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M L Higgins

Publications and source records attributed to M L Higgins.

At least 55 records · Page 3Linked to original sources
PubMed1982

Membrane-DNA attachment sites in Streptococcus faecalis cells grown at different rates.

The M-band technique was used to assess the number of attachment points of DNA to the cell membrane of Streptococcus faecalis grown at three different rates. Cells were X irradiated in liquid nitrogen and then analyzed simultaneously for the introduction of double-strand breaks into the chromosome and the degree of removal of DNA from the cell membrane (M band). Consideration of the data from these experiments and of the topology of the bacterial chromosome resulted in a reevaluation of former quantitative models. Our results are consistent with a semiquantitative model in which the bacterial chromosome is organized around a core structure. We interpret our data to mean that the core is attached to the membrane and that the complexity of the core changes more drastically with growth rate than does the number of membrane-DNA attachment points. An alternative model in which RNA hybridizes with DNA containing single- and double-strand breaks is also discussed. In any event, the complexity of these interactions precludes a reliable estimate of the number of membrane-DNA attachment sites.

Binding Sites↗
PubMed1981

Surface tension-like forces determine bacterial shapes: Streptococcus faecium.

The same tendency that causes soap bubbles to achieve a minimum surface area for the volume enclosed seems to account for many of the features of growth and division of bacteria, including both bacilli and cocci. It is only necessary to assume that growth takes place in zones and that only in these zones does the tension caused by hydrostatic pressure create the strain that forces the cell to increase the wall area. The stress developed by osmotic pressure creates strains that significantly lower the free energy of bond splitting by hydrolysis or transfer. We believe this is sufficient to make growing wall have some of the properties ordinarily associated with surface tension. The feature common to all bacterial cell wall growth is that peptidoglycan is inserted under strain-free conditions. Only after the covalent links have been formed are the intervening stressed peptide bonds cleaved so that the new unit supports the stress due to hydrostatic pressure. The present paper analyses the growth of Streptococcus faecium in these terms. This is a particularly simple case and detailed data concerning morphology are available. The best fit to the data is achieved by assuming that growth takes place in a narrow region near the splitting septum and that the septal material is already under tension as it is externalized and is twice as thick as the external wall throughout the development of the nascent poles. Constancy of the ratio of hydrostatic pressure to the effective surface tension, P/T, is also consistent with electron microscopic observations.

Cell Division↗
PubMed1981

Nucleoid structure in freeze fractures of Streptococcus faecalis: effects of filtration and chilling.

With the techniques used in this study, the nucleoid of Streptococcus faecalis could not be seen in freeze-etch preparations unless glutaraldehyde had been added to cultures of cells before they were frozen. With time, the nucleoid became visible as a network of fibers, apparently as a result of the aggregation of individual chromosomal elements in the presence of glutaraldehyde. When glutaraldehyde was added to undisturbed cultures, the fibers that became visible were observed in small patches that were seemingly scattered throughout the cytoplasm. However, if cells were chilled or placed on filters before glutaraldehyde was added, the fibers which then developed were seen in large central areas. The appearance of centralized nucleoids in freeze fractures of cells that had been chilled or filtered could be correlated with a decrease in the central density of the cytoplasm, as seen by light microscopy, in cells embedded in gelatin or bovine serum albumin. These observations are discussed in relation to a model for the normal structure of the nucleoid which suggests that the treatments routinely used to study the morphology-physiology of cells (chilling, filtration, and fixation) result in a reorganization of the cytoplasm, leading to an increase in the centralization of nuclear material.

Cell Nucleus↗
PubMed1980

Evidence that Streptococcus mutans constructs its membrane with excess fluidity for survival at suboptimal temperatures.

When cells from cultures of Streptococcus mutans strain FA-1 grown at 37 degrees C were exposed to incubation temperatures of 26 degrees C or less for 5 min or more, an extensive aggregation of particles was observed on the convex fracture faces of their freeze-cleaved membranes. Aggregation of particles was accompanied by a parallel increase in the activation energy for growth. By shifting the growth temperature from 37 to 24 degrees C for one doubling of culture mass, the transition temperature for membrane particle aggregation could be lowered from about 26 to 0 degrees C. Although membrane lipids became enriched with unsaturated fatty acids during this period of growth at 24 degrees C, this enrichment was not accompanied by an increased growth rate of the culture. However, the period of growth at 24 degrees C did result in bacteria that could grow more rapidly at 10 degrees C than could bacteria directly transferred from cultures grown at 37 degrees C. These observations suggest that the increase in membrane fluidity that occurs when bacteria are grown at 24 degrees C doses not allow bacteria to grow faster at 24 degrees C, but rather allows them to adapt more readily to further decreases in growth temperature.

