cell growth and was characterized by a high protein content (about 40% dry weight). Teichoic and teichuronic acids were absent and the neutral sugar content low. Insoluble peptidoglycan represented only 10-12% of the cell envelope (dry weight basis); it contained glucosamine, muramic acid, alanine, diaminopimelic acid and glutamic acid (molecular ratio 1/1/2/1/1). SDS-PAGE revealed the presence of about 50 proteins in this cell envelope; however, one high molecular weight protein was largely predominant. They were not covalently bound to the peptidoglycan and their relative amounts were practically constant through cell growth and with various extraction treatments. A brief heat treatment of whole cells in PBS caused selective release of the major cell envelope proteins together with flagellin; this method was used to characterize these proteins in 37 strains of C. tyrobutyricum and some other clostridia. The major envelope proteins had molecular weights ranging from 96 to 145 Kd and the flagellins from 32 to 72 Kd.

In vivo target of benzylpenicillin in Gaffkya homari.

It has been established that the DD-carboxypeptidase is the primary in vitro target of benzylpenicillin in Gaffkya homari (W. P. Hammes, Eur. J. Biochem. 70:107-113, 1976). To determine whether this enzyme is also the primary target of benzylpenicillin in vivo, we compared the effects of this beta-lactam, cefmenoxime, cephalothin, and cefoxitin on growth with their acylation of penicillin-binding protein (PBP) 9, the DD-carboxypeptidase. Results of three types of experiments with membrane-walls indicated that PBP 9 is this enzyme and that it is the primary in vitro target of these beta-lactams in the synthesis of sodium dodecyl sulfate (SDS)-insoluble peptidoglycan. First, the acylation of PBP 9 by these beta-lactams paralleled the inhibition of DD-carboxypeptidase and the inhibition of SDS-insoluble peptidoglycan synthesis. Second, the rate of benzylpenicillin release from PBP 9 correlated with the recovery of DD-carboxypeptidase. Third, DD-carboxypeptidase activity was detected in a protein with the same apparent molecular weight as PBP 9 after elution from an SDS-polyacrylamide gel. When intact cells were treated with benzylpenicillin, the minimum growth inhibitory concentration (MGIC) correlated with the concentration of [35S]benzylpenicillin required to acylate PBPs 6 and 9 by 50%. When intact cells were treated with cefmenoxime, cephalothin, or cefoxitin, the MGICs correlated with the concentration of unlabeled beta-lactam required to reduce the subsequent binding of [35S]benzylpenicillin by 50% (ED50) for PBP 6. In contrast, the MGICs of these beta-lactams did not correlate with the ED50s for PBP 9. PBP 9 was not acylated by cefmenoxime or cephalothin at their MGICs, whereas this PBP was fully acylated by cefoxitin at one-tenth of its MGIC. It is suggested that PBP 6 may be a primary target of growth inhibition by benzylpenicillin, cefmenoxime, cephalothin, and cefoxitin; PBP 9, the DD-carboxypeptidase, is dispensable for growth under laboratory conditions; and PBP 9 does not appear to be a primary in vivo target of these beta-lactams, even though this PBP is their primary target in vitro.

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship.

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.

Evidence for the synthesis of peptidoglycan by spheroplasts of Mycobacterium smegmatis ATCC-14468

Evidence for the synthesis of insoluble peptidoglycan by spheroplasts of Mycobacterium smegmatis ATCC-14468 was obtained by inhibition of synthesis by vancomycin and cycloserine and no inhibition of synthesis by tetracycline. Radioactivity in the peptidoglycan fraction decreased when treated with lysozyme. The ability of spheroplasts to synthesize macromolecular peptidoglycan was half that of whole cells. A relationship was obtained between the amount of intact cell wall present and peptidoglycan synthesized: as the amount of intact cell wall decreased, the ability to synthesize macromolecular peptidoglycan also decreased.

Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes.

