Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.     

Photoreactivation, photoproduct formation, and deoxyribonucleic acid state in ultraviolet-irradiated sporulating cultures of Bacillus cereus

Photoreactivation of ultraviolet-irradiated Bacillus cereus T declined markedly during the development of stage IV forespores. During ultraviolet irradiation of a culture containing early and late stage IV forespores, both vegetative- and spore-type photoproducts were formed. The formation of vegetative-type photoproducts (mainly thymine dimers) decreased to nearly half during late stage IV, remaining constant until lysis of the mother cells began, when it fell to zero. Spore-type photoproducts were first observed during late stage IV and increased with the increase in numbers of late stage IV forespores. The occurrence of spore-type photoproducts preceded the development of refractile forespores by about 1 h. At stage III the nuclear material occupied a central position, and the ribosomes were at the periphery of the forespore protoplast. During stage IV the deoxyribonucleic acid (DNA) occurred in a peripheral position, and bundles of fibers ("transition" DNA) could be seen. By stage V, all of the DNA appeared to be of the spore type and was peripheral, and the forespore protoplast center was packed with ribosomes. Forespore stages II, III, and IV were classified by light and electron microscopy. The curve for electron microscope classifications preceded that for light microscope classifications by approximately one stage. The formation of spore-type photoproducts preceded differentiation of DNA by about 1 h, the latter coinciding with the development of refractility. Spore-type photoproducts have been associated with DNA in the A state, and the progressive change of the forespore DNA into this state is discussed in relation to the spore differentiation process.     

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.     

Bacillus subtilis deoxyribonucleic acid gyrase

Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics. The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B. subtilis and was functionally characterized in vitro. The genetic loci nalA and novA but not novB were shown to code for portions of the functional gyrase. Enzyme from the antibiotic-resistant mutants was resistant to the drug in vitro. The most striking observation was the remarkable similarity between the B. subtilis enzyme and other DNA gyrases, especially with respect to the oxolinic acid-induced DNA cleavage in the presence of sodium dodecyl sulfate. All of the enzymes appeared to possess the same specificity of cutting sites regardless of the source or type of DNA used. This result implies that gyrase binding to DNA is highly specific.     

Interaction of iodine with Bacillus subtilis spores and spore forms

Buffered solutions of iodine (pH 7.0) were effective against Bacillus subtilis spores, but concentrations and contact times for effective sporicidal action were relatively high. Concentrations of 500 to 1000 ppm available iodine with a contact time of 30–45 min were required to produce a 3–5 log reduction. Treatment of spores with agents which caused progressive extraction of coat protein and cortex hexosamine was associated with increased sensitivity to iodine. Treatment of spores with iodine produced extraction of spore coat protein which was potentiated in the presence of NaOH, but there was no evidence of breakdown of cortex hexosamines or release of dipicolinic acid, either from intact spores or spore protoplasts. Sporicidal concentrations of iodine stimulated the uptake of (³²P) phosphate over an initial period of 30–40 min, but phosphate then leaked from the cells; 1000 ppm available iodine produced total loss within 60 min. Results of this investigation are consistent with previous findings which suggest that the resistance of spores to biocides is related to the barrier properties of the spore outer layers and that the sporicidal action of halogen-releasing agents is related to their ability to cause coat and cortex degradation, leading to rehydration of the spore protoplast and allowing diffusion to their site of action on the underlying protoplast.     

Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme

Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms. The resistance of these to sodium hypochlorite (1000 parts/10(6) free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated. Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release. A spore lytic enzyme was extracted and examined in respect of hypochlorite activity. The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore.     

Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme

G orman , S.P., S cott , E.M. & H utchinson , E.P. 1984. Hypochlorite effects on spores and spore forms of- Bacillus subtilis and on a spore lytic enzyme. Journal of Applied Bacteriology 56 , 295–303.
Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms. The resistance of these to sodium hypochlorite (1000 parts/10⁶ free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated. Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release. A spore lytic enzyme was extracted and examined in respect of hypochlorite activity. The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.     

Characterization of spores of Bacillus subtilis which lack dipicolinic acid

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores. The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-gamma. Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.     

Acid-soluble spore proteins of Bacillus subtilis

Acid-soluble spore proteins (ASSPs) comprise about 5% of the total protein of mature spores of different Bacillus subtilis strains. They consist of three abundant species, alpha, beta, and gamma, four less abundant species, and several minor species, alpha, beta, and gamma make up about 18, 18 and 36%, respectively, of the total ASSPs of strain 168, have molecular weights of 5,900, 5,9000, and 11,000, respectively, and resemble the major (A, C, and B) components of Bacillus megaterium ASSPs in several respects, including sensitivity to a specific B. megaterium spore endopeptidase. However, they have pI's of 6.58, 6.67, and 7.96, all lower than those of any of the B. megaterium ASSPs. Although strains varied in the proportions of different ASSPs, to overall patterns seen on gel electrophoresis are constant. ASSPs are located interior to the cortex, presumably in the spore cytoplasm, and are synthesized during sporulation and degraded during germination.