DNA repair and photochemistry in Bacillus subtilis spores

Abstract

Bacterial endospores are 10 to 20 times more resistant to ultraviolet radiation than their vegetative counterparts, due to two interlocking mechanisms: the DNA photochemistry in spores, and the presence of two DNA repair systems. Spore DNA is closely associated with small acid soluble spore proteins (SASP) which change the conformation of DNA from the B form to an A-like form. When spores are subjected to UV radiation, SASP-bound DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally referred to as spore photoproduct (SP). Spores possess two DNA repair pathways that repair SP, the general nucleotide excision repair (NER) pathway encoded by the uvr genes and the SP-specific SP lyase repair system encoded by the splB gene. Most of the information regarding spore UV resistance has traditionally been obtained using commercial UV lamps that emit predominantly 254-nm UV (UV-C). In contrast, solar UV radiation that reaches the Earth's surface spans 290 to 400-nm wavelengths, the so-called UV-B and UV-A portions of the UV spectrum, whereas the UV-C portion of solar UV is mainly filtered by the stratospheric ozone layer. Ten percent of bacterial spore dry mass consists of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA). DPA has been implicated in triggering germination in germination-deficient mutant B. subtilis spores. DPA has also been shown to photosensitize spore DNA to UV radiation. In this dissertation the SP lyase repair system, spore DNA damage cause by environmental UV, and the role of DPA in the survival of spores to UV radiation were investigated. SplB protein containing an N-terminal 10-Histidine tag [(10His) SplB] was over-expressed and purified from Escherichia coli. The purified (10 His) SplB was used for characterizing the binding of the enzyme to its substrate, SP. A 35-bp oligonucleotide (oligo) was constructed with a single pair of adjacent thymidines on one strand. The oligo was irradiated with 254-nm UV under conditions to produce either SP or cyclobutane pyrimidine dimers (Py<>Py). By DNase I protection, (10His) SplB was shown to bind specifically to the SP-containing oligo and not to the oligo containing Py<>Py or the unirradiated oligo. (10His) SplB bound to the oligo containing SP exhibited a 9-bp DNase I footprint with two hypersensitive sites within the footprint. Bacillus subtilis spores were exposed to UV-C, UV-B, solar UV-A and full spectrum sunlight. Chromosomal DNA was then extracted and probed for the presence of damage using a combination of enzymatic and electrophoretic treatments. Spores were shown to accumulate Py<>Py, single stranded breaks and double stranded breaks in addition to SP. No apurinic/apyrimidinic sites were detected under any irradiation conditions used. Mutant spores that make DPA (DPA⁻) or that were DPA-deficient (DPA⁻) were exposed to UV-C, UV-B, solar UV-A, and full spectrum sunlight as a dried-film or in suspension. When irradiated as a dried-film DPA⁻ spores were the most sensitive followed by the DPA⁻ spores and wild-type spores under all irradiation conditions except for solar UV-A where the DPA⁻ spores were the most resistant. On the other hand, DPA⁻ spores irradiated with UV-C in suspension were 8 times more resistant in comparison to the same spore irradiated as a dried-film.