Cell Membrane↗
PubMed1980

Structure of the nucleoid in cells of Streptococcus faecalis.

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.

Cell Nucleus↗
PubMed1980

Effect of macromolecular synthesis and lytic capacity on surface growth of Streptococcus faecalis.

Exposure of exponential-phase cultures of Streptococcus faecalis to any of three inhibitors of protein synthesis was accompanied by an increase in the average distance that the cross wall extended into the cytoplasm. This resulted in: (i) an increase in the average surface area of the cross wall (Sa) and (ii) septation occurring in the envelope growth sites that were much smaller than the controls. However, although at the concentrations used, all three antibiotics inhibited protein synthesis and autolytic capacity to the same extent and with the same kinetics, cells treated with these agents showed large differences in the rate at which Sa values increased above those of the untreated cells. The largest increases in Sa were observed in cells that synthesized the least amount of cytoplasmic macromolecules (deoxyribonucleic acid, plus ribonucleic acid, plus protein). The observations were interpreted in terms of a model in which a decreased lytic capacity reduces the rate of splitting of the nascent cross wall into two layers of peripheral wall, preferentially using wall precursors to close open cross walls. However, the extent to which centripetal growth occurs would be inversely related to the rate at which cytoplasmic macromolecules are synthesized. In contrast, inhibition of deoxyribonucleic acid synthesis was accompanied by decreased extension of the leading edge of the cross wall into the cytoplasm, thus antagonizing septation. These findings are discussed in relation to the normal cell division cycle of S. faecalis.

Azacitidine↗
PubMed1980

Unit cell hypothesis for Streptococcus faecalis.

The mass doubling times of exponential-phase cultures of Streptococcus faecalis were varied from 30 to 110 min by omitting glutamine from a defined growth medium and providing different concentrations of glutamate (ranging from 300 to 14 mug/ml). After Formalin fixation, cells were dried by the critical point method, and carbon-platinum replicas were prepared. The surface area and volume of cell poles seen in these replicas were estimated by a computer-assisted, three-dimensional reconstruction technique. It was found that the amount of surface area and volume of poles seen in these replicas were independent of the growth rate of culture from which the samples were taken. These observations were consistent with the unit cell model hypothesis of Donachie and Begg, in which a small number of surface sites would produce a constant amount of new cell surface regardless of the mass doubling time of the culture. However, measurements of the thickness of the cell wall taken from thin sections of the same cells showed that the cell wall increased in thickness as a function of the increase in cellular peptidoglycan content which occurs when the growth rate of this organism is slowed down by a decrease in glutamate concentration. Thus, it would seem that although the size of polar shells made by S. faecalis is invariant with growth rate, the amount of wall precursors used to construct these shells is not.

Cell Division↗
PubMed1980

Morphological effect of cerulenin treatment on Streptococcus faecalis as studied by ultrastructure reconstruction.

Exponential-phase cells of Streptococcus faecalis ATCC 9790 were treated with a concentration of cerulenin (5 micrograms/ml) that has been shown to block both lipoteichoic acid and lipid synthesis and cell division within 10 min. The morphological effect of this treatment was studied by making three-dimensional reconstructions of cells based on measurements taken from axial thin sections. This analysis indicated that cerulenin interferes with cell division by inhibiting normal constriction of the division furrow and centripetal growth of the cross wall in envelope growth sites. Rather than dividing, many of the sites in treated cells apparently continue to elongate and produce abnormally large amounts of peripheral wall surface. These observations were interpreted in terms of a previously proposed model in which cerulenin would prevent the synthesis of a lipid-containing inhibitor of autolytic enzyme activity needed for division. In addition, measurements showed that the average number of envelope growth sites per cell increased during treatment, suggesting that although cerulenin treatment blocks division, it does not interfere with the formation of new envelope growth sites. It was also observed that the size and frequency of mesosomes did not decline during the 60-min period of drug treatment. This tends to decrease the likelihood that mesosomes are formed from a pool of intracellular membrane precursors that would be depleted during a period of restricted lipid biosynthesis.

Antifungal Agents↗
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↗
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

Study of pole assembly in Bacillus subtilis by computer reconstruction of septal growth zones seen in central, longitudinal thin sections of cells.

