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Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective ␣/␤-type small, acid-soluble, spore proteins (termed ␣ ؊ ␤ ؊ spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or ␣ ؊ ␤ ؊ spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.

Previous work has shown that the katX gene encodes the major catalase in dormant spores of Bacillus subtilis but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a katX-lacZ fusion began at approximately h 2 of sporulation, and >75% of thekatX-driven β-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor ςF abolishedkatX-lacZ expression, while mutations in genes encoding ςE, ςG, and ςK did not. Induction of ςF synthesis in vegetative cells also resulted in katX-lacZ expression, while induction of ςG expression did not; the katX-lacZ fusion was also not induced by hydrogen peroxide. Upstream of the in vivokatX transcription start site there are sequences with good homology to those upstream of known ςF-dependent start sites. These data indicate that katX is an additional member of the forespore-specific ςF regulon. A mutant in the katA gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while akatX mutant was not. However, outgrowth of katXspores, but not katA spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.

Although the implications of calcium carbonate (CaCO(3)) precipitation by microorganisms in natural environments are quite relevant, the physiology and genetics of this phenomenon are poorly understood. We have chosen Bacillus subtilis 168 as our model to study which physiological aspects are associated with CaCO(3) (calcite) formation during biofilm development when grown on precipitation medium. A B. subtilis eftA mutant named FBC5 impaired in calcite precipitation was used for comparative studies. Our results demonstrate that inactivation of etfA causes a decrease in the pH of the precipitation medium during biofilm development. Further analysis demonstrated that eftA extrudes an excess of 0.7 mol H(+) L(-1) with respect to B. subtilis 168 strain. Using media buffered at different pH values, we were able to control calcite formation. Because etfA encodes the alpha-subunit of a putative flavoprotein involved in fatty acid metabolism, we compared the intracellular levels of NADH among strains. Our physiological assay showed that FBC5 accumulated up to 32 times more NADH than the wild-type strain. We propose that the accumulation of NADH causes a deregulation in the generation of the proton motive force (DeltamicroH(+)) in FBC5 producing the acidification.