The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide. Both enzymes' K m values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately. V max values for hydrogen peroxide or cumene hydroperoxide reduction are >10,000 min ؊1 at 25°C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C340S. Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S).Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.We recently have isolated a new group of facultatively anaerobic bacteria from alkaline compost (1). The bacteria have unique phenotypic and chemotaxonomic characteristics (2) as well as bioenergetic properties (3) and were named Amphibacillus xylanus (2). A. xylanus, lacking a respiratory system and hemeproteins, catalase, and peroxidase, grows well and has the same growth rate and cell yield under strictly anaerobic and aerobic conditions (2). This growth characteristic of A. xylanus is due to the presence of anaerobic and aerobic pathways producing similar amounts of ATP (4). Under aerobic conditions, NADH is thought to be responsible for maintenance of the intracellular redox balance (4).A flavoprotein functional as NADH oxidase was purified from aerobically grown A. xylanus (5). The flavoprotein is a homotetramer composed of subunits (M r ϭ 56,000) containing 1 mol of FAD and also catalyzes a thiol-disulfide interchange reaction, NADH:DTNB 1 oxidoreductase (6). The complete reduction of enzyme by dithionite requires 6 electrons/subunit (6). Such behavior indicates the presence of redox centers in addition to the FAD, and these were postulated to be disulfides (6). To assess the catalytic role of disulfide...

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...

Three strains of gram-positive, facultatively anaerobic, sporeforming, rod-shaped bacteria were isolated from composts of manure with grass and rice straw. These organisms grew well in an alkaline medium and digested xylan both in strictly anaerobic cultures when titanium(III) citrate was used as a reducing agent and in aerobic cultures with shaking. The cells contained meso-diaminopimelic acid, and their cellular fatty acids consisted of iso-and anteiso-branched acids and considerable amounts of straight-chain acids. The DNA base composition of these strains ranged from 36 to 38 mol% guanine plus cytosine. Cytochromes, isoprenoid quinones, and catalase activity were not detected. DNA-DNA homology determinations did not show relatedness to strains of representative species of the genera Bacillus, Clostridium, and Sporolactobacillus. Considering the uniqueness of the characteristics, the sequence of the 5s rRNA, and the unique metabolic pathways, we propose Amphibacillus xylanus gen. nov., sp. nov., for these strains. The type strain is strain EpOl (= JCM 7361).

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.