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G D Shockman

Publications and source records attributed to G D Shockman.

At least 91 records · Page 5Linked to original sources
PubMed1975

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.

Acetates↗
PubMed1975

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.

Acetates↗
PubMed1975

Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis.

Protoplasts (autoplasts) of Streptococcus faecalis were produced by the action of native autolytic N-acetylmuramidase in the absence of added peptidoglycan hydrolases and were grown in osmotically stabilized medium containing L-[3H]lysine and D-[14C]alanine. To reduce the level of muralytic hydrolysis of glycan chains during growth, heat-inactivated cell walls were added to the medium to bind autolytic enzyme, and tetracycline (1 mug/ml) was added to inhibit further enzyme synthesis. Under these conditions, protoplasts synthesized newly labeled peptidoglycan in the form of soluble, infrequently peptide cross-linked glycan chains which were released into the supernatant medium. These relatively large glycan chains were not transferred to exogenously added cell walls.

Alanine↗
PubMed1974

Measurement of intracellular iodophilic polysaccharide in two cariogenic strains of Streptococcus mutans by cytochemical and chemical methods.

Quantitative cytological and chemical methods have been developed to study the intracellular iodophilic polysaccharide (IPS) content of two strains of Streptococcus mutans. The cytological method uses a periodic acid-chlorite treatment of thin sections to increase the affinity of IPS for uranyl and lead salts. This results in the IPS appearing as individual electron-dense granules which can be counted for quantitative studies. As a basis for these quantitative studies, IPS was measured chemically by dissolving whole cells with hot KOH and quantitating spectrophotometrically the amount of iodine-polysaccharide complex formed.

Bacteriological Techniques↗
PubMed1974

Effect of inhibition of deoxyribonucleic acid and protein synthesis on the direction of cell wall growth in Streptococcus faecalis.

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both.

Azacitidine↗
PubMed1974

Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis 9790: release of cell wall, autolysin, and formation of stable autoplasts.

A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.

Acetates↗
PubMed1973

Some properties of the autolytic N-acetylmuramidase of Lactobacillus acidophilus.

The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 mug/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.

Acetates↗
PubMed1973

Sites of cellular autolysis in Lactobacillus acidophilus.

Ultrastructural changes which occur during cellular autolysis of Lactobacillus acidophilus strain 63AM Gasser in 0.05 M citrate buffer, pH 5.0, were examined. Early in the process, randomly distributed electron-dense patches were seen on the wall surface, along with an accompanying eversion of mesosomes. Later, after a loss of about 20% of the initial cellular turbidity, dissolution from the outside of nascent cross walls was seen. This observation was related to the normal process of cell separation. After this stage, short lengths of the cylindrical portion of the wall appeared to be completely removed in a random manner over the entire surface. This dissolution produced gaps in the wall which allowed the extrusion of membrane and cytoplasm. Although membrane was usually extruded through one major, polar, subpolar, or septal site, other secondary points of membrane extrusion were also frequently seen in the same cell section.

Autolysis↗
PubMed1972

Some properties of two autolytic-defective mutants of Streptococcus faecalis ATCC 9790.

The isolation and some properties of two mutants of Streptococcus faecalis ATCC 9790 (S. faecium) which autolyze at a much slower rate than the wild type are described. Compared with the wild type, mutant E71 autolyzed more slowly, contained less active but more latent autolysin in the isolated wall fraction, and possessed a wall of very similar chemical composition and degree of cross-bridging. Ultrastructural studies of exponential phase cells showed that cells of E71 were on the average slightly longer and had slightly thickened walls compared to the wild type. Mutant E81 autolyzed much more slowly, grew exponentially in long chains (8 to 40 cells compared with mainly diplococci), contained much less active and latent autolysin in the wall, and possessed a wall of very similar chemical composition but with about twice the content of N-terminal groups. Mutant E81 walls were more susceptible to isolated autolysin but possessed an autolysin of the same specificity as the wild type. Ultrastructurally E81 cells were, on the average, significantly longer and had thicker walls than the wild type. Mutant E71 may be partially blocked at either transport of autolysin to the wall or in conversion of latent to active autolysin. The pleitropic effects noted in mutant E81 have been taken to suggest a possible membrane defect and to support the role of the autolysin in cell separation.

Alanine↗
PubMed1972

Influence of macromolecular biosynthesis on cellular autolysis in Streptococcus faecalis.

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.

Alcohols↗
PubMed1971

Reinitiation of cell wall growth after threonine starvation of Streptococcus faecalis.

Cultures of Streptococcus faecalis ATCC 9790 were starved of threonine for 10 hr and then allowed to reinitiate growth in a fresh complete medium. On regrowth, culture turbidity began to increase within 10 min, but the ability of cells to autolyze did not begin to increase until after 30 min. Ultrastructural studies of regrowth of the initially thick-walled cells showed, at about 30 min, centripetal linear extension of new thin cross wall. This was followed, at about 40 min, by a notching, splitting, and peeling apart of the base of the cross wall. After this, extension of new thin peripheral wall from the nascent cross wall appeared to push old thick wall toward the poles. After the first cell division, asymmetric cells with one initial generation thick-walled pole and one second generation thin-walled pole were seen. After two divisions, thick-walled hemispheres were still seen, suggesting conservation of old wall during this time. A small fraction of the initial cell population exhibited aberrations and difficulties in reinitiating linear wall extension and were useful in the establishment of a model for the reinitiation of linear wall extension.

Bacteriolysis↗
PubMed1971

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.

Ammonium Chloride↗