Sodium acetate enhances hydrogen peroxide production in Weissella cibaria.

AIMS: To investigate hydrogen peroxide production by lactic acid bacteria (LAB) and to determine the key factors involved. METHODS AND RESULTS: Six strains of Weissella cibaria produced large amounts (2.2-3.2 mmol l(-1)) of hydrogen peroxide in GYP broth supplemented with sodium acetate, but very low accumulations in glucose yeast peptone broth without sodium acetate. Increased production of hydrogen peroxide was also recorded when strains of W. cibaria were cultured in the presence of potassium acetate, sodium isocitrate and sodium citrate. Oxidases and peroxidases were not detected, or were present at low levels in W. cibaria. However, strong nicotinamide adenine dinucleotide (NADH) oxidase activity was recorded, suggesting that the enzyme plays a key role in production of hydrogen peroxide by W. cibaria. CONCLUSIONS: Weissella cibaria produces large quantities of hydrogen peroxide in aerated cultures, in a process that is dependent on the presence of acetate in the culture medium. NADH oxidase is likely the key enzyme in this process. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing that sodium acetate, normally present in culture media of LAB, is a key factor for hydrogen peroxide production by W. cibaria. The exact mechanisms involved are not known.

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

AIMS: The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. METHODS AND RESULTS: Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. CONCLUSION: A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1.

AIMS: The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. METHODS AND RESULTS: Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. CONCLUSION: A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.

Hydrogen peroxide-forming NADH oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria.

Amphibacillus xylanus and Sporolactobacillus inulinus NADH oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, AhpC (peroxiredoxin). In order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. AhpC-linked alkyl hydroperoxide reductase activities were detected in most of the tested strains, and especially high activities were shown in six bacterial species that grow well under aerobic conditions, including aerobic bacteria (Alcaligenes faecalis and Bacillus licheniformis) and facultatively anaerobic bacteria (Amphibacillus xylanus, Sporolactobacillus inulinus, Escherichia coli, and Salmonella enterica serovar Typhimurium). In the absence of AhpC, the purified enzymes from A. xylanus and S. inulinus catalyze the NADH-linked reduction of oxygen to hydrogen peroxide. Similar activities were observed in the cell extracts from each of these six strains. The cell extract of B. licheniformis revealed the highest AhpC-linked alkyl hydroperoxide reductase activity in the four strains, with V(max) values for hydrogen peroxide and alkyl hydroperoxides being similar to those for the enzymes from A. xylanus and S. inulinus. Southern blot analysis of the three strains probed with the A. xylanus peroxiredoxin reductase gene revealed single strong bands, which are presumably derived from the individual peroxiredoxin reductase genes. Single bands were also revealed in other strains which show high AhpC-linked reductase activities, suggesting that the NADH oxidases belonging to the peroxiredoxin oxidoreductase family are widely distributed and possibly play an important role both in the peroxide-scavenging systems and in an effective regeneration system for NAD in aerobically growing bacteria.

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus.

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.

Real-time fluorescence analysis on molecular mechanisms for regulation of cytochrome P450scc activity upon steroidogenic stimulation in adrenocortical cells.

Real-time fluorescence analysis revealed that the activity of cytochrome P450scc was related to Ca2+ signals arising from extracellular NADPH, ACTH and ATP stimulation in adrenocortical fasciculata cells. The side-chain cleavage reaction by cytochrome P450scc was measured with 3beta-hydroxy-22,23-bisnor-5-cholenyl ether (cholesterol-resorufin) by observing the distinct increase in fluorescence upon conversion of cholesterol-resorufin to resorufin and pregnenolone. Adrenocorticotropic hormone (ACTH) induced a relatively small stimulation of the P450scc activity. A significant production of resorufin was revealed after stimulation of cell cultures with 100 pM, 1 nM of ACTH for 3 h. On the other hand, extracellular NADPH was found to rapidly and greatly stimulate the resorufin production in intact cells immediately after the addition of 50-500 microM NADPH. The extracellular NADPH stimulation was prevented by the addition of thapsigargin and EGTA which abolished Ca2+ oscillations induced by NADPH. Suramin, a specific antagonist of the P2y type ATP receptor, also completely abolished the NADPH-induced cholesterol-resorufin conversion. These results imply that extracellular NADPH (membrane impermeable) produced Ca2+ oscillations through its binding to ATP receptor thereby stimulating the activity of P450scc. The application of 45-500 microM extracellular ATP to cells did not, however, significantly increase the resorufin production. These three stimulators produced very different types of Ca2+ signals. ACTH induced mainly a series of Ca2+ spikes superimposed on a long-lasting basal Ca2+ elevation. The Ca2+ signals induced by NADPH showed predominantly a series of Ca2+ spikes without elevation of the basal Ca2+ concentration. Only long-lasting Ca2+ elevation was induced by extracellular ATP. The stimulation of cytochrome P450scc may thus be correlated with the different patterns of Ca2+ signals.

