Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.O ne major aspect of space biological research is the investigation of the responses of bacterial, viral, archaeal, and fungal species, as well as biomolecules, to simulated Martian conditions and their evaluation as potential forward contamination risks in the context of planetary protection (2, 4-7, 11, 12, 24, 30, 37-40, 44-46). For this reason, Mars environmental simulation experiments have been conducted to estimate (i) the survival rates of terrestrial microorganisms and (ii) the persistence of organic molecules on Mars (3,4,10,15,25,29,30,37,38,45). Historically, several studies have explored the resistance of bacterial spores to simulated Martian conditions (8,10,12,14, and references therein); these studies have concentrated mainly on the survival of spores of wild-type strains of various spore-forming species (10,32,(38)(39)(40). More recent experiments have attempted to better understand the molecular factors causing spore resistance to environmental extremes, in which mainly spores of the model organism Bacillus subtilis that carry mutations affecting spore protective factors or spore DNA repair systems have been used (9, 18-23; reviewed in references 16, 28, 41, and 42). Spores of B. subtilis possess brownish pigmentation, 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 (Ca-DPA) and ␣/-type small, acid-soluble spore proteins (SASP), all factors which have been previously found to contribute to spore resistance (reviewed in references 16, 28, 41, and 42). However, the possible roles of these factors in spore resistance to the extreme environmental conditions prevailing on the surface of Mars have not been explored, which is the subject of the present work.The B. subtilis strains used in this study are listed in Table 1, and all of the strains used were congenic to their respective wild-type strains. Spores were obtained by cultivation under vigorous aeration in double-strength liquid Schaeffer sporulation medium (36) under identical conditions for each strain, purified, and stored as described previously (31). When appropriate, chloramphenicol (5 g/ml), erythromycin (1 g/ml), spectinomycin (100 g/ml), or tetracycline (10 g/ml) was added to the medium. Spore preparations consisted of single spores with no detectable clumps and were Ͼ99% free of growing cells, germinated spores, and cell debris, as seen in a phase-contrast microscope. Triplicate air-dried spore samples with a thickness of approximately 25 spore layers (for the 5 ϫ 10 8 spore concentration) on presterilized sp...