Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance

Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).