Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2). When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparged with 3% O(2)/97% N(2) mixed carrier-gas) growth conditions in the mid exponential phase (OD(660)=1.0). When the strain grew under 3% O(2)/97% N(2), the medium remains anoxic. Thirty minutes after beginning aeration with 3% O(2), the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H(2)O, and the estimated K(m) for oxygen was 61.9 microM. These data demonstrate that an O(2)-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.

Clostridium acetobutylicum DSM792 ( ¼ ATCC824), a solvent producing obligate anaerobe, grew well after a shift in growth conditions from anoxic to microoxic at the mid exponential phase. In two-dimensional gel electrophoresis, a spot migrating at 45 kDa and three spots at 23 kDa accumulated after 30 min of flushing with 5% O 2 /95% N 2 . Based on peptide mass fingerprints, the 45 kDa polypeptide was determined to be NP_347663 (A-type flavoprotein homologue) and the 23 kDa polypeptides were determined to be NP_350180 or NP_350181 (novel type rubrerythrin homologue). Northern blot analysis indicated that the expressions of these peptide transcripts were upregulated within 10 min after flushing with 5% O 2 /95% N 2 .