Biochim Biophys Acta, 1995 Jun 1, 1230(1-2), 74 - 6 Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c; Witt H et al.; Site-directed mutagenesis in subunit II of the cytochrome c oxidase (haem aa3) from Paracoccus denitrificans reveals that two carboxylic residues, Glu-246 and Asp-206 (corresponding to 198 and 158 in the bovine subunit II), are involved in the binding of cytochrome c . Spectrophotometric and polarographic measurements with the isolated enzymes of both mutant strains show a strongly reduced activity compared to wild-type oxidase, with the overall catalytic capacity (kcat/KM) of both mutants decreased about 8-fold . EPR spectra reveal no significant differences between the wild-type and the mutant enzymes, indicating that neither residue contributes significantly to the structure of the CuA centre . We conclude that Glu-246 and Asp-206 constitute an essential part of the binding site for cytochrome c. Arch Microbiol, 1995 Jun, 163(6), 418 - 23 Purification and properties of benzyl alcohol dehydrogenase from a denitrifying Thauera sp; Biegert T et al.; Toluene and related aromatic compounds are anaerobically degraded by the denitrifying bacterium Thauera sp . strain K172 via oxidation to benzoyl-CoA . The postulated initial step is methylhydroxylation of toluene to benzyl alcohol, which is either a free or enzyme-bound intermediate . Cells grown with toluene or benzyl alcohol contained benzyl alcohol dehydrogenase, which is possibly the second enzyme in the proposed pathway . The enzyme was purified from benzyl-alcohol-grown cells and characterized . It has many properties in common with benzyl alcohol dehydrogenase from Acinetobacter and Pseudomonas species . The enzyme was active as a homotetramer of 160 kDa, with subunits of 40 kDa . It was NAD(+)-specific, had an alkaline pH optimum, and was inhibited by thiol-blocking agents . No evidence for a bound cofactor was obtained . Various benzyl alcohol analogues served as substrates, whereas non-aromatic alcohols were not oxidized . The N-terminal amino acid sequence indicates that the enzyme belongs to the class of long-chain Zn(2+)-dependent alcohol dehydrogenases, although it appears not to contain a metal ion that can be removed by complexing agents. Microbiology, 1995 Jun, 141 ( Pt 6), 1469 - 77 Paracoccus thiocyanatus sp . nov., a new species of thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus versutus comb . nov . with emendation of the genus; Katayama Y et al.; A facultatively chemolithotrophic thiocyanate-degrading bacterium, strain THI 011T, which was previously isolated from activated sludge and tentatively named Thiobacillus sp., was studied taxonomically and phylogenetically . This bacterium utilizes thiocyanate as sole energy source and the specific growth rate for chemolithoautotrophic growth with thiocyanate was 0.059 h-1 . Molecular phylogenetic relationships of strain THI 011T to Thiobacillus versutus and members of the genus Paracoccus were elucidated by comparing 16S rRNA gene sequences . Binary sequence comparisons showed that strain THI 011T was most related to Paracoccus aminophilus, at a similarity level of 97.0%, and T . versutus was most similar to Paracoccus denitrificans, at a level of 99.1% . A neighbour-joining phylogenetic tree showed that strain THI 011T formed a cluster together with T . versutus and known species of the genus Paracoccus within the alpha-3 subclass of the Proteobacteria . DNA-DNA hybridization assays and phenotypic studies indicated that strain THI 011T differed from T . versutus and known species of the genus Paracoccus . On the basis of these results, we propose to classify strain THI 011T into a new species of the genus Paracoccus with the name Paracoccus thiocyanatus sp . nov . We also propose to transfer T . versutus to the genus Paracoccus and present an emended description of the genus. Eur J Biochem, 1995 May 15, 230(1), 359 - 63 Immunoelectron microscopy and epitope mapping with monoclonal antibodies suggest the existence of an additional N-terminal transmembrane helix in the cytochrome b subunit of bacterial ubiquinol:cytochrome-c oxidoreductases; Kleymann G et al.; The topology of the ubiquinol:cytochrome-c oxidoreductase (cytochrome bc1 complex) from Paracoccus denitrificans was investigated by immunoelectron microscopy with sequence-specific murine monoclonal antibodies . Epitope mapping with synthetic peptides and enzymic proteolytic cleavage of the cytochrome bc1 complex were employed to localize precisely the respective antibody epitopes on the subunits of this membrane protein complex . Localization of defined epitopes on the cytochrome bc1 complex by immunoelectron microscopy clearly demonstrates that the N-terminus of the cytochrome b subunit is exposed to the periplasmic space . This finding is in agreement with a nine-transmembrane-helices topology model (I-IX) as predicted before for cytochrome b . However, due to other published evidence we favour the existence of an additional transmembrane helix (helix 0) complementing a more recently published eight-helices model (A-C,cd, D-H), at least for prokaryotes. Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4626 - 30 Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli; Leon-Del-Rio A et al.; Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells . Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm . We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase . Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid . The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759 . Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues . Human HCS shows specific regions of homology with the BirA protein of E . coli and the presumptive biotin ligase of Paracoccus denitrificans . Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites . A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation . We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed. Biochemistry, 1995 May 2, 34(17), 5824 - 30 Electron transfer between cytochrome c and the isolated CuA domain: identification of substrate-binding residues in cytochrome c oxidase; Lappalainen P et al.; Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA . The central part of the cytochrome c binding site is thought to reside in this domain . We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy . The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site . In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively . The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex . The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site . Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding . Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation . The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate . Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain. J Bacteriol, 1995 May, 177(10), 2628 - 36 Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans; Malki S et al.; A genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced . This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD . Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively . HndB does not show any significant homology with any known protein . HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B . taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three {4Fe-4S} clusters . HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D . vulgaris Hildenborough, respectively . The 4.5-kb length of the transcripts expressed in D . fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit . The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E . coli, using the T7 promoter/polymerase system . The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively . One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis . Immunoblotting studies carried out on cell extracts from D . fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only . In soluble extracts from D . fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H(2) was present, but no H(2)-dependent NAD reduction ever occurred . This H(2)-dependent NADP reductase activity disappeared completely in extracts from SM3 . These results indicate that the hnd operon actually encodes an NAdP-reducing hydrogenase in D . fructosovorans. New Horiz, 1995 May, 3(2), 352 - 64 Nitric oxide in the gut; Salzman AL; Nitric oxide (NO.) plays a central role in the physiology of the gastrointestinal tract and its response to critical illness . Potential sources of NO . in the gut include: intrinsic intestinal tissue (mast cells, epithelium, smooth muscle, neural plexus), resident and/or infiltrating leukocytes (neutrophils, monocytes), reduction of luminal gastric nitrate, and denitrification by commensal anaerobes . The brain and endothelial isoforms of nitric oxide synthase are expressed under resting conditions, whereas inflammatory stimuli are required for the induction of the inducible type . Under resting conditions, mucosal perfusion is regulated by NO . derived from the vascular endothelium of the mesenteric bed . During inflammation, excessive NO . production from the inducible synthase may contribute to mucosal hyperemia . Coordination of peristalsis and sphincteric action is mediated by the release of NO., which acts as the principal neurotransmitter of the nonadrenergic, noncholinergic enteric nervous system . Alterations in bowel motility, such as ileus, result from excessive concentrations of NO . generated during endotoxicosis and inflammatory bowel disease . The role of NO . in the regulation of salt and water secretion is poorly understood . Endotoxin-induced inhibition of gastric acid secretion appears to be mediated by the action of NO . on parietal cells . NO . may protect the gastrointestinal mucosa from a variety of stimuli (caustic ingestion, ischemia, ischemia/reperfusion injury, early endotoxic shock) by maintaining mucosal perfusion, inhibiting neutrophil adhesion to mesenteric endothelium, blocking platelet adhesion, and preventing mast cell activation . Excessive NO., however, may directly injure the mucosa . Barrier function of the intestinal mucosa is protected by NO . in the early stages of injury, when neutrophil adhesion, ischemia, and mast cell activation are relevant . Inhibition of NO . synthesis ameliorates barrier dysfunction during more advanced stages of inflammation, when activation of inducible NOS yields toxic concentrations of NO. . At high concentrations, NO . disrupts the actin cytoskeleton, inhibits ATP formation, dilates cellular tight junctions, and produces a hyperpermeable state . Selective inhibition of the inducible isoform of NOS and maintenance of the constitutive types may be therapeutic. Arch Biochem Biophys, 1995 Apr 20, 318(2), 322 - 32 Molecular cloning and expression of biotin sulfoxide reductase from Rhodobacter sphaeroides forma sp . denitrificans; Pollock VV et al.; Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin . Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp . denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R . sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E . coli biotin sulfoxide reductase and R . sphaeroides dimethyl sulfoxide reductase . Screening of a genomic cosmid library, prepared from R . sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R . sphaeroides BSO reductase which has been sequenced . The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da . The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop . The R . sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E . coli enzyme . In addition, there were only two conserved cysteines between the two BSO reductase sequences . The R . sphaeroides gene was demonstrated, by complementation, to rescue a mutant E . coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin . When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay. Eur J Biochem, 1995 Apr 1, 229(1), 148 - 54 The oxidation of methylamine in Paracoccus denitrificans; De Gier JW et al.; The in vivo oxidation of methylamine has been studied in Paracoccus denitrificans . Four components are involved in the electron transfer from methylamine to oxygen; methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome-c oxidase . In P . denitrificans, MADH and its electron acceptor amicyanin are indispensable for growth on methylamine . In the present study, site-directed mutants have been used to demonstrate participation of cytochrome c550 and the aa3-type cytochrome-c oxidase . Moreover, evidence is provided for the operation of alternative routes, branching from amicyanin, in which at least cytochrome c1 and the cbb3-type cytochrome-c oxidase are involved. Int J Syst Bacteriol, 1995 Apr, 45(2), 327 - 33 Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp . nov., and