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G D Shockman

Publications and source records attributed to G D Shockman.

At least 55 records · Page 3Linked to original sources
PubMed1980

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.

Autolysis↗
PubMed1980

Soluble group- and type-specific antigens from type III group B Streptococcus.

Two soluble polysaccharide antigens of a type III group B Streptoccus were isolated from the culture medium after growth of strain M732 in a chemically defined broth supplemented with acid-hydrolyzed casein. The type- and group- specific antigens were isolated from the culture supernatant by anion-exchange chromatography with diethylaminoethyl-Sephacel. Two carbohydrate-containing peaks, which had serological reactivity with group B or type III antiserum, respectively, were eluted with a linear NaCl gradient and further purified by gel filtration. The type III polysaccharide was found to contain glucose, galactose, glucosamine, and sialic acid, whereas the group B polysaccharide contained galactose, glucosamine, and rhamnose. For the type III polysaccharide, sialic acid was shown to be the major immunodeterminant, and for the group B polysaccharide, rhamnose was the immunodominant sugar. Both the type III and group B polysaccharides were obtained in high yields without employing harsh physical or chemical treatment and both were immunologically distinct. By immunoelectrophoresis or counterimmunoelectrophoresis, type III antigen failed to react with group-specific antiserum and the group B antigen failed to react with type III antiserum.

Antigen-Antibody Reactions↗
PubMed1980

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.

Lysine↗
PubMed1980

Lipids and lipoteichoic acid of autolysis-defective Streptococcus faecium strains.

Two of four previously isolated autolysis-defective mutants of Streptococcus faecium (Streptococcus faecalis ATCC 9790) incorporated substantially more [14C]glycerol into lipids and lipoteichoic acid than did the parent strain. Consistent with increased accumulation of lipids and lipoteichoic acid, significantly higher levels of phosphorus were found in the corresponding fractions of the two mutant strains than in the wild type. Although the autolysis-defective mutant strains contained the same assortment of lipids as the wild type, the relative amount of [14C]glycerol incorporated into diphosphatidylglycerol increased, accompanied by a decreased fraction of phosphatidylglycerol. These results suggested that increased cellular content of two types of substances, acylated lipoteichoic acid and lipids (notably diphosphatidylglycerol), which previously had been shown to be potent inhibitors of the N-acetylmuramoylhydrolase of this species, contributed to the autolysis-defective phenotype of these mutants. Consistent with this interpretation are observations that (i) cerulenin inhibition of fatty acid synthesis increased the rates of benzylpenicillin-induced cellular lysis and that (ii) Triton X-100 or Zwittergent 3-14 treatment could reveal the presence of otherwise cryptic but substantial levels of the active form of the autolysin in cells of three of four mutants and of the proteinase-activable latent form in all four mutants.

Bacteriolysis↗
PubMed1980

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.

Bacteriological Techniques↗
PubMed1980

Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods.

Strains of Streptococcus mutans are very susceptible to growth inhibition by benzylpenicillin, but are tolerant to lysis when exposed to even high concentrations of this drug. These properties enabled this study of S. mutans GS-5 surface growth and peptidoglycan, ribonucleic acid, protein, and deoxyribonucleic acid syntheses in the absence of osmotic stabilization. Inhibition of syntheses of peptidoglycan, ribonucleic acid, and protein was dose dependent. Synthesis of peptidoglycan was most susceptible. Substantial but less severe inhibitions of ribonucleic acid and protein syntheses rapidly followed decreased peptidoglycan synthesis, whereas inhibition of deoxyribonucleic acid synthesis was delayed and minimal. Computer-assisted reconstructions of surface growth zones and poles observed in electron micrographs of replicas were performed and indicated that at low concentrations of benzylpenicillin (0.03 micrograms/ml), growth sites reached abnormally large sizes and surface/volume ratios. The observed shifts in surface/volume ratio were attributed to an inhibition of the normal constrictive division mechanism. The poles of these cells also increased in size over those of the controls, but the relatively smaller change in surface/volume ratio confirmed the visual impression that the shape of the poles was much less altered than the shape of the growth sites. As the concentration of benzylpenicillin used was raised from 0.03 to 2 micrograms/ml, the ability of growth sites and poles to enlarge was restricted in a manner that most closely agreed with the extent of inhibition of peptidoglycan (rather than deoxyribonucleic acid, ribonucleic acid, or protein) synthesis. This correlation suggested that increases in cell size may be regulated by the supply of peptidoglycan precursors.

Bacterial Proteins↗
PubMed1979

Precursor-product relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium.

Exponential biosynthesis and excretion of lipoteichoic acid (LTA) during the exponential phase of growth, and continued synthesis and excretion during valine starvation of Streptococcus faecium (S. faecalis ATCC 9790), were shown. During exponential growth, extracellular LTA (LTAx) accounted for approximately 13% of the total LTA in cultures, whereas during valine starvation, this percentage increased to approximately 60% within 4 h. LTAx was present in a low-molecular-weight, apparently deacylated form, whereas intracellular (LTAi) was present primarily in an apparently high-molecular-weight, acylated and micellar form. Experiments utilizing chases of either fully equilibrated or short pulses of [14C]- or [3H]glycerol were used to demonstrate that LTAx was derived directly from LTAi.

Acetates↗
PubMed1979

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.

Enterococcus faecalis↗
PubMed1979

Morphological and physiological study of autolytic-defective Streptococcus faecium strains.

Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.

Anti-Bacterial Agents↗
PubMed1979

Release of autolytic enzyme from Streptococcus, faecium cell walls by treatment with dilute alkali.