The septal growth of Bacillus subtilis 168/s has been studied by making a number of observations from thin sections of cells from exponentially growing cultures. The process was initiated by the formation of a new cross wall under a preexisting layer of cylindrical wall. An annular notch appeared to cut through the overlying wall and presumably allowed the cross wall to split into two layers of peripheral wall. During this initial notching process, two raised bands of wall material were produced which resembled those previously observed in morphological studies of Streptococcus faecalis. Through an improved fixation technique, it was possible to preserve the bands seen in B. subtilis to the extent that they were used as markers to study the subsequent stages of septal growth. These stages included (i) the continued displacement of the two bands from the cross wall (as the two nascent polar surfaces enlarged and as the diameter of the cross wall decreased), (ii) the closure of the cross wall, and (iii) the final severance of the common cross wall connection between two completed poles. To study this process in a more quantitative manner, three-dimensional reconstructions of the envelope observed between pairs of the raised bands were made from axial thin sections of cells. The process of reconstruction was based on a technique by which x, y coordinates were taken from thin sections and were rotated around the cell's central axis. These reconstructions were used to estimate the surface area or volume of the reconstructed zones or their parts. A round of septal growth was then simulated by arranging 118 reconstructions in order of increasing surface area or volume. The topology of the process was studied by noting how various measurements of septal thickness, length, surface area, and volume varied as a function of increasing septal zone size. This analysis was based on several assumptions, of which three of the most important are: (i) the bands produced by the initial notching process are markers which separate septal from cylindrical wall growth; (ii) a septal zone observed between pairs of bands is made up of two nascent poles and a single cross wall; and (iii) as septal zones develop in terms of relative age they increase in size (volume or surface area) or amount of wall. The data suggested that the S. faecalis model of surface growth (in which polar growth occurs through a regulated constrictive separation and expansion of a cross wall) also seems applicable to the pattern of septal growth observed here for B. subtilis. This was indicated from measurements which showed that increases in the size of nascent polar surfaces were correlated with decreases in cross wall diameter. An explanation of these observations may be that decreases in cross wall diameter were due to a progressive splitting of the cross wall that removed surface from the outer circumference of the cross wall and converted it into new polar surface. Calculations further suggested that if the poles of B. subtilis were made by this model a sizeable and variable increase in surface area of the cross wall would also be required to convert these separating cross wall layers into two curved polar structures. Measurements of wall thickness taken from various locations within septal zones indicated that while the thickness of the polar wall of B. subtilis was constant over its surface, the width of the cross wall varied considerably during a round of synthesis. Again, one of the simplest explanations compatible with these observations and those previously made in S. faecalis is that the B. subtilis cross wall is brought to a constant thickness (possibly by remodeling or precursor addition) before or during separation. Although most observations made from the reconstruction of the septal zones of B. subtilis may fit the S. faecalis model of surface growth, differences in the pattern of septal growth were seen when the two organisms were compared. These have been discussed in terms of differences in the regulation of their respective septal growth sites and basic mechanisms of wall assembly and modification.

Bacillus subtilis↗
PubMed1978

Effects of addicting and nonaddicting drugs on HeLa cells.

After 70 subcultivations of HeLa cells in the presence of amphetamine, diphenhydramine hydrochloride, caffeine, codeine, or morphine, cells were obtained with increased tolerance to the respective drug. Withdrawal of amphetamine, diphenyhydramine hydrochloride or morphine from cells cultivated 645 days in the presence of the drug yielded cells that had either a decreased growth rate in the absence of drug or superior plating efficiencies with low drug concentration when compared to untreated HeLa cells. These observations would be expected from addicted cells. Cinematographic observations showed cell membrane undulation decreased in the presence of morphine. The membrane undulations of cells tolerant to morphine were less sensitive to that drug.

Cell Count↗
PubMed1978

A quantitative ultrastructural and chemical investigation of the accumulation of iodophilic polysaccharide in two cariogenic strains of Streptococcus mutans.

The amount of intracellular, iodophilic, glycogen-like polysaccharide (IPS) present in cells of two strains of Streptococcus mutans at various stages of growth in a chemically defined medium was determined by quantitative electron microscopy. The results obtained were then compared with the chemically determined, iodophilic polysaccharide content of cultures. The ultrastructural method used determined the fraction of area of central longitudinal sections of individual cells occupied by stained granules, and was therefore capable of determining amounts of polysaccharide in cells starved of glucose. Although the results of the two methods showed very good quantitative correlation, the ultrastructural method allowed study of glycogen synthesis on a cellular basis, and detected some heterogeneity in amounts of IPS stored by individual cells. The ultrastructural method also permits the detection of much smaller amounts of stored IPS than does the chemical method.

Cytoplasmic Granules↗