Membranes from Gaffkya homari reactivated by freezing and thawing were used to study the processing events involved in the assembly of both sodium dodecyl sulfate (SDS)-insoluble peptidoglycan (PG) and SDS-soluble PG. The ability to reactivate membranes for the synthesis of these polymers provided an opportunity to monitor those events that are not influenced by wall-linked PG. In G. homari, processing for the formation of cross-links requires the selective actions of DD-carboxypeptidase, LD-carboxypeptidase, and NE-(DAla)-Lys transpeptidase. Time courses of cross-link formation, as measured by the amounts of amidated bisdisaccharide peptide dimer and nonamidated bisdisaccharide peptide dimer, showed a lack of correlation with those for the synthesis of SDS-insoluble PG. SDS-soluble PG, which is significantly cross-linked when synthesized in the absence of penicillin G, was a precursor of the SDS-insoluble PG. In the presence of penicillin G, un-cross-linked SDS-soluble PG was synthesized. This PG was also utilized and processed for the synthesis of cross-linked SDS-insoluble PG after removal of the beta-lactam. This protocol provided a method for separating stages in the synthesis and elongation of PG from those involved in processing. Cross-linkage in the various PG fractions ranged from 0 to 19% in SDS-soluble PG and from 2 to 24% in SDS-insoluble PG. Thus, the results indicated that there is no direct correlation between SDS insolubility and the degree of cross-linkage. Instead, they suggested that additional features may contribute to the insolubility of PG in SDS.

Effects of mecillinam and cefoxitin on growth, macromolecular synthesis, and penicillin-binding proteins in a variety of streptococci.

Although some strains of streptococci seem to be virtually inert to mecillinam, the growth of other strains, notably certain viridans streptococci (Streptococcus mutans and Streptococcus sanguis) was inhibited by relatively low concentrations of the drug. Inhibition of the synthesis of peptidoglycan, RNA, protein, and DNA in two tolerant strains, S. mutans FA-1 and GS-5, was studied over a wide range of concentrations of mecillinam, benzylpenicillin, and cefoxitin. The responses of both strains to all three beta-lactams were very similar; that is, synthesis of insoluble peptidoglycan was most susceptible. Inhibition of peptidoglycan synthesis was followed rapidly and sequentially by substantial but less severe inhibitions of RNA and protein synthesis. Significant inhibition of DNA synthesis was not observed. Binding studies with [14C]benzylpenicillin alone or after preexposure of membrane preparations to benzylpenicillin, mecillinam, or cefoxitin suggest that reasonably selective binding of a beta-lactam antibiotic to one or two of the major penicillin-binding proteins (PBP 1 or PBP 4) of S. mutans GS-5 and FA-1 may be the initial step in the series of events that results in the inhibition of growth and in the inhibition of insoluble peptidoglycan assembly and of RNA and protein synthesis.

Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth.

The progress of incorporation of radioactive glucosamine into the O-acetylated and non-O-acetylated sub-units of the insoluble peptidoglycan of growing Neisseria gonorrhoeae was examined. More than 80% of the final degree of cross-linking was achieved within 3 min; the remainder of the process took much longer. Rapid O-acetylation occupied up to 10 min, at which time only about 65% of the maximum value had been reached. There was thus evidence for maturation of peptidoglycan both in regard to cross-linking and to O-acetylation.

Peptidoglycan loss during hen egg white lysozyme-inorganic salt lysis of Streptococcus mutans.

