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.     

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.     

Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis; location of active and latent autolysin.

Over 80% of the active and porteinase-activatable, latent forms of the autolytic N-acetylmuramide glycanhydrolase of Streptococcus faecalis ATCC 9790 were released to the supernatant buffer during the autolytic formation of protoplasts (autoplasts) in the presence of absence of trypsin. Autolysin activity was not found in association with released mesosomal vesicles and had little affinity for binding to membranes or to the outer surface of the wall. Isolated walls were able to bind over four times as much autolysin activity as that present on wall exponential-phase cells. Using a rapid technique for wall isolation, evidence was obtained that the latent form (as well as the active form) was wall bound in intact cells. In addition, isotope labeling and ultrastructural studies were able to show that latent autolysin was concentrated in the newer, septally associated portion of the wall.     

Transformation and fusion of Streptococcus faecalis protoplasts.

Nonconjugative plasmids were transferred by protoplast fusion among Streptococcus faecalis strains and from Streptococcus sanguis to S. faecalis. S. faecalis protoplasts were also transformed with several different plasmids, including the Tn917 delivery vehicle pTV1. Transformation was reproducible, but low in frequency (10(-6) transformants per viable protoplast). A new shuttle vector (pAM610), able to replicate in Escherichia coli and S. faecalis, was constructed and transformed into S. faecalis protoplasts. pAM610 was mobilized by the conjugative plasmid pAM beta 1 in matings among S. faecalis strains and from S. sanguis to S. faecalis. Chimeric derivatives of pAM610 were also transformed into S. faecalis.     

Formation of the glycan chains in the synthesis of bacterial peptidoglycan

The main structural features of bacterial peptidoglycan are linear glycan chains interlinked by short peptides. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), all linkages between sugars being beta,1-->4. On the outside of the cytoplasmic membrane, two types of activities are involved in the polymerization of the peptidoglycan monomer unit: glycosyltransferases that catalyze the formation of the linear glycan chains and transpeptidases that catalyze the formation of the peptide cross-bridges. Contrary to the transpeptidation step, for which there is an abundant literature that has been regularly reviewed, the transglycosylation step has been studied to a far lesser extent. The aim of the present review is to summarize and evaluate the molecular and cellullar data concerning the formation of the glycan chains in the synthesis of peptidoglycan. Early work concerned the use of various in vivo and in vitro systems for the study of the polymerization steps, the attachment of newly made material to preexisting peptidoglycan, and the mechanism of action of antibiotics. The synthesis of the glycan chains is catalyzed by the N-terminal glycosyltransferase module of class A high-molecular-mass penicillin-binding proteins and by nonpenicillin-binding monofunctional glycosyltransferases. The multiplicity of these activities in a given organism presumably reflects a variety of in vivo functions. The topological localization of the incorporation of nascent peptidoglycan into the cell wall has revealed that bacteria have at least two peptidoglycan-synthesizing systems: one for septation, the other one for elongation or cell wall thickening. Owing to its location on the outside of the cytoplasmic membrane and its specificity, the transglycosylation step is an interesting target for antibacterials. Glycopeptides and moenomycins are the best studied antibiotics known to interfere with this step. Their mode of action and structure-activity relationships have been extensively studied. Attempts to synthesize other specific transglycosylation inhibitors have recently been made.     

Enzymatic deacylation of lipoteichoic acid by protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790).

High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the former was incubated in the presence of growing protoplasts of Streptococcus faecium (S. faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid LTA(X) from this organism or from chemically deacylated LTA. Native LTA(X) was shown to contain less than one fatty acid equivalent per 18 LTA(X) molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA.     

Balanced macromolecular biosynthesis in "protoplasts" of Streptococcus faecalis

Osmotically fragile forms of Streptococcus faecalis 9790 were grown in 0.5 m sucrose- or 0.5 m NH(4)Cl-stabilized medium. The "protoplast" cultures exhibit an average growth rate constant of 0.66 to 0.94 mass doublings/hr. In a variety of experiments, turbidity and the net content of protein, ribonucleic acid (RNA) and deoxyribonucleic acid increase at the same rate, indicating balanced macromolecular biosynthesis. A total of two to three mass doublings was obtained, with no evidence of cell division. After osmotic shock, "protoplast" cultures released 93 to 94% of their RNA content in a form not sedimentable at 12,800 x g for 15 min, in contrast to streptococci, which released 7% of their RNA content after the same treatment.     

Synthesis of the L-alanyl-L-alanine cross-bridge of Enterococcus faecalis peptidoglycan

The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(2) side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-d-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(2))-d-Ala-d-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two l-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the dd-transpeptidases in the cross-linking reaction. However, the bppA2 deletion impaired expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.     

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus.

The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and high-molecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecular-weight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.     

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.     

Protein Synthesis in Cell-Free Extracts of Streptococcus faecalis

If a cell-free extract of Streptococcus faecalis is fractionated by way of a 10 to 70% sucrose gradient, at least three areas are found capable of protein synthesis having a density greater than can be accounted for by association of individual ribosomes. These areas represent distinct “polysome” peaks rather than random distribution of polymers of varied length. They appear to be membrane subunits. In addition there is a further particle with a density of about 150S, incapable of protein synthesis but which is capable of stimulating protein synthesis in some of the larger fractions found by gradient centrifugation.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

Kinetic analysis of tetracycline accumulation by Streptococcus faecalis.

Tetracycline transport by Streptococcus faecalis occurs by an energy-dependent, carrier-mediated process. The Michaelis constant for transport was unchanged, but the maximal velocity was increased when an energy source, glucose, was present. Accumulation levels, sometimes 25-fold greater than the external concentration, were controlled by the transport system.     

Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

Inhibition of protein synthesis in Streptococcus faecalis by ochratoxin A.

A non-competitive inhibition of binding of cAMP to bovine protein kinase by ochratoxin A (OTA) is shown. Preliminary evidence of a protein kinase in Streptococcus faecalis is presented. The cAMP stimulation of this kinase is also inhibited by OTA. At the lowest OTA concentrations, RNA and protein synthesis are inhibited in S. faecalis. The inhibition of RNA synthesis is secondary, as in the presence of chloramphenicol no inhibition occurs for 10 min after the addition of OTA. The synthesis but not the induction of beta-P-galactosidase is inhibited by OTA. The polysomes of S. faecalis are stabilized after addition of OTA, showing an inhibition of peptide elongation. The model of action of OTA in bacteria is discussed and it is concluded that inhibition of protein synthesis is the process which might be closest to the primary target of OTA.