Biomedical subjects
Publications and source records attributed to D J Tipper.
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A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable of lysing cells of Staphylococcus aureus was purified approximately 500-fold by passage through diethylaminoethyl cellulose and chromatography on carboxymethyl-cellulose. The purified enzyme was shown to act as an endopeptidase, cleaving the pentaglycine cross-bridges of the cell wall peptidoglycan at d-alanyl-glycine and glycyl-glycine linkages with the release of di-, tri-, and tetraglycine fragments. Release of NH(2)-alanine indicated weak N-acetylmuramyl-l-alanine amidase activity, but most of the residual peptide remained attached to the glycan. No hydrolysis of the glycan occurred. The lytic spectrum of the enzyme toward a variety of other cell walls of known peptidoglycan composition indicated relatively high specificity for peptidoglycans with polyglycine bridges.
Autolysis of isolated cell walls of Staphylococcus aureus strain Copenhagen was accompanied by the release of 1 mole of N-terminal alanine per mole of glutamic acid. No other N-terminal amino acids and no C-terminal amino acids were released. These observations indicated that complete hydrolysis of N-acetylmuramyl-l-alanine linkages ("amidase" action) had occurred. This was confirmed by fractionation and analysis of the products. Hydrolysis of 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages also occurred to a variable extent; on one occasion, complete degradation to disaccharides and hexosamine-free polypeptides (with intact pentaglycine cross-bridges) occurred. In one other instance, hydrolysis within pentaglycine bridges also occurred. Analyses of intact cell walls indicated that, in vivo, glycine endopeptidase activity was negligible and amidase activity was low, but that endo-beta-N-acetylglucosaminidase hydrolysed about 8% of the N-acetylglucosaminyl-N-acetylmuramic acid linkages. Autolysis of isolated cell walls was too slow for the enzymes isolated with them to have significant action during this isolation. The possible functions of these autolytic activities are discussed.
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The chemical nature and distribution of the peptidoglycan in Myxococcus xanthus at various stages of the cellular life cycle were investigated. Vegetative cells and microcysts contained approximately 0.6% by weight of peptidoglycan. The overall composition of the peptidoglycan was similar in both cell types and was approximately 1 glutamic acid, 1 diaminopimelic acid, 1.7 alanine, 0.75 N-acetylglucosamine, and 0.75 N-acetylmuramic acid. (We have assumed that all the hexosamines are N-acetylated.) The sizes of the subunits (estimated by gel filtration) solubilized by muramidases were considerably larger (tetramer and oligomer) in the microcysts than in the vegetative cells (mostly dimer). There was a transient decrease in cross-linking (measured as an increase in the amount of free amino group of diaminopimelic acid) during the stage of microcyst formation when the cells converted from ovoids to spheres. At the same time, there occurred a large and rapid increase in a galactosamine derivative which may have reflected the synthesis of capsular material. Immediately prior to this period of morphogenesis, the cells became resistant to penicillin but remained sensitive to d-cycloserine. The walls of vegetative cells were completely disaggregated by trypsin and sodium lauryl sulfate, suggesting a discontinuous peptidoglycan layer. This was no longer apparent after the ovoid-sphere stage of microcyst formation. The relationship to morphogenesis of the chemical changes in the cell wall is discussed.
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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.
Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C- and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one l-alanine residue. In sphere cell walls, l-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C- and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall.
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Biochemical investigations of the mode of action of beta-lactam antibiotics have focused on the interaction of these drugs with sensitive enzymes and penicillin-binding proteins in vitro and on the correlation of these data with physiological responses to the drugs. The classical response is inhibition of growth and cell death followed by lysis; however, the bacteriostatic response to penicillins, which is seen in certain bacterial species such as Streptococcus mutans and in mutants of species such as Streptococcus pneumoniae, is also described. The biochemical data remain consistent with the acyl-D-alanyl-D-alanine donor substrate analogue theory, but multiple targets with differential sensitivity to different beta-lactam antibiotics exist. The relationship of these targets to penicillin-binding proteins and their possible functions in cell growth and division are discussed.