Stability of water-soluble and lipid-soluble paramagnetic probes in Bacillus subtilis

Batch cultures of the Gram-positive bacterium Bacillus subtilis PB19 have been investigated for their metabolic action to electron spin resonance (ESR) probes. Five- and six-membered water-soluble and lipid-soluble nitroxides have been used, which were reduced most probably to the corresponding hydroxylamine derivatives. The reduction was followed by the ESR signal intensity and found to be dependent on chemical structure and stability, lipophilic/hydrophilic character, charge, concentration, and temperature. Water-soluble nitroxides did not show apparent toxicity towards B. subtilis, in contrast with n-DXSA (n=5, 12, 16) which were found to be strongly cytotoxic. The cytotoxicity depended on the position of the doxyl unit along the hydrocarbon chain. The hydrophilic nitroxides were reduced at a much slower rate relative to the lipophilic ones. Membrane diffusion was suggested to be a slower process relative to chemical reduction for water-soluble nitroxides. The lipophilic nitroxides were solubilized into the membrane where they were rapidly reduced with a reduction maximum at 303-310 K, which is the optimal growth temperature of B. subtilis, while an inactivation at higher temperatures was observed. Both toxicity and reduction rates of nitroxides strongly indicated that the reduction was an enzyme-mediated process taking place near the outer surface of the periplasmic membrane.