Evidence for two restriction-modification systems in Halobacterium cutirubrum.

Data from plating experiments indicated that Halobacterium cutirubrum NRC34001 has at least two separate restriction-modification systems. A spontaneous or induced loss of one or both systems resulted in four restriction-modification phenotypes. There was a positive correlation between changes in gas vacuolation phenotypes and either restriction-modification system.

Bacteriophages of Halobacterium halobium: virion DNAs and proteins.

The DNAs from bacteriophages Hh-1 and Hh-3 that infect Halobacterium halobium were characterized. Both phages contain linear double-stranded DNA and show no relatedness based on restriction endonuclease fragment patterns. Hh-1 DNA has 67.05% guanine plus cytosine (G+C) and a molecular weight of 24.6 X 10(6), whereas Hh-3 DNA has 62.15% G+C and a molecular weight of 19.4 X 10(6). Polyacrylamide gel electrophoresis indicated that the major proteins of the two phages are of different molecular weights.

Recovery from nutrient starvation by a marine Vibrio sp.

A marine psychrophilic Vibrio sp., Ant-300, recovered from starvation after the addition of 1 volume of complete nutrient medium to 9 volumes of starvation menstruum. Turbidity (measured by optical density), viable cell counts, cell size (measured from electron micrographs), and cellular concentrations of protein, DNA, and RNA were monitored with recovery time. The usual growth curve of bacterial cultures was observed. On a per viable cell basis, protein, DNA, and RNA increased to maximum values just before cell division and then returned to close to the initial starved-cell value during the stationary phase. Cells under complete starvation conditions or missing only one nutrient in the stationary phase responded with cell division resulting in many smaller cells. The length of the lag phase during recovery was directly proportional to the length of the prior starvation period, even when identical numbers of cells were used for recovery. Cells appeared to pass more deeply into dormancy with starvation time.

Starvation-survival processes of a marine Vibrio.

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.

Bacteriophages of Halobacterium halobium: isolated from fermented fish sauce and primary characterization.

BActeriophages infecting extremely halophilic bacteria of the genus Halobacterium have been isolated from fermented anchovy sauce. Two distinct phages, designated Hh-1 and Hh-3, have been characterized. Both Hh-1 and Hh-3 are more tolerant of suspension in solutions of low ionic strength that their host bacteria. Both Hh-1 and Hh-3 have the ability to establish a carrier state upon infection of sensitive cells of H. halobium. Bacterial cells infected with phage in the carrier state are viable, produce phages, are immune to superinfection with homologous phages, yet remain fully capable of supporting heterologous phages. These properties suggest that the halophages are well adapted to survival in environments in which the salinity is subject to rapid changes of considerable magnitude.

Induction of the SOS system by DNA ligase-deficient growth of Escherichia coli.

When Escherichia coli carrying a thermosensitive mutation in DNA ligase was grown at the restrictive temperature, several functions associated with the SOS system were induced. These included lambda prophage induction, W-reactivation and W-mutagenesis of ultraviolet-irradiated lambda phage, and recA protein synthesis, all of which were lexA+ recA+ recB+ dependent and chloramphenicol sensitive, and lexA+-dependent filamentation. These results indicate that ligase-deficient growth leads to the induction of the SOS system, and that all the above functions may respond to common induction signals.

Effect of chloramphenicol and the recB gene product on DNA metabolism in Escherichia coli K12 strains defective in DNA ligase.

We have examined DNA strand breakage, DNA degradation, and the rate of DNA synthesis in lig and lig-recB strains of Escherichia coli K12 incubated in the presence and absence of 3 mug/ml chloramphenicol. Substantial DNA strand breakage and DNA degradation is observed in the lig strain upon growth at 40 degrees C; however, such strand breakage and DNA degradation is not observed in th lig-recB strainl Incubation of the lig strain at 40 degrees C in the presence of 3 mug/ml chloramphenicol reduces the amount of DNA strand breakage and DNA degradation to the level observed in the lig-recB strain. Together, these results demonstrate that exonuclease V (the recBC gene product) is responsible for the increased DNA degradation associated with DNA ligase deficiency.

Properties of a DNA ligase mutant of Escherichia coli: introduction of strand breaks in DNA.

