Protection of DNA by alpha/beta-type small,acid-soluble proteins from Bacillus subtilis spores against cytosine deamination

Spores of Bacillus subtilis contain high levels of proteins, termed alpha/beta-type small, acid-soluble proteins (SASP), that protect the spore's DNA against different types of DNA damage. We tested one such protein, SspC, and two of its variants for their ability to protect plasmid DNA against hydrolytic deamination of cytosine to uracil. If unrepaired, such damage to DNA causes C to T mutations. We found that one SspC variant, SspC(Delta 11-D13K), protected DNA against cytosine deamination at two different temperatures (45 and 70 degrees C) and pH values (5.2 and 7.9), reducing the rate of deamination by as much as 10-fold. At 70 degrees C, pH 7.9, the wild-type SspC and its variant, SspC(Delta 11), provided little protection against deamination but were effective in protecting DNA at 45 degrees C, pH 7.9. Parallel studies of the abilities of these proteins to protect DNA against restriction digestion revealed that there was a good correlation between the abilities of the proteins to protect against restriction endonucleases and reductions in cytosine deaminations. These results show that the binding of SspC variants to DNA can prevent attack on DNA bases by water and suggest a new general mechanism by which DNA-binding proteins in cells may be able to protect chromosomes from endogenous and exogenous reactive chemicals by excluding them from the vicinity of DNA.     

Synthesis of acid-soluble spore proteins by Bacillus subtilis.

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.     

Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro.

alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.     

Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective alpha/beta-type small acid-soluble proteins

Dipicolinic acid (DPA) comprises approximately 10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective alpha/beta-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-gamma. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most alpha/beta-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to alpha/beta-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.     

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.     

Binding to DNA protects alpha/beta-type, small, acid-soluble spore proteins of Bacillus and Clostridium species against digestion by their specific protease as well as by other proteases.

Binding of alpha/beta-type, small, acid-soluble proteins from Bacillus subtilis and Clostridium bifermentans to DNA protected these proteins against cleavage by their specific protease (GPR) as well as by trypsin and chymotrypsin. These data suggest that alpha/beta-type, small, acid-soluble protein binding to DNA (i) may result in a structural change in these proteins, giving a more compact protein structure, and (ii) may be important in slowing the degradation of these proteins by GPR, in particular during sporulation.     

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.     

Heat inactivation of Bacillus subtilis spores lacking small, acid-soluble spore proteins is accompanied by generation of abasic sites in spore DNA.

Previous work has shown that lethal heat treatment of Bacillus subtilis spores lacking the major DNA-binding proteins SASP-alpha and -beta (alpha-beta- spores) causes significant DNA damage, including many single-strand breaks. In this work we have used a reagent specific for aldehydes present in abasic sites in DNA to show that DNA from wild-type spores killed by heat treatment to levels of < 0.05% survival had at most two aldehydes (i.e., abasic sites) per 10(4) nucleotides, while DNA from alpha(-)beta- spores killed to similar levels had 7 to 20 times as many abasic sites per 10(4) nucleotides. These data were generally consistent with the level of single-strand breaks in DNA from these heated spores and strongly suggest that a major mechanism responsible for the heat killing of alpha(-)beta- (but not wild-type) spores is DNA depurination followed by strand breakage at the resultant abasic site. In contrast, hydrogen peroxide killing of alpha(-)beta - spores was not accompanied by generation of a high level of DNA aldehydes.     

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small,acid-soluble spore proteins

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective alpha/beta-type small, acid-soluble spore proteins (SASP) (termed alpha(-)beta(-) spores). These four treatments kill alpha(-)beta(-) spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal(r)) mutants generated by these treatments has now shown that the nal(r) mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal(r) mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in alpha(-)beta(-) spores are similar to those in spontaneous nal(r) mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of alpha(-)beta(-) spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and alpha(-)beta(-) spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.     

Effect of a small, acid-soluble spore protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores

Alpha/beta-type small, acid-soluble spore proteins (SASP) are essential for the resistance of DNA in spores of Bacillus species to damage. An alpha/beta-type SASP, Ssp2, from Clostridium perfringens was expressed at significant levels in B. subtilis spores lacking one or both major alpha/beta-type SASP (alpha- and alpha- beta- strains, respectively). Ssp2 restored some of the resistance of alpha- beta- spores to UV and nitrous acid and of alpha- spores to dry heat. Ssp2 also restored much of the resistance of alpha- spores to nitrous acid and restored full resistance of alpha- spores to UV and moist heat. These results further indicate the interchangeability of alpha/beta-type SASP in DNA protection in spores.     

Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light.

