Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation

Abstract

Aag from Bacillus subtilis has been implicated in in vitro removal of hypoxanthine and alkylated bases from DNA. The regulation of expression of aag in B. subtilis and the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptional aag-lacZ fusion expressed low levels of ␤-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably, aag-lacZ expression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in a sigG-deficient background, suggesting a forespore-specific mechanism of aag transcription. Two additional findings supported this suggestion: (i) expression of an aag-yfp fusion was observed in the forespore, and (ii) in vivo mapping of the aag transcription start site revealed the existence of upstream regulatory sequences possessing homology to G -dependent promoters. In comparison with the wild-type strain, disruption of aag significantly reduced survival of sporulating B. subtilis cells following nitrous acid or methyl methanesulfonate treatments, and the Rif r mutation frequency was significantly increased in an aag strain. These results suggest that Aag protects the genome of developing B. subtilis sporangia from the cytotoxic and genotoxic effects of base deamination and alkylation. IMPORTANCEIn this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.T he integrity of genomes of organisms is constantly compromised by intracellular and extracellular factors that have the potential to generate different base modifications, including, oxidations, alkylations, and deaminations (1). These types of nonbulky genetic insults are detected primarily by specific DNA glycosylases and eliminated through the base excision repair (BER) pathway (2). DNA deamination is a major type of spontaneous genetic damage with which cells must contend (3), and the spontaneous loss of the exocyclic amino groups in cytosine, guanine, and adenine yields the bases uracil, xanthine, and hypoxanthine (HX), respectively (4, 5). HX in DNA is potentially mutagenic, since it can pair not only with thymine but also with cytosine and therefore would result in ATto-GC transitions after DNA replication (6). Organisms such as Escherichia coli and Saccharomyces cerevisiae employ the 3-methyladenine DNA glycosylases AlkA and MAG, respectively, to process HX and the modified bases 3-methyladenine, 7-methylguanine, and 7-methyladenine (7,8). Other enzymes of mammalian origin, which are structurally unrelated to E. coli AlkA, include alkyl-adenine-DNA glycosylas...

Transcriptional coupling (Mfd) and DNA damage scanning (DisA) coordinate excision repair events for efficient Bacillus subtilis spore outgrowth

“…Of note, mfd and uvrA are initially expressed in both sporangial compartments, but only in the forespore in the last stages of sporulation (Ramírez‐Guadiana et al., ). A similar pattern of transcription was reported for repair proteins that are packaged in spores and employed to eliminate DNA lesions during spores’ return to vegetative growth (Ayala‐García et al., ; Pedraza‐Reyes, Gutiérrez‐Corona, & Nicholson, ; Pedraza‐Reyes et al., ). Our results showed that, whereas the disruption of mfd alone did not affect spore outgrowth or chromosomal replication, such processes were significantly altered after disrupting mfd in DisA‐deficient spores.…”

“…Aliquots (10 μl) of this cell suspension were supplemented with 2 μg ml −1 of 4′, 6′‐diamidino‐2‐phenylindole (DAPI) to stain the chromosome and with FM4‐64 (5 μg ml −1 ) to stain cell membranes and stained samples kept at room temperature for 15 min. For microscopy, cell samples were prepared as previously described (Ayala‐García, Valenzuela‐García, Setlow, & Pedraza‐Reyes, ). Fluorescence microscopy was performed with a ZEISS Axioscope A1 microscope equipped with an AxioCam ICc1 camera.…”

YwqL (EndoV), ExoA and PolA act in a novel alternative excision pathway to repair deaminated DNA bases in Bacillus subtilis

“…A recent report revealed that in addition to Ung and Bs EndoV, B . subtilis employs Aag, an alkyl-adenine DNA-glycosylase, to help counteract the noxious effects of base deamination [20].…”

Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine

“…In bacteria, hypoxanthine has been mainly studied in relation to DNA damage and mutagenesis due to spontaneous deamination of adenine, which yields hypoxanthine and leads to AT-to-GC transitions after DNA replication (45). In B. subtilis, hypoxanthine has been studied in relation to both purine metabolism (38) and DNA damage and repair (46). To the best of our knowledge, this is the first time that hypoxanthine has been reported as a mediator of interactions in B. subtilis biofilms.…”