The major acidic glycolipids from the kidney of the Pacific salmon (Oncorhynchus keta): characterization of a novel ganglioside, fucosyl-N-acetylgalactosaminyl-GM1.

Two fractions of a major ganglioside from the kidney of the pacific salmon, Oncorhynchus keta, were eluted from a DEAE-Sephadex column in the monosialosyl fraction. The faster moving ganglioside (X1) on TLC was separated from the slower moving one (X2) by HPLC using a silica beads column. By methylation analysis, chemical and enzymatic degradation, reaction with monoclonal antibodies, LSIMS, and (1)H-NMR spectroscopy, X1 was determined to be a monosialosyl ganglioside belonging to the ganglio-series with a unique Fucalpha1-3GalNAc linkage at the nonreducing terminal: Fucalpha1-3GalNAcbeta1-3Galbeta1-3GalNAcbeta1-4[ NeuAcalpha2-3]Galbeta 1-4Glcbeta1-1Cer. Analysis of the lipophilic moiety indicated predominance of 24:1 fatty acid in combination with sphingenine. X2 was found to have a glycon structure identical to X1. The ceramide of X2 consisted predominantly of saturated fatty acids (18:0 and 16:0). The tissue concentrations of X1 and X2 in kidney were 3.7 and 2.8 nmol/g, respectively.

Module-intron correlation and intron sliding in family F/10 xylanase genes.

Xylanases are classified into two families, numbered F/10 and G/11 according to the similarity of amino acid sequences of their catalytic domain (Henrissat, B., Bairoch, A., 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781-788). Three-dimensional structure of the catalytic domain of the family F/10 xylanase was reported (White, A., Withers, S.G., Gilkes, N.R., Rose, D.R., 1994. Crystal structure of the catalytic domain of the beta-1,4-glycanase Cex from Cellulomonas fimi. Biochemistry 33, 12546-12552). The domain was decomposed into 22 modules by centripetal profiles (Go, M., Nosaka, M., 1987. Protein architecture and the origin of introns. Cold Spring Harbor Symp. Quant. Biol. 52, 915-924; Noguti, T., Sakakibara, H., Go, M., 1993. Localization of hydrogen-bonds within modules in barnase. Proteins 16, 357-363). A module is a contiguous polypeptide segment of amino acid residues having a compact conformation within a globular domain. Collected 31 intron sites of the family F/10 xylanase genes from fungus were found to be correlated to module boundaries with considerable statistical force (p values <0.001). The relationship between the intron locations and protein structures provides supporting evidence for the ancient origin of introns, because such a relationship cannot be expected by random insertion of introns into eukaryotic genes, but it rather suggests pre-existence of introns in the ancestral genes of prokaryotes and eukaryotes. A phylogenetic tree of the fungal and bacterial xylanase sequences made two clusters; one includes both the bacterial and fungal genes, but the other consists of only fungal genes. The mixed cluster of bacterial genes without introns and the fungal genes with introns further supports the ancient origin of introns. Comparison of the conserved base sequences of introns indicates that sliding of a splice site occurred in Aspergillus kawachii gene by one base from the ancestral position. Substrate-binding sites of xylanase are localized on eight modules, and introns are found at both termini of six out of these functional modules. This result suggests that introns might play a functional role in shuffling the exons encoding the substrate-binding modules.

Stimulation of peroxidase activity by decamerization related to ionic strength: AhpC protein from Amphibacillus xylanus.

AhpC protein, purified from Amphibacillus xylanus with a molecular mass of 20.8 kDa, protects cells against oxidation damage. The enzyme catalyses the reduction of hydroperoxides in cooperation with the 55 kDa flavoprotein, A. xylanus NADH oxidase (NADH oxidase-AhpC system). A. xylanus AhpC has two disulfide linkages between monomers and can act in the homodimer form. Gel-filtration column chromatography and dynamic light scattering (DLS) suggest that A. xylanus AhpC also forms a large oligomeric assembly (10-12 mers). A. xylanus AhpC was crystallized and X-ray diffraction data were collected to 3.0 A. The self-rotation function revealed fivefold and twofold axes located perpendicularly to each other, suggesting that the molecular assembly of A. xylanus AhpC is composed of ten monomers. The oligomerization of A. xylanus AhpC is affected by ionic strength in the DLS measurements. The H(2)O(2) reductase activity of the A. xylanus NADH oxidase-AhpC system is also affected by ionic strength, and it was found that the decamerization of AhpC might be required for the activation of the NADH oxidase-AhpC system.