Strand breaks accumulated in the DNA of a temperature-sensitive DNA ligase mutant of Escherichia coli growing at the restrictive temperature, as detected by zone sedimentation through alkaline sucrose density gradients. The rate of strand breakage was increased by concomitant thymine starvation. Rifampicin and chloramphenicol inhibited the accumulation of strand breaks in the DNA. There was a correlation between the accumulation of strand breaks in the DNA and lethality, suggesting that such breaks are the basis for lethality at the restrictive temperature.

Induction of error-prone repair as a consequence of DNA ligase deficiency in Escherichia coli.

DNA ligase deficiency is shown to induce generalized mutator activity in E. coli. This mutator activity is unaffected by 3 mug/ml of chloramphenicol but is abolished both in lig-recA double mutants and by incubation with 20 mug/ml of chloramphenicol. Dna ligase deficiency is also shown to reactivate ultraviolet light-irradiated phage lambda and T7 and to increase both spontaneous and ultraviolet light-induced mutagenesis in phage lambda, all of which are abolished in lig-recA strains. Interaction occurs between the molecular events of mutagenesis induced by ultraviolet irradiation and those induced by DNA ligase deficiency. These observations suggest a common pathway, coordinately expressed with the inducible repair mode, that is responsible for mutagenesis.

Two mechanisms of near-ultraviolet lethality in Saccharomyces cerevisiae: a respiratory capacity-dependent and an irreversible inactivation.

Near-ultraviolet irradiation of actively growing yeast cells leads to cell death by two distinct mechanisms. The first type of cell death is evident after low doses of near-ultraviolet light (3 times 10-4 ergs times mm- minus 2) and is due to a reversible inactivation of the respiratory capacity of the cell. In studies with yeast mitochondrial membranes the quinones were identified as the site of inactivation by determining the relative levels of the following oxidase activities after irradiation: exogenous NADH, endogenous NADH (via isocitrate dehydrogenase), succinate, and D-lactate oxidases. A second type of cell death is caused after high doses (1.8 times 10-5 ergs times mm- minus 2) and is irreversible. The mechanism of this inactivation is unknown.

Thymineless mutagenesis in Escherichia coli.

To clarify the relationship between thymineless death and thymineless mutagenesis, the induction of arginine revertants of Escherichia coli TAU-bar by thymine starvation was examined in physiological terms. Induced revertants were detectable both on minimal medium lacking arginine and minimal medium supplemented with 1 mug of arginine per ml. Substantial thymineless mutagenesis occurred during the period before the onset of thymineless death. Mutagenesis and loss of viability were observed upon incubation in medium lacking thymine and arginine, and both were inhibited upon incubation in medium lacking thymine and uracil. Mutagenesis also occurred during thymine starvation at 25 C, where there was relatively little loss of viability. At 37 C thymineless mutagenesis did not require complete thymine starvation, and the induction of revertants appeared to be initiated at the same suboptimal thymine concentration at which lethality was first detectable. Mutagenesis was found not to occur preferentially at the growing point of deoxyribonucleic acid replication. These results suggest that thymineless mutagenesis does not involve simply errors in base pairing due to the absence of thymine. The data also suggest that the induction of mutations and thymineless death are due to the same primary event but that mutagenesis is the more sensitive response.

Survival and macromolecular synthesis during incubation of Escherichia coli in limiting thymine.

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.

Properties of a deoxyribonucleic acid ligase mutant of Escherichia coli: x-ray sensitivity.

A deoxyribonucleic acid ligase-deficient mutant is X-ray sensitive relative to the parent strain, suggesting that deoxyribonucleic acid ligase functions in repair of X-ray-induced, single-strand scissions.

Properties of a temperature-sensitive, radiation-sensitive mutant of Escherichia coli. II. DNA replication.

We have described a temperature-sensitive, radiation-sensitive mutant of Escherichia coli. In vitro assays have demonstrated that DNA ligase activity is markedly reduced in this strain relative to that of the parent strain. Okazaki fragments of newly replicated DNA accumulate in this mutant strain upon growth at the restrictive temperature. These results imply that the same enzyme, DNA ligase, functions both in normal semiconservative replication and in nonconservative repair replication of DNA.