Genetically controlled removal of "spore photoproduct" from deoxyribonucleic acid of ultraviolet-irradiated Bacillus subtilis spores

Previous genetic analysis indicated that at least two genes determine the ultraviolet (UV) sensitivity of Bacillus subtilis spores. The present study shows that these genes independently control two distinguishable processes for removing UV-induced spore photoproduct (5-thyminyl-5,6-dihydrothymine, or TDHT) from spore deoxyribonucleic acid. The first, is a spore repair mechanism by which TDHT is removed rapidly without appearing in acid-soluble form. This mechanism, which is demonstrated in both UV-resistant and excision-deficient strains, operates to a certain extent during germination without requiring vegetative growth. The second, demonstrated in a mutant which lacks the first mechanism, removes TDHT relatively slowly and only if germinated spores are allowed to develop toward vegetative cells. The latter mechanism appears identical to excision-resynthesis repair, since the mutation abolishing it renders the irradiated vegetative cells incapable of removing cyclobutane-type pyrimidine dimers. Blocking either one of these mechanisms only slightly affects the UV sensitivity of spores, but blocking both prevents TDHT removal and gives high UV sensitivity.     

Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.     

Photoreactivation of ultraviolet-irradiated, plasmid-bearing, and plasmid-free strains of Bacillus anthracis.

The effects of toxin- and capsule-encoding plasmids on the kinetics of UV inactivation of various strains of Bacillus anthracis were investigated. Plasmids pXO1 and pXO2 had no effect on bacterial UV sensitivity or photoreactivation. Vegetative cells were capable of photoreactivation, but photo-induced repair of UV damage was absent in B. anthracis Sterne spores.     

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

Cells of Bacillus subtilis recA1 are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system.     

Respiratory system of vegetative and sporulating Bacillus cereus.

The composition and organization of the Bacillus cereus respiratory system were studied. The abolition of NADH-dependent respiration in vegetative and sporulating cell membranes by near-UV light (360 nm) indicated that electrons reduce oxygen only through a quinone-cytochrome pathway. Difference spectroscopy demonstrated the presence of cytochromes b555, c548, aa3, b562, and a2. This composition and studies with respiratory inhibitors suggested that cytochromes are organized in at least two branches, one being highly sensitive to cyanide.     

Repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Haemophilus influenzae

Ultraviolet-sensitive and wild-type Haemophilus influenzae cells were exposed to irradiated and unirradiated transforming deoxyribonucleic acid (DNA) containing a marker which can be linked to another marker in the cells. Lysates were made after various times of incubation and assayed for transforming activity on an excisionless recipient. Repair can be noted as an increase in activity from the irradiated donor DNA after its linkage to the recipient DNA. No repair can be observed in a mutant which is unable to integrate transforming DNA. There is a little repair in another mutant which is unable to excise pyrimidine dimers. H. influenzae cells also repair nondimer damage, as judged by the increase in activity observed in lysates made with irradiated and maximally photoreactivated DNA.     

Regulation of dipicolinic acid biosynthesis in sporulating Bacillus cereus. Characterization of enzymic changes and analysis of mutants

Some of the early enzymes in the lysine-biosynthetic pathway also function for dipicolinic acid synthesis in sporulating Bacillus cereus T. 1. The first enzyme, aspartokinase, loses its sensitivity to feedback inhibition by lysing. This change occurs before the time of dipicolinic acid synthesis but at a time when diaminopimelic acid is required for spore cortex formation. 2. A possible regulatory change at a branch point in the pathway was studied by examining the properties of a key enzyme, dihydrodipicolinic acid reductase. No alteration in the feedback sensitivity or sedimentation rate of this enzyme could be detected during sporulation. 3. Two mutants producing heat-sensitive spores were analysed. Both produced spores that contained decreased amounts of dipicolinic acid. Although neither was a lysine auxotroph, they both had greatly decreased activities of certain lysine-biosynthetic enzymes in sporulating cells. 4. Starvation of cells for calcium also results in the production of spores that are heat-sensitive and contain less dipicolinic acid than the control. A decreased content of one of the lysine-biosynthetic enzymes, dihydrodipicolinic acid synthetase, in calcium-starved cells could account for the lower concentration of dipicolinic acid in the spores.     

Ampicillin induced septum formation in Bacillus cereus

Cells of Bacillus cereus grown in the presence of subinhibitory concentrations of ampicillin at either 30 degrees or 45 degrees C exhibited an increase in the numbers of centres of septum formation per unit cell length. Under identical conditions of cultivation, cells of Escherichia coli grew as aseptate filaments. In general, untreated B. cereus cells grown at 45 degrees C were longer than those grown at 30 degrees C. The strain of E. coli used was unaffected in terms of filamentation by elevated growth temperature. Results are discussed in terms of the presence and availability of penicillin binding proteins and autolysins involved in cell growth, division and separation.     

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.     

Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli.

A system of cells made permeable by treatment with high concentrations of surcrose (plasmolysis) has been exploited to study the excision repair of ultraviolet-irradiated deoxyribonucleic acid in Escherichia coli. It is demonstrated that adenosine 5'-triphosphate is required for incision breaks to be made in the bacterial chromosome as well as in covalently closed bacteriophage lambda deoxyribonucleic acid. After plasmolysis, uvrC mutant strains appear as defective in the incision step as the uvrA-mutated strains. This is in contrast to the situation in intact cells where uvrC mutants accumulate single-strand breaks during postirradiation incubation. These observations have led to the proposal of a model for excision repair, in which the ultraviolet-specific endonuclease, coded for by the uvrA and uvrB genes, exists in a complex with the uvrC gene product. The complex is responsible for the incision and possibly also the excision steps of repair. The dark-repair inhibitors acriflavine and caffeine are both shown to interfere with the action of the adenosine 5'-triphosphate-dependent enzyme.     

Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum.

A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostelium discoideum. Analysis of the frequency of T4 endonuclease V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (rad+) removed greater than 98% of the dimers induced by irradiation at 40 J/m2 (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8% were removed during the 10- to 15-min period immediately after irradiation, whereas NC4 excised 64% during this interval. HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic acid at the sites of pyrimidine dimers.