Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.     

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24, 39-41). Encoded by the dnaN gene, the β clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The β clamp is a head-to-tail, ring-shaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the β clamp is proliferating cell nuclear antigen (PCNA) (15, 28, 40, 41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40, 41). Although the primary structures of the β clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the β clamp is not limited to its well-defined role in genome replication. The Escherichia coli β clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19, 37, 43). This allows the β clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the β clamp binds YabA, a negative regulator of DNA replication initiation (12, 29, 52). It has also been suggested that the B. subtilis β clamp sequesters DnaA from the replication origin during the cell cycle through the binding of DnaA to YabA and the binding of YabA to the β clamp (70). Thus, it is hypothesized that in E. coli and B. subtilis, the β clamp influences the frequency of replication initiation through interactions with Hda and YabA, respectively.The E. coli and B. subtilis β clamp has an important role in translesion DNA synthesis during the replicative bypass of noncoding bases by specialized DNA polymerases belonging to the Y family (20, 33). The roles of the E. coli β clamp in translesion synthesis are well established (5, 8, 30, 31). Binding sites on the E. coli β clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD₂′C) have been identified, and the consequence of disrupting their association with the β clamp has illustrated the critical importance of the β clamp to the activity of both of these polymerases (4, 5, 8, 26, 30, 31, 48, 49).In addition to the involvement of the β clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis β clamp also functions in DNA mismatch repair (MMR) (45, 46, 64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13, 25, 63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a “matchmaker,” coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60). MutS and MutL of E. coli and B. subtilis physically interact with the β clamp (45, 46, 51, 64). Interaction between the B. subtilis β clamp and MutS is important for efficient MMR and organization of MutS-green fluorescent protein (GFP) into foci in response to replication errors, while the function of MutL binding to the β clamp is unknown (64).These studies show that the β clamp is critical for several aspects of DNA metabolism in E. coli and B. subtilis. In E. coli, many dnaN alleles have been examined and used to define the mechanistic roles of the β clamp in vivo (5, 18, 24, 30, 31, 48, 49, 73). A limitation in studying the mechanistic roles of the B. subtilis β clamp is that only two dnaN alleles (β clamp) are available, dnaN5 and dnaN34 (36) (www.bgsc.org/), and both of these alleles do not support growth at temperatures above 49°C, suggesting that they may cause similar defects (36) (www.bgsc.org/). Of these two dnaN alleles, only dnaN5 has been investigated in any detail (36, 53, 64). The mutant β clamp encoded by dnaN5 contains a G73R substitution [dnaN5(G73R)] in a surface-exposed residue located on the outside rim of the β clamp (53, 64). Our previous studies with this allele showed that dnaN5(G73R) confers an increase in mutation frequency at 30°C and 37°C (64). Further characterization of dnaN5(G73R) showed that the increased mutation frequency is caused by a partial defect in MMR (64). Additionally, dnaN5(G73R)-containing cells have a reduced ability to support MutS-GFP focus formation in response to mismatches (64). These results support the hypothesis that G73R in the β clamp causes a defect in DNA replication at 49°C (36) and impaired MMR manifested by a defect in establishing the assembly of MutS-GFP foci in response to replication errors (64).To understand the roles of the B. subtilis β clamp in MMR and DNA replication, we examined the dnaN5 and dnaN34 alleles. We found that the nucleotide sequences of dnaN5 and dnaN34 and the phenotypes they produce were identical, both producing the G73R missense mutation. We analyzed in vivo β clamp^G73R protein levels and found that the β clamp^G73R protein accumulated to wild-type levels at elevated temperatures. To identify amino acid residues that would restore DNA replication at elevated temperatures, we isolated three intragenic suppressors of dnaN5(G73R) that conferred growth of B. subtilis cells at 49°C. Epistasis analysis and determination of the mutation spectrum showed that each dnaN allele isolated in this study caused an MMR-dependent increase in mutation frequency. Additionally, we found that the β clamp binding protein YabA can reduce the efficiency of MMR in vivo when yabA expression is induced. Thus, we have identified residues in the β clamp that are critical for DNA replication and MMR in B. subtilis. We also found that a β clamp binding protein, YabA, can reduce the efficiency of MMR in vivo.