Azoarcus, as Azoarcus evansii sp . nov., respectively, members of the beta subclass of the Proteobacteria; Anders HJ et al.; In the past workers have isolated several pseudomonad strains which have been used for studies of anaerobic aromatic metabolism . The best studied of these strains are strains KB 740T (T = type strain) and K 172T . The taxonomic positions of these two organisms were determined by classical methods, including experiments to determine substrate spectrum, quinone type, and total fatty acid composition . Our results clearly excluded these strains from the authentic genus Pseudomonas, which belongs to the gamma subclass of the Proteobacteria . Instead, the properties of these organisms indicated that they belong to the beta subclass of the Proteobacteria . The sequences of the 16S ribosomal DNA genes confirmed this conclusion and indicated that strain K 172T represents a new species of the genus Thauera, Thauera aromatica, and that strain KB 740T represents a new species of the genus Azoarcus, Azoarcus evansii. Int J Syst Bacteriol, 1995 Apr, 45(2), 290 - 6 Roseobacter algicola sp . nov., a new marine bacterium isolated from the phycosphere of the toxin-producing dinoflagellate Prorocentrum lima; Lafay B et al.; We describe a new species on the basis of phenotypic characteristics and the results of an analysis of small-subunit rRNA sequences . Three strains of this organism were isolated from a culture of the toxin-producing dinoflagellate Prorocentrum lima . These bacteria are gram-negative, strictly aerobic, ovoid organisms that are motile by means of one or two subpolar flagella . They grow at temperatures ranging from 10 to 37 degrees C and in the presence of NaCl concentrations ranging from 0.1 to 2 M and have an absolute requirement for sodium ions . They are strictly aerobic with a nonfermentative type of metabolism and are not able to grow anaerobically in presence or absence of nitrate . They do not denitrify . They exhibit oxidase, catalase, gelatinase, esculinase, beta-galactosidase, and (to a lesser extent) amylase activities . The three strains which we examined require thiamine and biotin for growth . They grow only when glucose, trehalose, saccharose, fructose, maltose, pyruvate, malate, citrate, esculin, 2-ketoglutarate, 5-ketogluconate, glutamate, or shikimate is present as a sole carbon source . The three strains have identical small-subunit rRNA sequences . A phylogenetic analysis of these sequences revealed that these bacteria belong to the alpha subdivision of the Proteobacteria and that they form a distinct and robust monophyletic group with Roseobacter denitrificans and Roseobacter litoralis . This result and the general phenotypic characteristics of the organisms place them in the genus Roseobacter, although they do not produce bacteriochlorophyll a, in contrast to previously described Roseobacter species.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Mar, 177(6), 1564 - 9 Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii); Sattler I et al.; We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme . The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans . In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P . freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme . Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P . freudenreichii . Uroporphyrinogen III methyltransferase of P . freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway . The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism . A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis . These two genes do not appear to constitute part of an extensive cobalamin operon. Appl Environ Microbiol, 1995 Mar, 61(3), 883 - 9 Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor; Usuda K et al.; We examined the denitrification system of the fungus Cylindrocapon tonkinense and found several properties distinct from those of the denitrification system of Fusarium oxysporum . C . tonkinense could form N2O from nitrite under restricted aeration but could not reduce nitrate by dissimilatory metabolism . Nitrite-dependent N2O formation and/or cell growth during the anaerobic culture was not affected by further addition of ammonium ions but was suppressed by respiration inhibitors such as rotenone or antimycin, suggesting that denitrification plays a physiological role in respiration . Dissimilatory nitrite reductase and nitric oxide reductase (Nor) activities could not be detected in cell extracts of the denitrifying cells . The Nor activity was purified and found to depend upon two isoenzymes of Cytochrome P-450nor (P-450nor), which were designated P-450nor1 and P-450nor2 . These isozymes differed in the N-terminal amino acid sequence, isoelectric point, specificity to the reduced pyridine nucleotide (NADH or NADPH), and the reactivity to the antibody to P-450nor of F . oxysporum . the difference between the specificities to NADH and NADPH suggests that P-450nor1 and P-450nor2 play different roles in anaerobic energy acquisition. Antimicrob Agents Chemother, 1995 Mar, 39(3), 771 - 4 Identification of a carbenicillin-hydrolyzing beta-lactamase in Alcaligenes denitrificans subsp . xylosoxydans; Decre D et al.; Eleven strains of Alcaligenes denitrificans subsp . xylosoxydans produced a beta-lactamase with a pI of 5.7 with kinetic data characteristic of a PSE-1-type enzyme . A CARB-type enzyme was identified by using an intragenic DNA probe of blaCARB . Hybridization of genomic DNA after XbaI restriction and pulsed-field electrophoresis suggested a chromosomal location for the gene. Microbiology, 1995 Mar, 141 ( Pt 3), 551 - 60 A gene from Alcaligenes denitrificans that confers albicidin resistance by reversible antibiotic binding; Basnayake WV et al.; Albicidin antibiotics specifically block prokaryote DNA replication . The albicidin resistance gene (albB) cloned from a soil isolate of Alcaligenes denitrificans encodes a 23 kDa protein capable of detoxifying albicidin by reversible binding . This mechanism operates intracellularly to protect DNA replication in albicidin-sensitive Escherichia coli expressing the cloned resistance gene, which can be induced fivefold in the presence of 1.5 micrograms albicidin ml-1 in the surrounding medium . The coding region of 621 bp has regions with partial DNA sequence homology to an albicidin resistance gene (albA) from Klebsiella oxytoca, but