Streptococcus mutans BHT was grown in Todd-Hewitt dialysate medium containing N-acetyl[(14)C]glucosamine for 6 to 11 generations. After treatment with cold and hot trichloroacetic acid and trypsin, 52 to 65% of the radioactivity remained present in insoluble peptidoglycan-containing residues. Hen egg white lysozyme or mutanolysin treatment of the peptidoglycan residues resulted in the release of 80 and 97%, respectively, of the (14)C label to the supernatant fraction. Hydrochloric acid hydrolysates of such supernatants showed that essentially all of the radioactivity present in insoluble peptidoglycan fractions was present in compounds that comigrated on paper chromatography with glucosamine ( approximately 60%) or muramic acid ( approximately 30%). Treatment of whole cells with low and high concentrations of lysozyme alone resulted in losses of 45 and 70% of the insoluble peptidoglycan, respectively, yet release of deoxyribonucleic acid from cells was not detected. Sequential addition of appropriate concentrations of selected inorganic salts after lysozyme treatment did result in the liberation of deoxyribonucleic acid. Deoxyribonucleic acid release was correlated with a further release of peptidoglycan from the insoluble fraction. However, the total amount of peptidoglycan lost effected by the low concentration of lysozyme and NaSCN (lysis) was significantly less than the amount of peptidoglycan hydrolyzed by high concentrations of lysozyme alone (no lysis), suggesting that the overall amount of peptidoglycan lost did not correlate well with cellular lysis. The total amount of insoluble peptidoglycan lost at the highest salt concentrations tested was found to be greater than could be accounted for by lysozyme-sensitive linkages of the peptidoglycan, possibly implicating autolysins. The results obtained suggested that hydrolysis of peptidoglycan bonds in topologically localized, but strategically important, sites was a more significant factor in the sequence that results in loss of cellular integrity (lysis).

Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis.

Turnover of the cell wall peptidoglycan fraction of six different strains of Streptococcus mutans and eight different strains of Streptococcus sanguis was examined. Cells were grown in the presence of [3H]lysine and [14C]leucine for at least eight generations and then chased in growth medium lacking the two labels. At intervals during the chase, samples of cultures were removed, and the amounts of the two labeled precursors remaining in the peptidoglycan and protein fractions were quantitated. Similar experiments were done in which the pulse-labeling technique was used. In addition, cells were labeled in the presence of tetracycline or penicillin, chased with growth medium containing no inhibitor, and assayed at intervals during the chase for the amount of [3H]lysine present in peptidoglycan fractions. Studies of cultures of S. mutans strains FA-1, OMZ-61, OMZ-176, 6715, GS-5, and Ingbritt and of S. sanguis strains 10558, M-5, Wicky, DL-101, DL-1, 71X26, and 71X48 maintained in the exponential phase of growth in a chemically defined medium failed to show evidence of loss of insoluble peptidoglycan via turnover. Similarly, for the strains of S. mutans, insoluble peptidoglycan assembled during 2 h of benzylpenicillin or tetracycline treatment was also conserved during recovery from growth inhibition.

Inhibition of peptidoglycan, ribonucleic acid, and protein synthesis in tolerant strains of Streptococcus mutans.

Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 mug/ml). However, in contrast to some other streptococcal species, growth inhibition was not accompanied by cellular lysis or by a rapid loss of viability. In both strains, synthesis of insoluble cell wall peptidoglycan was very sensitive to Pen G inhibition and responded in a dose-dependent manner to concentrations of about 0.2 and 0.5 mug/ml for strains GS-5 and FA-1, respectively. Higher Pen G concentrations failed to inhibit further either growth or insoluble peptidoglycan assembly. Somewhat surprisingly, Pen G also inhibited both ribonucleic acid (RNA) and protein syntheses, each in a dose-dependent manner. Compared with inhibition of peptidoglycan synthesis, inhibition of RNA and protein syntheses by Pen G was less rapid and less extensive. Maximum amounts of radiolabeled Pen G were specifically bound to intact cells upon exposure to about 0.2 and 0.5 mug/ml of Pen G for strains GS-5 and FA-1, respectively, concentrations consistent with those that resulted in maximum or near-maximum inhibitions of the synthesis of cellular peptidoglycan, RNA, and protein. Five polypeptide bands that had a very high affinity for [(14)C]Pen G were detected in a crude cell envelope preparation of strain FA-1. After exposure of cultures of strain FA-1 to the effects of saturating concentrations of the drug for up to 3 h, addition of penicillinase was followed by recovery of growth after a lag. The length of the lag before regrowth depended on both Pen G concentration and time of exposure. On the basis of these and other observations, it is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly.

Peptidoglycans synthesized by a membrane preparation of Micrococcus luteus.

By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsin-solubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala leads to L-Lys and D-Ala leads to L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.

