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Biomedical subjects

J N Reeve

Publications and source records attributed to J N Reeve.

At least 127 records · Page 7Linked to original sources
PubMed1977

Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis.

Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.

Acetylglucosamine↗
PubMed1975

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.

Bacillus subtilis↗
PubMed1974

Physiological studies of Bacillus subtilis minicells.

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.

Adenosine Triphosphate↗
PubMed1974

Growth of the Bacillus subtilis cell surface.

A double mutant strain, Bacillus subtilis div IV B1 dal trp, has been constructed which grows as filamentous cells and minicells and requires d-alanine for growth. Removal of d-alanine from a growing population of cells resulted in cell bulging 25% of the cell length from one cell pole, followed by cell lysis. Little ultrastructural change in the cell envelope could be detected during bulge formation.

Alanine↗
PubMed1973

Minicells of Bacillus subtilis.

After nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis, two Bacillus subtilis mutants (div IV-A1 and div IV-B1) were isolated that are defective in the location of division site along cell length. Both mutations were transferred into strain CU403 by transformation, and their properties were studied in the CU403 genetic background. Location of divisions in close proximity to cell pole regions in both mutants results in minicell production. Purified minicells contain a ratio of ribonucleic acid to protein comparable to that found in the parent cells. Autoradiographs of (3)H-thymine incorporation into deoxyribonucleic acid (DNA), thymine-2-(14)C incorporation into DNA, electron micrographs, and chemical analyses for DNA all fail to demonstrate DNA in the minicells. Minicells produced by both mutants are highly motile, an indication of functional energy metabolism. Electron micrographs reveal that minicells are produced by a structurally normal division mechanism and that minicells contain a normal cell surface. The div IV-A1 mutation has been mapped by PBS1 transduction linked to ura. The div IV-B1 mutation is closely linked to pheA by both PBS1 transduction and by co-transformation.

Autoradiography↗
PubMed1972

Cell division of Escherichia coli BUG-6: effect of varying the length of growth at the nonpermissive temperature.

When Escherichia coli BUG-6 is grown at 42 C and then returned to 30 C, the division kinetics during the recovery at 30 C are dependent on the length of time the cells were grown at 42 C. If chloramphenicol is added when the cells are shifted from 42 to 30 C, no division occurs if the period at 42 C is less than 4 min or more than 110 min. Maximum division occurs when the period at 42 C is 50 min. A discussion of these results with reference to a previously proposed model is presented.

Cell Division↗
PubMed1972

Cell division of Escherichia coli BUG-6: effect of varying the temperature used as the nonpermissive growth condition.

When Escherichia coli BUG-6 is shifted from 30 C to 36 or 38 C, division does not stop, but the rate of division of the cell population is initially decreased followed by a period of increased rate of division before the rates characteristic of growth at 36 and 38 C are obtained. After a shift from 30 to 40 C, the rate of cell division gradually decreases over a 10-min period and then stops. The inhibition continues for 25 min, and then the cells divide rapidly before the division rate characteristic of 40 C is obtained. If filaments produced by 45 min of growth at 42 C are temporarily replaced at 30 C and then returned to 42 C, division occurs at 42 C. The amount of division is dependent on the length of the period at 30 C and can be decreased by a 3-min pulse of chloramphenicol immediately before the 42 to 30 C shift.

Cell Division↗
PubMed1970

Regulation of Cell Division in Escherichia coli: Characterization of Temperature-Sensitive Division Mutants.

A temperature-sensitive division mutant of Escherichia coli was isolated by using differential filtration to select for filaments at 42 C and normal cells at 30 C. Cells shifted from 30 to 42 C stop dividing almost immediately, suggesting the temperature-sensitive element is required for cell division late in the cell cycle. Cells returned to 30 from 42 C divide abruptly, suggesting accumulation of division potential at 42 C. Inhibitors of protein, deoxyribonucleic acid, and ribonucleic acid synthesis do not block division during the recovery period at 30 C. Cycloserine does not stop cell division, vancomycin shows some effect on cell division, whereas penicillin completely stops cell division during this period. The addition of high concentrations of NaCl to filaments at 42 C results in a burst of cell division. The final cell number is equivalent to the control which is grown at 30 C if sufficient salt is added (11 g/liter, final concentration). After the original burst, cell division ceases at the nonpermissive temperature even at increased osmolality. Chloramphenicol, puromycin, vancomycin, and penicillin prevent division during the recovery in the presence of NaCl. Kinetic data indicate division potential decays to a reversible inactive intermediate which rapidly decays to an irreversible inactive form. Conversion of division potential to the inactive form is correlated with a 100- to 1,000-fold derepression of the synthesis of division potential. The mutation appears to involve a stage in cross-wall synthesis which is required during the terminal stages of division.

Journal Article↗