Peroxide reductase activity of NADH dehydrogenase of an alkaliphilic Bacillus in the presence of a 22-kDa protein component from Amphibacillus xylanus.

The NADH oxidase of Amphibacillus xylanus shows high NADH-peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of a 22-kDa disulfide-containing protein component (Y. Niimura, L. B. Poole, and V. Massey, J. Biol.Chem. 270, 25645-25650, 1995). It was found that the membrane-bound NADH dehydrogenase of an alkaliphilic Bacillus (YN-1) involved in the respiratory chain also exhibits reductase activity for hydrogen peroxide and cumene hydroperoxide in the presence of the 22-kDa component from Amphibacillus xylanus. Vmax values for these substrates were as high as those of the NADH oxidase of A. xylanus. Although the 38-kDa protein produced by trypsin treatment of NADH dehydrogenase retains NADH dehydrogenase activity, it exhibited no peroxide reductase activity in the presence of the 22-kDa component from A. xylanus. The NADH dehydrogenase of YN-1 might not only catalyze electron flow from NADH to the respiratory chain, but also function for scavenging peroxide.

Time resolved study of effect of chlorpromazine on mobility of cytochrome P-450 and phospholipids in the inner membrane of adrenocortical mitochondria.

The effects of chlorpromazine on the mobility of cytochrome P-450 and the fluidity of lipid membranes have been investigated in bovine adrenocortical submitochondrial particles (SMP). Rotational diffusion of the cytochrome was measured by observing the decay of absorption anisotropy, ra(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of ra(t) was based on a 'rotation-about-membrane-normal' model. The anisotropy decayed within 2 ms to a time independent value r3. The presence of chlorpromazine decreased the mobile population of cytochrome P-450 from 28 to 23%. The rotational relaxation time phi a of the mobile population (approximately 1100 microseconds) was, however, not significantly changed by chlorpromazine. The lipid fluidity was examined by observing time-resolved fluorescence anisotropy, rf(t), of 1,6-diphenyl 1,3,5-hexatriene (DPH). The anisotropy rf(t) decayed within 70 ns to a time independent value r infinity. The motion of DPH was analyzed based on a 'wobbling-in-cone' model. The presence of chlorpromazine decreased the cone angle from 42 degrees to 39 degrees, while the rotational relaxation time phi f (approximately 2 ns) was not significantly changed by the presence of chlorpromazine. These results demonstrate that chlorpromazine decreased the mobility of not only lipids but also membrane proteins.

Reaction mechanism of Amphibacillus xylanus NADH oxidase/alkyl hydroperoxide reductase flavoprotein.

NADH oxidase from Amphibacillus xylanus is a potent alkyl hydroperoxide reductase in the presence of the small disulfide-containing protein (AhpC) of Salmonella typhimurium. In the presence of saturating AhpC, kcat values for reduction of hydroperoxides are approximately 180 s-1, and the double mutant flavoprotein enzyme C337S/C340S cannot support hydroperoxide reduction (Niimura, Y., Poole, L. B., and Massey, V. (1995) J. Biol. Chem. 270, 25645-25650). Kinetics of reduction of wild-type and mutant enzymes are reported here with wild-type enzyme; reduction by NADH was triphasic, with consumption of 2.6 equivalents of NADH, consistent with the known composition of one FAD and two disulfides per subunit. Rate constants for the first two phases (each approximately 200 s-1) where FAD and one disulfide are reduced are slightly greater than kcat values for AhpC-linked hydroperoxide reduction. The rate constant for the third phase (reduction to the 6-electron level) is too small for catalysis. Only the first phase of the wild-type enzyme occurs with the mutant enzyme. These results and the stoichiometry of NADH consumption indicate Cys337 and Cys340 as the active site disulfide of the flavoprotein and that electrons from FADH2 must pass through this disulfide to reduce the disulfide of AhpC.

Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component.

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' Km values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately. Vmax values for hydrogen peroxide or cumene hydroperoxide reduction are > 10,000 min(-1) at 25 degrees 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/C40SC337S/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.

Role of cysteine 337 and cysteine 340 in flavoprotein that functions as NADH oxidase from Amphibacillus xylanus studied by site-directed mutagenesis.

A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The Km for NADH and oxygen and the kcat value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.

Purification and analysis of a flavoprotein functional as NADH oxidase from Amphibacillus xylanus overexpressed in Escherichia coli.