The autolytic enzyme (endo-beta-1,4-N-acetylmuramoylhydrolase) of Streptococcus faecium (S. faecalis ATCC 9790) was released in a soluble form from insoluble cell wall-autolytic enzyme complexes by treatment with dilute NaOH at 0 degree C. Treatment of cell wall-enzyme complexes, obtained from either exponential- or stationary-phase cells, with 0.008 to 0.01 N NaOH gave maximum yields of autolytic enzyme activity. At a fixed concentration of NaOH, the yield of autolysin increased with increasing wall densities and was accompanied by the release of methylpentose and phosphorus in amounts proportional to the autolysin. Since extraction of wall-enzyme complexes with 4.5 M LiCl at 0 degree C also removed methylpentose and phosphorus, release of enzyme with NaOH did not appear to result from hydrolysis of covalent linkages. The autolytic enzyme activity released from intact cells, or cell walls, was predominantly in the later (proteinase activable) form which could be activated by trypsin or a proteinase present in commerical bovine plasma albumin.

Cell Wall↗
PubMed1978

Structural arrangement of polymers within the wall of Streptococcus faecalis.

The structure of the cell wall of Streptococcus faecalis was studied in thin sections and freeze fractures of whole cells and partially purified wall fractions. Also, the structures of wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan wall polymers were compared with wall preparations that possess a full complement of accessory polymers. The appearance of the wall varied with the degree of hydration of preparations and physical removal of the cell membrane from the wall before study. Seen in freeze fractures of whole cells, the fully hydrated wall seemed to be a thick, largely amorphic layer. Breaking cells with beads caused the cell membrane to separate from the wall and transformed the wall from a predominantly amorphic layer to a structure seemingly made up of two rows of "cobblestones" enclosing a central channel of lower density. Dehydration of walls seemingly caused the cobblestones to be transformed into two bands which continued to be separated by a channel. This channel was also observed in isolated wall preparations treated with hot trichloroacetic acid to remove non-peptidoglycan polymers. These observations are consistent with the interpretation that both peptidogylcan and non-peptidoglycan polymers are concentrated at the outer and inner surfaces of cell walls. These observations are discussed in relation to possible models of wall structure and assembly.

Cell Wall↗
PubMed1978

Autolytic defective mutant of Streptococcus faecalis.

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.

Bacteriolysis↗
PubMed1978

Cellular autolytic activity in synchronized populations of Streptococcus faecium.

The autolytic capacity of Streptococcus faecium (S. faecalis ATCC 9790) varied during synchronous cell division. This phenomenon was initially observed in rapidly dividing populations (TD=30 to 33 min) synchronized by a combination of induction and size selection techniques. To minimize the problems inherent in studies of cells containing overlapping chromosome cycles and possible artifacts generated by induction techniques, the autolytic capacities of slowly dividing populations (TD=60 to 110 min) synchronized by selection only were examined. Although the overall level of cellular autolytic capacity was observed to decline with decreasing growth rate, sharp, periodic fluctuations in cellular autolytic capacity were seen during synchronous growth at all growth rates examined. On the basis of similar patterns of cyclic fluctuations in autolytic capacity of cultures synchronized by (i) selection, (ii) amino acid starvation followed by size selection, and (iii) amino acid starvation followed by inhibition of DNA synthesis, a link of such fluctuations with the cell division cycle has been postulated.

Amino Acids↗
PubMed1978

Relationship between cellular autolytic activity, peptidoglycan synthesis, septation, and the cell cycle in synchronized populations of Streptococcus faecium.

Synchronized, slowly growing (TD = 70 to 80 min) cultures were used to study several wall-associated parameters during the cell cycle: rate of peptidoglycan synthesis, septation, and cellular autolytic activity. The rate of peptidoglycan synthesis per cell declined during most of the period of chromosome replication (C), but increased during the latter part of C and into the period between chromosome termination and cell division (D). An increase in cellular septation was correlated with the increased rate of peptidoglycan synthesis. Cellular autolytic capacity increased during the early portion of C, reached a maximum late in C or early in D, and declined during D. Inhibition of DNA synthesis during C prevented the decline in autolytic capacity at the end of the cell cycle, caused a slight reduction in the rate of peptidoglycan synthesis, delayed but did not prevent septation, and prevented the impending cell division by inhibiting cell separation. Inhibition of DNA synthesis during D did not prevent the increase in autolytic capacity during the next C phase, but, once again, prevented the decline at the end of the subsequent cycle. Thus, increased autolytic capacity at the beginning of the cell cycle did not seem to be related to chromosome initiation, whereas decreased autolytic capacity at the end of the cell cycle seemed to be related to chromosome termination. The data presented are consistent with the role of autolytic enzyme activity in the previously proposed model for cell division of S. faecium (G.D. Shockman et al., Ann. N.Y Acad. Sci. 235:161-197, 1974).

Cell Cycle↗
PubMed1978

Approximation of the cell cycle in synchronized populations of Streptococcus faecium.

Slowly growing populations (TD = 70 to 80 min) of Streptococcus faecium (S. faecalis ATCC 9790) were synchronized by selection after sucrose gradient fractionation. The cell cycle was approximated by correlating the patterns of DNA accumulation and cell division. More specifically, the beginning of cell cycle was equated with the beginning of a rapid linear increase in DNA accumulation. The DNA content of the culture approximately doubled during the period of accumulation, which lasted about 51 min. The period of rapid DNA accumulation, was followed by a period of reduced accumulation that lasted about 24 min. During synchronized growth, cell numbers increased rapidly in coordination with the period of rapid DNA accumulation and exhibited a plateau during the period of reduced DNA accumulation. In contrast, RNA and protein appeared to accumulate exponentially throughout the cell cycle at the same rate as culture mass.

Bacterial Proteins↗
PubMed1978

Cellular lysis of Streptococcus faecalis induced with triton X-100.

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.

Bacterial Proteins↗