Clostridium acetobutylicum and Clostridium aminovalericum, both obligatory anaerobes, grow normally after growth conditions are changed from anoxic to microoxic, where the cells consume oxygen proficiently. In C. aminovalericum, a gene encoding a previously characterized H 2 O-forming NADH oxidase, designated noxA, was cloned and sequenced. The expression of noxA was strongly upregulated within 10 min after the growth conditions were altered to a microoxic state, indicating that C. aminovalericum NoxA is involved in oxygen metabolism. In C. acetobutylicum, genes suggested to be involved in oxygen metabolism and genes for reactive oxygen species (ROS) scavenging were chosen from the genome database. Although no clear orthologue of C. aminovalericum NoxA was found, Northern blot analysis identified many O 2 -responsive genes (e.g., a gene cluster [CAC2448 to CAC2452] encoding an NADH rubredoxin oxidoreductase-A-type flavoprotein-desulfoferrodoxin homologue-MerR family-like protein-flavodoxin, an operon [CAC1547 to CAC1549] encoding a thioredoxin-thioredoxin reductase-glutathione peroxidase-like protein, an operon [CAC1570 and CAC1571] encoding two glutathione peroxidase-like proteins, and genes encoding thiol peroxidase, bacterioferritin comigratory proteins, and superoxide dismutase) whose expression was quickly and synchronously upregulated within 10 min after flushing with 5% O 2. The corresponding enzyme activities, such as NAD(P)H-dependent peroxide (H 2 O 2 and alkyl hydroperoxides) reductase, were highly induced, indicating that microoxic growth of C. acetobutylicum is associated with the expression of a number of genes for oxygen metabolism and ROS scavenging.Bacteria belonging to the genus Clostridium are classified as obligatory anaerobes (26,62) and are widely used in the field of solvent fermentation, biodegradation, and microbial energy production. Oxygen has a crucial effect on the growth of clostridia, but the mechanisms of growth inhibition, as well as the existence of O 2 metabolic systems, remain unknown. Some hypotheses to explain aerobic growth inhibition in anaerobes were proposed, such as the possibility that oxygen attacks oxygen-sensitive enzymes causing metabolic cessation or that anaerobes lack the ability to decompose active oxygen species, such as catalase, which cause irreversible oxidative damage to DNA and lipid molecules (2,27,49,63,67). O'Brien and Morris proposed that NAD(P)H oxidation systems react with oxygen to cause oxidation of the electron donor, i.e., NAD(P)H, which is required for the central pathway for anaerobic metabolism; this then leads to the eventual inability of clostridia to maintain their internal redox balance (51, 55). However, many questions remain about the mechanisms of aerobic growth inhibition in clostridia (50).Most Clostridium species do not form colonies in the presence of 1% oxygen (2, 62); however, they can accept microoxic conditions when grown in liquid medium (32-35, 39, 51, 55). Based on physiological examination, clostridia possess systems to met...

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 .

Clostridium acetobutylicum, an obligatory anaerobe, is able to grow microoxically with the accumulation of two functionally unknown O 2 -induced proteins identified by two-dimensional electrophoresis. One was determined to be a novel type rubrerythrin-like protein, named rubperoxin (Rpr) in this study, that conserves one rubredoxin-type Fe(SCys) 4 site per polypeptide in the N-terminus. Recombinant rubperoxin expressed in E. coli purified in its oxidized form is a dimer with optical absorption maxima at 492, 377, and 277 nm. Reduced rubperoxin is rapidly and fully oxidized by a half molar ratio of H 2 O 2 per mole protein, and slowly oxidized by t-butyl hydroperoxide and O 2 . Cell-free extracts from microoxically grown cells efficiently reduce rubperoxin when NAD(P)H is used as the electron donor (preferentially reduced by NADH). These results strongly suggest that rubperoxin is involved in NAD(P)H-dependent H 2 O 2 detoxification in vivo.