with rearrangements and frame-shifts resulting in loss of protein homology . There is a short region of N-terminal homology between the albicidin resistance (Albr) proteins from A . denitrificans and K . oxytoca, although the two genes use different codons for shared amino acids . The N-terminal homology suggested a common functional domain; this was confirmed by deletion analysis, translational fusions and albicidin binding by a synthetic oligopeptide . Antibiotic binding provides a high level of albicidin resistance in E . coli . The gene appears to be a useful candidate for transfer to plants to protect plastid DNA replication from inhibition by albicidin phytotoxins involved in sugarcane leaf scald disease. FEBS Lett, 1995 Feb 27, 360(2), 151 - 4 Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators; Van Spanning RJ et al.; The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans . We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions . The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N-terminal cysteines . A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase . Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb3-type cytochrome c oxidase were not affected by mutation of this gene . These results suggest that denitrification in P . denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms. J Biol Chem, 1995 Feb 24, 270(8), 4146 - 51 The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum; Kobayashi M et al.; A copper-containing nitrite reductase (Cu-NiR) was purified to homogeneity from the denitrifying fungus Fusarium oxysporum . The enzyme seemed to consist of two subunits with almost the same M(r) value of 41,800 and contains two atoms of copper per subunit . The electron paramagnetic resonance spectrum showed that both type 1 and type 2 copper centers are present in the protein, whereas the visible absorption spectrum exhibited a sole and strong absorption maximum at 595 nm, causing a blue but not green color . The reaction product due to the Cu-NiR was mainly nitric oxide (NO), whereas a stoichiometric amount of nitrous oxide (N2O) was formed when cytochrome P-450nor was further added to the assay system . Therefore, the denitrifying (N2O forming) nitrite reductase activity that we had detected in the cell-free extract of the denitrifying cells (Shoun, H., and Tanimoto, T . (1991) J . Biol . Chem . 266, 11078-11082) could be reconstituted upon combination of the purified Cu-NiR and P-450nor . The Km for nitrite and specific activity at pH 7.0 were estimated as 49 microM and 447 mumol NO.min-1.mg protein-1, respectively . Its activity was strongly inhibited by cyanide, carbon monoxide, and diethyldithiocarbamate, whereas enormously restored by the addition of cupric ions . An azurin-like blue copper protein (M(r) = 15,000) and a cytochrome c were also isolated from the same fungus, both of which together with cytochrome c of the yeast Saccharomyces cerevisiae were effective in donating electrons to the fungal Cu-NiR . The result suggested that the physiological electron donor of the Cu-NiR is the respiratory electron transport system . The intracellular localization of Cu-NiR was investigated, and it was suggested that the Cu-NiR localizes in an organelle such as mitochondrion . These findings showed the identity in many aspects between the fungal nitrite reductase and bacterial dissimilatory Cu-NiRs . This is the first isolation of dissimilatory NiR from a eukaryote. Gene, 1995 Feb 3, 153(1), 75 - 9 Genes encoding RubisCO in Pseudomonas hydrogenothermophila are followed by a novel cbbQ gene similar to nirQ of the denitrification gene cluster from Pseudomonas species; Yokoyama K et al.; The cbbL and cbbS genes, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), were cloned and sequenced from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1 . The cbbL gene encoded a 474-amino-acid (aa) protein (53,285 Da); cbbS encoded a 124-aa protein (14,656 Da) . An ORF found downstream from the cbbLS genes encoded a 267-aa protein (29,565 Da) which had no similarity to cbbX located downstream from cbbLS from Alcaligenes eutrophus and Xanthobacter flavus . This gene, called cbbQ, was highly similar to the nirQ gene of the denitrification gene cluster from P . aeruginosa and P . stutzeri. J Bacteriol, 1995 Feb, 177(4), 1116 - 8 Identification of alcaligin as the siderophore produced by Bordetella pertussis and B . bronchiseptica; Moore CH et al.; The siderophores produced by iron-starved Bordetella pertussis and B . bronchiseptica were purified and were found to be identical . Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans. Bioorg Med Chem, 1995 Feb, 3(2), 109 - 12 Enantioselective deoxygenation of alkyl aryl sulfoxides by DMSO reductase from Rhodobacter sphaeroides f.s . denitrificans; Abo M et al.; The substrate specificity and enantioselectivity of DMSO reductase from Rhodobacter sphaeroides f.s . denitrificans were studied on a series of alkyl aryl sulfoxides as substrate . The enzyme was found to catalyze deoxygenation of (S)-sulfoxides predominantly . (R)-Sulfoxides were recovered with a high enantiomeric excess. Arch Microbiol, 1995 Feb, 163(2), 96 - 103 Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria; Rabus R et al.; Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate . Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively . For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively . Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis . The strains exhibited different specific capacities for degradation of alkylbenzenes . In addition to ethylbenzene, strain EbN1 utilized toluene, but not propylbenzene . In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene . Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene . Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO2 by strain EbN1 . Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone . In contrast to the other isolates, stain mXyN1 did not grow on benzyl alcohol . Benzyl alcohol (also m-methyl-benzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1 . None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor . However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions . All four isolates grew anaerobically on crude oil . Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene. New Horiz, 1995 Feb, 3(1), 33 - 45 Nitric oxide in the gut; Salzman AL; Nitric oxide (NO.) plays a central role in the Physioliology of the gastrointestinal tract and its response to critical illness . Potential sources of NO . in the gut include: intrinsic intestinal tissue (mast cells, epithelium, smooth muscle, neural plexus), resident and/or infiltrating leukocytes (neutrophils, monocytes), reduction of luminal gastric nitrate, and denitrification by commensal anaerobes . The brain and endothelial isoforms of nitric oxide synthase are expressed under resting conditions, whereas inflammatory stimuli are required for the induction of the inducible type . Under resting conditions, mucosal perfusion is regulated by NO . derived from the vascular endothelium of the mesenteric bed . During inflammation, excessive NO . production from the inducible synthase may contribute to mucosal hyperemia . Coordination of peristalsis and sphincteric action is mediated by the release of NO., which acts as the principal neurotransmitter of the nonadrenergic, noncholinergic enteric nervous system . Alterations in bowel motility, such as ileus, result from excessive concentrations of NO . generated during endotoxicosis and inflammatory bowel disease . The role of NO . in the regulation of salt and water secretion is poorly understood . Endotoxin-induced inhibition of gastric acid secretion appears to be mediated by the action of NO . on parietal cells . NO . may protect the gastrointestinal mucosa from a variety of stimuli (caustic ingestion, ischemia, ischemia/reperfusion injury, early endotoxic shock) by maintaining mucosal perfusion, inhibiting neutrophil adhesion to mesenteric endothelium, blocking platelet adhesion, and preventing mast cell activation . Excessive NO., however, may directly injure the mucosa . Barrier function of the intestinal mucosa is protected by NO . in the early stages of injury, when neutrophil adhesion, ischemia, and mast cell activation are relevant . Inhibition of NO . synthesis ameliorates barrier dysfunction during more advanced stages of inflammation, when activation of inducible NOS yields toxic concentrations of NO. . At high concentrations, NO . disrupts the actin cytoskeleton, inhibits ATP formation, dilates cellular tight junctions, and produces a hyperpermeable state . Selective inhibition of the inducible isoform of NOS and maintenance of the constitutive types may be therapeutic. Biochemistry, 1995 Jan 31, 34(4), 1238 - 43 Roles of dipolar effects and local charge in the ionic strength dependence of redox reactions between c-type cytochromes; Davidson VL et al.; Redox reactions between different c-type cytochromes were monitored by stopped-flow spectroscopy . Second-order rate constants were determined at different ionic strengths for the reactions of Paracoccus denitrificans cytochrome c-551i with its physiologic redox partner cytochrome c-550, and with the nonphysiologic partner horse heart cytochrome c . The latter two cytochromes are structurally quite similar and exhibit identical redox potentials but bear net charges of -7 and +7, respectively . Despite these opposite overall charges, the ionic strength dependencies for the reaction of each with the acidic cytochrome c-551i were very similar . The observed decrease in reaction rate with increasing ionic strength that was observed with cytochromes c-550 and c-551i, the latter of which bears a net charge of -20, cannot be explained simply on the basis of monopole-monopole interactions . These data were analyzed by two different methods: one which treats proteins as both monopoles and dipoles and considers the net charge; and another which neglects dipolar effects and considers only the local charge of the reactive site of the protein rather than net charge . The applicability of each method to the analysis of these data and to protein electrostatic interactions in general is discussed. Eur J Biochem, 1995 Jan 15, 227(1-2), 43 - 60 Metabolic pathway for biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from 4-hydroxybutyrate by Alcaligenes eutrophus; Valentin HE et al.; Various aerobic Gram-negative bacteria have been examined for their ability to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth . Alcaligenes eutrophus strains H16, HF39, PHB-4 and Pseudomonas denitrificans 'Morris' were not able to grow with 1,4-butanediol or 4-hydroxybutyrate . From A . eutrophus HF39 spontaneous primary mutants (e.g . SK4040) were isolated which grew on 4-hydroxybutyrate with doubling times of approximately 3 h . Tn5::mob mutagenesis of mutant SK4040 led to the isolation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4-hydroxybutyrate . Mutants exhibiting the phenotype 4-hydroxybutyrate-negative did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydroxybutyrate . Hybridization experiments led to the identification of a 10-kbp genomic EcoRI fragment of A . eutrophus SK4040, which was altered in mutants with the phenotype 4-hydroxybutyrate-negative, and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4-hydroxybutyrate-leaky . This 10-kbp EcoRI fragment was cloned from A . eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A . eutrophus H16 conferred the ability to grow with 4-hydroxybutyrate to the wild type . DNA-sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A . eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-negative mutants . Enzymic studies also provided evidence for the presence of an active succinate-semialdehyde dehydrogenase in 4-hydroxybutyrate-grown cells . This indicated that degradation of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle . NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulated from 4-hydroxy {1-13C}butyrate or 4-hydroxy{2-13C}butyrate as substrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic analysis. Eur J Biochem, 1995 Jan 15, 227(1-2), 161 - 8 Benzoyl-coenzyme-A 3-monooxygenase, a flavin-dependent hydroxylase . Purification, some properties and its role in aerobic benzoate oxidation via gentisate in a denitrifying bacterium; Niemetz R et al.; A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate {Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H . & Fuchs, G . (1993) New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J . Bacteriol . 