Bacillus subtilis strains containing deletions in the genes coding for one or two of the major small, acid-soluble spore proteins (SASP; termed SASP-alpha and SASP-beta) were constructed. These mutants sporulated normally, but the spores lacked either SASP-alpha, SASP-beta, or both proteins. The level of minor SASP did not increase in these mutants, but the level of SASP-alpha increased about twofold in the SASP-beta- mutant, and the level of SASP-beta increased about twofold in the SASP-alpha- mutant. The growth rates of the deletion strains were identical to that of the wild-type strain in rich or poor growth media, as was the initiation of spore germination. However, outgrowth of spores of the SASP-alpha(-)-beta- strain was significantly slower than that of wild-type spores in all media tested. The heat resistance of SASP-beta- spores was identical to that of wild-type spores but slightly greater than that of SASP-alpha- and SASP-alpha(-)-beta- spores. However, the SASP-alpha- and SASP-alpha(-)-beta- spores were much more heat resistant than vegetative cells. The UV light resistances of SASP-beta- and wild-type spores were also identical. However, SASP-alpha(-)-beta- spores were slightly more sensitive to UV light than were log-phase cells of the wild-type or SASP-alpha(-)-beta- strain (the latter have identical UV light resistances); SASP-alpha- spores were slightly more UV light resistant than SASP-alpha(-)-beta- spores. These data strongly implicate SASP, in particular SASP-alpha, in the UV light resistance of B. subtilis spores.     

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.     

Binding of DNA in vitro by a small, acid-soluble spore protein from Bacillus subtilis and the effect of this binding on DNA topology.

The DNA within spores of Bacillus subtilis is complexed with a large amount of alpha/beta-type small, acid-soluble spore protein (SASP). Measurement of the interaction of a purified alpha/beta-type SASP with DNA in vitro by a filter binding assay showed that the binding saturated at one molecule of SASP per approximately 5 bp. SASP-DNA binding did not require a divalent cation, was optimal at pH 6.7, and was unaffected by salt up to 400 mM. Binding of SASP to relaxed plasmid DNA in the presence of topoisomerase I resulted in the introduction of 18 (for plasmid pUC19) or 36 (for plasmid pUB110) negative supertwists, a superhelical density similar to that found in several plasmids isolated from spores. The SASP-dependent introduction of negative supertwists did not require a divalent cation, was unaffected by salt, and also gave a value of one molecule of SASP per approximately 5 bp at saturation. There was at least one slow step in the binding of SASP to DNA as seen in both the filter binding and supercoiling assays.     

Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B. subtilis mutants

The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins. Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B. megaterium C protein gene. The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Properties of Bacillus subtilis small, acid-soluble spore proteins with changes in the sequence recognized by their specific protease.

Alpha/beta-type small, acid-soluble proteins (SASP) of dormant spores of Bacillus subtilis bind to DNA and increase its resistance to a variety of damaging agents both in vivo and in vitro. When spores germinate, degradation of alpha/beta-type SASP is rapidly initiated by a sequence-specific protease, which is termed GPR. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the wild-type alpha/beta-type SASP SspCwt; all three mutations change residues in the highly conserved sequence recognized by GPR. In one mutant protein (SspCV), residue 33 (Ser) was changed to Val; in the second (SspCDL), residues 30 and 31 (Glu and Ile) were changed to Asp and Leu, respectively; and in the third mutant protein (SspCDLV), residues 30, 31, and 33 were changed to Asp, Leu, and Val. All three mutant proteins were rapidly degraded by GPR during spore germination, and SspCDL and SspCDLV were degraded by GPR in vitro at rates 8 to 9% of that for SspCwt, although not exclusively at the single site cleaved by GPR in SspCwt. These results indicate (i) that the sequence specificity of GPR is broader than originally imagined and (ii) that GPR can cleave the sequence in SspCDLV. Since the latter sequence is identical to that cleaved during the proteolytic activation of GPR, this result further supports an autoprocessing model for GPR activation during sporulation. The properties of these mutant proteins were also examined, both in vivo in B. subtilis spores and in Escherichia coli and in vitro with purified protein. SspC(v) interacted with DNA similarly to SspC(wt) in vivo, resorting UV and heat resistance to spores lacking major alpha/beta-type SASP to the same extent as SspC(wt). In contrasst, SspC(DL) had much less effect on DNA properties in vivo and bound strongly only to poly(dG) . poly(dC) in vitro; SspC(DLV) exhibited only weak binding to poly(dG).poly(dC) in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the binding of these proteins to spore DNA and alteration of DNA properties and show further that the GRP recognition region in alpha/beta-type SASP plays some role in DNA binding.