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.

Amphibacillus xylanus EpOl, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus EpOl.The A. xylanus enzyme is a homotetramer composed of a subunit (Mr 56,000) containing 1 mol of fiavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with ,-NADH as the preferred electron donor and exhibits no activity with NADPH. The Hlavoprotein gene of A. xylanus EpOl was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively.Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.We recently isolated a new group of facultatively anaerobic bacteria from an alkaline compost which have unique phenotypic and chemotaxonomic characteristics (17), as well as bioenergetic properties (10). The isolates were then recognized to belong to an new taxon and named Amphibacillus xylanus (16).In aerobic and facultatively anaerobic bacterial cells having a respiratory pathway, most of the NADH produced through glycolysis and the tricarboxylic acid cycle is effectively utilized to generate biological energy by the respiratory chain. A. xylanus, lacking a respiratory system, grows well and has the same growth rate and cell yield under strictly anaerobic conditions and aerobic conditions with shaking (16). This growth characteristic of A. xylanus appears to be due to the presence of pathways that function under both anaerobic and aerobic conditions and produce similar amounts of ATP; NADH produced anaerobically through glycolysis is reoxidized by NAD-linked aldehyde dehydrogenase and NAD-linked alcohol dehydrogenase (15). Under aerobic conditions, the high NADH oxidase activity which is detected in a cell extract should be responsible for maintenance of the intracellular redox balance and thus assuring fast aerobic growth by consuming NADH (15). * Corresponding author.NADH oxidase is found in several microorganisms (7,12,18,23) and has been purified from ...

We investigated the effects of O 2 on Bifidobacterium species using liquid shaking cultures under various O 2 concentrations. Although most of the Bifidobacterium species we selected showed O 2 sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O 2 conditions accompanied by the accumulation of H 2 O 2 in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O 2 conditions without H 2 O 2 accumulation, and growth was stimulated compared to anoxic growth. H 2 O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).Although anaerobes are defined as being unable to grow in the presence of O 2 , their degree of O 2 sensitivity exhibits wide variation (2,3,10,11,14,15,16,17,18,20,21,25,28). The genus Bifidobacterium is a well-investigated anaerobe known to be beneficial to human health. Its sensitivity to O 2 causes a loss of viability during manufacture and storage as well as after incorporation into the human body (36). The O 2 sensitivity differs among strains and species (13). de Vries and Stouthamer (7) classified Bifidobacterium species into three categories according to their sensitivities to O 2 . They proposed that some O 2 -sensitive species produce H 2 O 2 through NADH oxidase activity. Since then, there have been several approaches taken to investigate bifidobacterial oxidative growth inhibition (1,6,9,32,33,37); however, the mechanism of growth inhibition under oxic conditions remains unclear.In this study, microaerophilic Bifidobacterium species were found. The main objectives of the present study were to (i) determine growth characteristics with respect to O 2 using several Bifidobacterium species, (ii) determine the factor responsible for aerobic growth inhibition using O 2 -sensitive species, (iii) investigate the metabolic properties of O 2 -sensitive and microaerophilic Bifidobacterium species under oxic growth conditions, and (iv) investigate the properties of O 2 reduction systems that should differ between O 2 -sensitive and microaerophilic Bifidobacterium species.Effect of O 2 on the growth of Bifidobacterium species. Bifidobacterium species are classified as typical anaerobic bacteria; however, their differing degrees of O 2 sensitivity in liquid shaking culture are not well characterized. We selected several species by referring to reports concerning the physiological effects of O 2 . B. bifidum, B. longum, B. breve, and B. infantis were selected from among strains often used in milk products and intestinal probiotics. B. asteroides is reported to possess catalase (13, 30). B. indicum has characteristics similar to those of B. asteroides but shows catalase activity only when hemin is added to the medium (13, 30). B. boum, B. globosum, and B. thermophilum are reported to form colonies under atmospheric conditions of 90% air...

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