Wayne Nicholson

Bacterial endospores and their significance in stress resistance

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment.

Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168

Endospores of Bacillus spp. were purified from three Sonoran desert soil samples by Chelex extraction and NaBr density gradient centrifugation and their UV resistances compared with that of B. subtilis strain 168. Natural spore populations exhibited tight adherence to soil particles which was not readily overcome by the extraction and purification procedure. It was observed that spores purified from soil exhibited 2-3 fold higher resistance to UV (as measured by the 90% lethal dose, LD90) than did B. subtilis strain 168 grown on NSM, a standard laboratory sporulation medium, and purified by the same extraction procedure. Cultivation of spore-forming bacteria isolated from soil on NSM resulted in production of spores with essentially identical UV resistance as strain 168, suggesting that spore UV resistance is influenced by the environment in which spores are produced.

Using Thermal Inactivation Kinetics to Calculate the Probability of Extreme Spore Longevity: Implications for Paleomicrobiology and Lithopanspermia

Thermal inactivation kinetics with extrapolation were used to model the survival probabilities of spores of various Bacillus species over time periods of millions of years at the historical ambient temperatures (25–40 °) encountered within the 250 million-year-old Salado formation, from which the putative ancient spore-forming bacteriumSalibacillus marismortui strain 2-9-3 was recovered. The model indicated extremely low-to-moderate survivalprobabilities for spores of mesophiles, but surprisingly high survival probabilities for thermophilic spores. The significance of the results are discussed in terms of the survival probabilities of (i) terrestrial spores in ancient geologic samples and (ii) spores transported betweenplanets within impact ejecta.

Spore Photoproduct Lyase Operon (splAB) Regulation During Bacillus subtilis Sporulation: Modulation of splB-lacZ Fusion Expression by P1 Promoter Mutations and by an In-Frame Deletion of splA

Abstract. EσG-dependent transcription of the splAB operon in the forespore at stage III of Bacillus subtilis sporulation initiates from two promoters, P1 preceding splA (major) and P3 preceding splB (minor). To explore the possible role of splA in controlling splB-encoded spore photoproduct lyase expression, we measured β-galactosidase from splB-lacZ fusions integrated at the SPβ prophage locus which contained point mutations or deletions which either inactivated or physically removed P1 and/or splA. Paradoxically, inactivation of P1 by point mutation or its removal by deletion from upstream resulted in elevated β-galactosidase expression of the resulting splB-lacZ fusion, as did an in-frame deletion of splA which left P1 and P3 intact; however, expression of all fusions remained sporulation specific and EσG dependent.