The gene encoding the Amphibacillus xylanus flavoprotein has been cloned into pTTQ18 and overexpressed in Escherichia coli. The recombinant enzyme has been purified to homogeneity yielding 15 mg of pure enzyme/liter of cell culture. Recombinant flavoprotein is fully active and has an absorption spectrum identical to that of the enzyme purified from A. xylanus. The N-terminal sequence analysis and analytical gel filtration data confirm the structural identity of recombinant and A. xylanus enzymes. The Km value for oxygen and the Km value for NADH are 1.7 mM and 33.3 microM, respectively. In the presence of free additional FAD, however, the Km value for oxygen decrease dramatically. The NADH oxidase activity is accelerated markedly in the presence of additional FAD. The intracellular free FAD concentration of A. xylanus is calculated about 13 microM. This FAD concentration would be enough to accelerate the NADH oxidase activity of flavoprotein in cells of A. xylanus. Two-electron reduction of the enzyme FAD by the strong reductant dithionite occurs during the total uptake of 6 electrons. Such behavior usually indicates the presence of non-flavin redox centers. The high degree of homology between this enzyme and alkyl hydroperoxide reductase F52a protein and thioredoxin reductase suggests that these centers are the redox-active disulfide adjacent to the FAD and another disulfide, which is able to slowly interchange with the redox-active disulfide. The presence of two disulfides has been demonstrated.

Complete genomic sequence and patterns of transcription of a member of an unusual family of closely related, chromosomally dispersed Ig gene clusters in Raja.

Ig genes in cartilaginous fish are organized in clusters. This unique form of organization suggests major differences in the regulation of the segmental rearrangement mechanism from that found in mammals and other higher vertebrates. The complete DNA sequence of an IgX-type cluster in the species Raja eglanteria is defined, and shown to consist of four rearranging segmental elements and three constant region exons. Using fluorescence in situ hybridization it is shown that Raja clusters are present at multiple sites within the genome, and that there is no apparent relationship between the chromosomally dispersed IgX clusters and a second isotype (IgM type) in this species. Comprehensive examination of sequence motifs associated with transcription regulation reveals an abundance of short sequences closely resembling those found in higher vertebrate Ig and other genes. However, the linear relationship of these motifs differs markedly from that associated with regulation of expression of the mammalian Ig gene locus. Additional studies of the transcription products of the IgX gene loci emphasize the diversity of transcription and processing of these genes. Considerable variation was noted in the processing of putative IgX transcripts, including the detection of a heretofore unrecognized form containing at least four additional Ig-like domains. These results have profound significance in terms of understanding the selective expression and the evolutionary diversification of Ig genes.

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene.

Amphibacillus xylanus Ep01, 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 Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 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.

Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli.

The alpha-amylase genes of Streptococcus bovis 148 were cloned in Escherichia coli MC1061, using pBR322. The recombinant plasmids were classified into two groups on the basis of their restriction maps. Southern blot analysis did not show homology between the two types of alpha-amylase genes, and the two alpha-amylase genes existed on the chromosomal DNA of S. bovis 148. The enzymatic properties and N-terminal amino acid sequences of the two purified enzymes produced by the cloned E. coli strains were quite different from each other. Particularly, one alpha-amylase (Amy I) was adsorbed on raw corn starch and hydrolyzed raw corn starch, and another (Amy II) was not adsorbed on raw corn starch and did not hydrolyze raw corn starch. Amy I was considered to be the same as the extracellular alpha-amylase of S. bovis 148 in raw starch absorbability, ability to hydrolyze raw corn starch, enzymatic characteristics, N-terminal amino acid sequence, and mode of action on soluble starch. Amy II showed a unique pattern of oligosaccharide production from soluble starch compared with the extracellular alpha-amylase of S. bovis 148. Amy II was suggested to be an intracellular alpha-amylase of S. bovis 148.

Accumulation of sulfoglycolipids in hyperosmosis-resistant clones derived from the renal epithelial cell line MDCK (Madin-Darby canine kidney cell).

1. Two clones (osmR-A and osmR-B) resistant to hyperosmotic media of 700 and 800 mosmol/l, respectively, were selected from Madin-Darby canine kidney (MDCK) cells. 2. When cultured in isosmotic medium (300 mosmol/l), the concentration of galactosyl sulfatide and lactosyl sulfatide in these hyperosmosis-resistant clones was 3.4-5.9 times higher than in the wild-type MDCK. The rate of incorporation of [35S]sulfate into sulfolipids of osmR-A and osmR-B was 1.9-6.7 times higher than MDCK. 3. The stimulation of incorporation into sulfolipids by hyperosmotic culture was completely inhibited by cycloheximide. The pulse-chase studies indicated decreased turnover rate of sulfolipids in osmR-A.