Characterization of the presumed peptide cross-links in the soluble peptidoglycan fragments synthesized by protoplasts of Streptococcus faecalis.

Protoplasts of Streptococcus faecalis ATCC 9790 were produced with the aid of lysozyme, and the ability of these bodies to synthesize soluble, peptide cross-linked peptidoglycan (PG) fragments was examined. Lysozyme digests of PG isolated using gel filtration from the supernatant medium of protoplasts grown in the presence of [14C]acetate and L-[3H]lysine contained small amounts of PG having KD expected for peptide cross-linked dimers and trimers. Addition of benzyl penicillin (300 mug/ml) to growing protoplast cultures did not affect the net amount of PG fragments synthesized but resulted in inhibition of synthesis of dimer and trimer fractions by 27 and 59%, respectively. Failure of penicillin to completely inhibit the accumulation of the dimer fraction was attributed to the presence of atypical forms of dimer. In fact, the supernatant medium of penicillin-treated cultures did not contain detectable amounts of typical peptide cross-linked dimer. The degree of peptide cross-linkage of protoplast PG was at most only 13% of that found in walls isolated from intact streptococci. The relative amounts of monomers, dimers, and trimers synthesized during early and late stages of protoplast growth was approximately the same. Protoplasts synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci.

Studies of the formation of peptide cross-links in the cell wall peptidoglycan of Streptococcus faecalis.

A method was developed to label specifically the glycan chains of the cell wall peptidoglycan of Streptococcus faecalis ATCC 9790 with [14C]acetate. The formation of peptide cross-links (a) during exponential growth, (b) after valine starvation and wall thickening, and (c) during regrowth after 2 hours of valine starvation, was studied using continuous, pulse and pulse-chase labeling of the peptidoglycan with both [14C]acetate and [3H]lysine. After labeling, walls were isolated, digested with the muramidase of Chalaropsis B, and the "free" peptidoglycan fragments (75 to 90% of the total peptidoglycan) were then fractionated on columns of Sephadex G-50, G-50, and G-25 in series into disaccharide-peptide monomer and peptide cross-linked bisdisaccharide-peptide dimer, trisdisaccharide-peptide trimer, and higher oligomer fractions. Peptidoglycan made during valine starvation and wall thickening was found to be slightly more cross-linked than peptidoglycan made during exponential growth. Pulse and pulse-chase experiments indicated that peptide cross-linking continued for an unexpectedly long time after incorporation of precursors into insoluble peptidoglycan.

Turnover of the cell wall peptidoglycan of Lactobacillus acidophilus. The presence of a fraction immune to turnover.

Exponentially growing cultures of Lactobacillus acidophilus strain 60AM Gasser were previously shown to lose about one-third of their cell wall peptidoglycan per generation via turnover (Boothby, D., Daneo-Moore, L., Higgins, M. L., Coyette, J., and Shockman, G. D. (1973) J. Biol. Chem. 248, 2161-2169). We now show that 20 to 30% of the [3H]lysine initially present in insoluble peptidoglycan fractions was retained after 4 or more generations of continued exponential growth of cultures in the absence of label. Treatment of peptidoglycan fractions, before and after 6 or 8 generations of chase with lysozyme (EC 3.2.1.17), released soluble products containing [3H]lysine which had electrophoretic mobilities identical with the disaccharide-peptide derivatives obtained from the wall peptidoglycan of this species. Because protein is known to contaminate peptidoglycan residues, the double labeled technique was used to show that one-half or less of the label lysine present after 6 or 8 generations of chase could be attributed to protein contamination. This then left a minimum fraction of 10 to 20% of the peptidoglycan that was immune to turnover. The absence of turnover of peptidoglycan labeled during short pulses has now been quantitated to show that pulses shorter than 12% of a generation (6 to 7 min) did not turn over. This turnover-immune fraction is in reasonably good agreement with the immune fraction of 10 to 20% observed after long periods of chase of extensively labeled peptidoglycan.

Preparation of Cell Wall Antigens of Staphylococcus aureus

Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo-N-acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others.