175, 4851-4858)} . The initial reaction is catalyzed by benzoate-CoA ligase (AMP-forming), converting benzoate into benzoyl-CoA . The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase . This novel enzyme has been purified and studied . It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor . Only benzoyl-CoA, but not benzoate, is hydroxylated . The protein is a monomer of M(r) 65,000 and is induced when cells are grown aerobically with benzoate . 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxybenzoate (gentisate) . This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific specific with respect to the pyridine nucleotide . Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxybenzoate . Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced . The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate. J Biol Chem, 1995 Jan 13, 270(2), 573 - 80 Structure of metal site in azurin, Met121 mutants of azurin, and stellacyanin investigated by 111mCd perturbed angular correlation (PAC); Danielsen E et al.; The geometries of the metal sites in cadmium-substituted azurins have been investigated by 111mCd perturbed angular correlation (PAC) . The study includes wild type azurin as well as Met121 mutants of azurin, where methionine has been substituted by Ala, Asn, Asp, Gln, Glu, and Leu . The nuclear quadrupole interaction of wild type azurin analyzed in the angular overlap model is well described as coordination of His46, His117, and Cys112 and cannot be described by coordination of Met121 and/or Gly45 . For most of the mutants, there exist two coordination geometries of the cadmium ion . With the exception of the Glu and Asp mutants, one of the conformations is similar to the wild type conformation . The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid . These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site . The nuclear quadrupole interaction of stellacyanin was also measured . The value resembles the most prominent nuclear quadrupole interaction of the Met121-->Gln mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are similar. Acta Microbiol Pol, 1995, 44(2), 197 - 200 On the role of bacterial inoculum growth conditions on nitrate denitrification pathway; Blaszczyk M et al.; Activities of nitrite and nitrate reductases as well as the efficiencies of denitrification carried out by Pseudomonas aeruginosa and Pseudomonas stutzeri under different culture conditions have been compared . The inoculum growth conditions had no influence on denitrification pathway in either strain . Activities of both reductases were higher in Pseudomonas stutzeri than in Pseudomonas aeruginosa cultures. Biopolymers, 1995, 37(6), 401 - 10 Design of metal ion binding peptides; Fattorusso R et al.; Two cyclic and branched peptides (PLA and AZU) were synthesized with the aim of reproducing the active site of the blue copper proteins plastocyanin and azurin . Both peptides, designed on the basis of the x-ray structures of Poplar plastocyanin and Alcaligenes denitrificans azurin, contain the same coordinating residues of the parent native proteins . The visible spectra of PLA in the presence of equimolar amount of Cu(II) strongly support the interaction between the peptide and copper(II) ion . The CD titration of AZU with the Hg(II) ion indicates for the formation of two species, {AZUHg}+ and {AZUHg2}3+ having binding constants (Keq) of 3.10(6) and 2.10(4) M-1, respectively. Biometals, 1995 Jan, 8(1), 70 - 9 The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer, expression, regulation and DNA homologies to various nickel-resistant bacteria; Stoppel RD et al.; Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Goteborg, Sweden, was found to be nickel-resistant (tolerating 10 mM NiCl2 in non-complexing mineral-gluconate media; inducible resistance) . The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mM NiCl2 . A 4.3 kb HindIII fragment was cloned from the genomic DNA of K . oxytoca . Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mM Ni2+) in various E . coli strains . After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104 . With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K . oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb) . Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants . No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A . eutrophus CH34 or A . denitrificans 4a-2, were used as target DNA . Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A . xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5-5) isolated from nickel-rich soil in New Caledonia. J Bacteriol, 1995 Jan, 177(1), 247 - 51 Isolation, sequencing, and mutagenesis of the gene encoding NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) from Paracoccus denitrificans, in which GD-FALDH is essential for methylotrophic growth; Ras J et al.; NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) of Paracoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa . The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene . The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation . This finding indicates that GD-FALDH of P . denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth. Lett Appl Microbiol, 1995 Jan, 20(1), 65 - 8 Polymerase chain reaction amplification of the flaA gene for the rapid identification of Listeria spp; Gray DI et al.; The polymerase chain reaction was used to amplify specifically a 448 base pair internal fragment of the flaA gene from bacteria belonging to the genus Listeria . This gene encodes for the flagellin protein and allowed specific genus discrimination between other commonly found Gram-positive and Gram-negative bacteria and all Listeria spp . with the exception of Jonesia denitrificans. Mol Microbiol, 1995 Jan, 15(2), 319 - 31 Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop; Berks BC et al.; Three genes, narH, narJ and narI, of the membrane-bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced . The derived gene products show high sequence similarity to the equivalent (beta, putative delta and gamma) subunits of the two membrane-bound nitrate reductases of the enteric bacterium Escherichia coli . All iron-sulphur cluster ligands proposed for the E . coli beta subunits are conserved in T . pantotropha NarH . Secondary structure analysis of NarJ suggests that this protein has a predominantly alpha-helical structure . Comparison of T . pantotropha NarI with the b-haem-binding integral membrane subunits of the E . coli enzymes allows assignment of His-53, His-63, His-186 and His-204 (T . pantotropha NarI numbering) as b-haem axial ligands and the construction of a three-dimensional model of this subunit . This model, in which the two b-haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems . Similar movement of electrons is required in the membrane-bound uptake hydrogenases and membrane-bound formate dehydrogenases . We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer . One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate-reducing bacteria has structural resemblance to NarI. Mol Microbiol, 1995 Jan, 15(2), 307 - 18 Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product is required for assembly of all c-type cytochromes, including cytochrome c1; Page MD et al.; A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test . P . denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome c550, but the membrane-bound holocytochromes c1 and c552 were present at 5-10% of the levels observed in wild-type cells . DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P . denitrificans wild-type DNA library . A hybridizing 3.05 kb BamHI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced . One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum . Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected . Significant differences between the phenotypes of P . denitrificans DP108 and the B . japonicum cycH mutant COX3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor. Proteins, 1995 Jan, 21(1), 74 - 7 Crystals of an antibody Fv fragment against an integral membrane protein diffracting to 1.28 A resolution; Ostermeier C et al.; The Fv fragment of a monoclonal antibody, 7E2 (IgG1, kappa, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli . Crystals suitable for high-resolution X-ray analysis were obtained by microdialysis under low salt conditions . The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 51.51 A, b = 56.15 A, c = 99.86 A (1 A = 0.1 nm) and contain one Fv fragment per asymmetric unit . Using synchrotron radiation diffraction data were collected up to 1.28 A resolution . This high resolution is very unusual for a heterodimeric protein . The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. Biosens Bioelectron, 1995 Summer, 10(6-7), 569 - 76 Amperometric detection of histamine at a quinoprotein dehydrogenase enzyme electrode; Loughran MG et al.; Methylamine dehydrogenase, a tryptophan tryptophyl quinone (TTQ) containing quinoprotein, catalyzes the oxidation of a variety of primary aliphatic monomaines and diamines to their respective aldehydes and ammonia . This paper reports the construction and characterization of an enzyme electrode capable of detecting histamine and methylamine at +200 mV versus a saturated calomel reference electrode . The methylamine dehydrogenase isolated from Paracoccus denitrificans was used in conjunction with the insoluble mediator tetracyanoquinodimethane (TCNQ) to construct enzyme electrodes which will potentially provide simple rapid analysis of histamine without the need for the extensive sample pretreatments currently required in HPLC and GLC analysis . The linear response of this amperometric sensor, between 0 and 200 microM, correlates well with elevated histamine levels predominant in patients with chronic myelogenous leukaemia, whilst the observed limit of detection, 4.8 microM, compares favourably with the lower limits of detection reported for a potentiometric histamine sensitive enzyme electrode. J Biol Chem, 1994 Dec 23, 269(51), 32239 - 45 Expression and characterization of human and chimeric human-Paracoccus denitrificans electron transfer flavoproteins; Herrick KR et al.; Electron transfer flavoprotein (ETF) is a heterodimer that contains a single equivalent of FAD and accepts electrons from nine flavoprotein dehydrogenases in the mitochondrial matrix . Human ETF was expressed in Escherichia coli using the expression vector previously employed to express Paracoccus denitrificans ETF (Bedzyk, L . A., Escudero, K . W., Gill, R . E., Griffin, K . J., and Frerman, F . E . (1993) J . Biol . Chem . 268, 20211-20217) . cDNAs encoding the beta and alpha subunits of the human protein were inserted into the vector, mimicking the arrangement of the P . denitrificans genes in which coding sequences are joined by overlapping termination and initiation codons . A human ETF containing 30% P . denitrificans sequence at the amino terminus of the beta subunit was also expressed and purified . This chimeric ETF has 64% sequence identity with the human sequence in the substituted region . Kinetic constants of medium chain and short chain acyl-CoA dehydrogenases for the chimeric ETFs were slightly changed from those of human ETF; but, there are marked differences in the kinetic constants of sarcosine dehydrogenase and electron transfer flavoprotein-ubiquinone oxidoreductase with the two ETFs . Absorption spectra of the three redox states of human, chimeric, and P . denitrificans ETF flavins are identical . However, the flavin circular dichroism spectra of the three ETFs are characteristic for each species . The spectrum of the chimeric ETF has both human and P . denitrificans ETF features . The amplitude of the 436 nm band is identical to that of the of the human ETF flavin, but the amplitude of the 375 nm band is identical to that of the P . denitrificans ETF flavin . Thus, flavin in the chimeric ETF appears to be exposed to dipoles in the protein framework provided by human and bacterial sequences . These spectral data indicate that the flavin is located in the vicinity of the amino-terminal region of the beta subunit . The kinetic data suggest that the amino-terminal region of the beta subunit comprises part of the docking site for some primary dehydrogenases and electron transfer flavoprotein-ubiquinone oxidoreductase. J Biol Chem, 1994 Dec 23, 269(51), 32120 - 30 Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein; Chen D et al.; The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been identified, cloned, and sequenced . A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequences determined for the ETF subunits purified from W3A1 . The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fragments using this probe identified a 2.2-kilobase pair insert containing the complete coding sequences for both W3A1 ETF subunits . The genes are arranged in tandem in the genomic DNA with only 2 bases between the TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit . The deduced amino acid sequences of each of the W3A1 ETF subunits exhibit only approximately 30% identity with the corresponding subunits of the ETF from human, rat, and Paracoccus denitrificans, which as a group are greater than 50% identical . Thus, the ETF from W3A1 may exhibit some unique structural features that, like other differences in some of its physical and functional properties, may distinguish this ETF from others in this family . A highly homologous region near the COOH terminus of the large subunit in all the ETF proteins was found to contain a sequence that matches in several ways the ADP-binding motif of flavoproteins and other dinucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins . Under control of the tac promoter, the cloned ETF subunit genes were co-expressed in Escherichia coli producing the heterodimeric holoprotein with physical, spectral, and electron-accepting properties essentially identical to the ETF isolated from W3A1 . The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accumulating as the air-stable anionic semiquinone in the presence of excess trimethylamine . Fully reduced ETF could not be obtained even after prolonged enzymatic reduction. Eur J Biochem, 1994 Dec 15, 226(3), 841 - 6 Unique primary structure of 2-nitropropane dioxygenase from Hansenula mrakii; Tchorzewski M et al.; We have isolated the gene encoding 2-nitropropane dioxygenase from Hansenula mrakii, an FAD enzyme that catalyzes the oxygenative denitrification of various anionic nitroalkanes . The gene contained an open reading frame consisting of 1122 nucleotides corresponding to 374 amino acid residues . The protein molecular mass was estimated to be 41,466 Da, which was similar to the subunit molecular mass of the enzyme determined by SDS/PAGE . Several FAD enzymes such as D-amino acid oxidase and glucose oxidase also catalyze the oxidation of nitroalkanes as a side-reaction, although not so efficiently {Kido, T . & Soda, K . (1984) Arch . Biochem . Biophys . 234, 468-475} . However, we found no proteins in the databases (GenBank, EMBL, PIR and SWISS-PROT) which are homologous to 2-nitropropane dioxygenase of H . mrakii in primary structure . No protein motifs, including a nucleotide-binding motif, GXGXXG, were found in PROSITE, a database of biologically significant protein sites and patterns . Accordingly, 2-nitropropane dioxygenase is a new type of flavoprotein with a unique structure. Eur J Biochem, 1994 Dec 15, 226(3), 789 - 98 Mo(V) electron paramagnetic resonance signals from the periplasmic nitrate reductase of Thiosphaera pantotropha; Bennett B et al.; A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans . We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals . As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T . pantotropha upon treatment with excess dithionite . The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus . A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state . The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed. Appl Environ Microbiol, 1994 Dec, 60(12), 4567 - 72 Dechlorination of trichlorofluoromethane (CFC-11) by sulfate-reducing bacteria from an aquifer contaminated with halogenated aliphatic compounds; Sonier DN et al.; Groundwater samples were obtained from a deep aquifer contaminated with halogenated aliphatic compounds . One-milliliter samples contained 9.2 x 10(5) total bacteria (by acridine orange microscopic counts) and 2.5 x 10(3) sulfate-reducing bacteria (by most probable number analysis) . Samples were incubated anaerobically in a basal salts medium with acetate as the electron donor and nitrate and sulfate as the electron acceptors . Residual levels of trichlorofluoromethane (CFC-11) in samples were biotically degraded, while trichloroethylene was not . When successively higher levels of CFC-11 were added, increasingly rapid degradation rates were observed . Concomitant with CFC-11 degradation was the near stoichiometric production of fluorodichloromethane (HCFC-21); the production of HCFC-21 was verified by mass spectrometry . CFC-11 degradation was dependent on the presence of acetate (or butyrate) and sulfate but was independent of nitrate . Other carbon sources such as lactate and isopropanol did not support the degradation . The addition of 1 mM sodium sulfide completely inhibited CFC-11 degradation; however, degradation occurred in the presence of 2 mM 2-bromoethanesulfonic acid . These results indicate that the anaerobic dechlorination of CFC-11 is carried out by sulfate-reducing bacteria and not by denitrifying or methanogenic bacteria. Biodegradation, 1994 Dec, 5(3-4), 343 - 57 Transfer and expression of PCB-degradative genes into heavy metal resistant Alcaligenes eutrophus strains; Springael D et al.; Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals . The latter might inhibit the biodegradation of the organics and impair bioremediation . Chromosomally located polychlorinated biphenyl (PCB) catabolic genes of Alcaligenes eutrophus A5, Achromobacter sp . LBS1C1 and Alcaligenes denitrificans JB1 were introduced into the heavy metal resistant Alcaligenes eutrophus strain CH34 and related strains by means of natural conjugation . Mobile elements containing the PCB catabolic genes were transferred from A . eutrophus A5 and Achromobacter sp . LB51C1 into A . eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively . The PCB catabolic genes of A . denitrificans JB1 were transferred into A . eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation . The A . eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers . Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals. Appl Microbiol Biotechnol, 1994 Dec, 42(4), 631 - 5 Toxicity of 2-mercaptobenzothiazole towards bacterial growth and respiration; De Wever H et al.; Active sludge systems containing benzothiazoles may be intoxicated by 2-mercaptobenzothiazole (MBT) . This toxicity towards several bacteria is now confirmed and is situated at around 100 mg MBT l-1 . Octanol-water partition coefficients indicated that MBT might interact with membrane-bound systems . This was confirmed through experiments showing that bacterial cell respiration was inhibited using lactate or succinate as substrates . Using these substrates and also NADH, it was found that their oxidation was also inhibited using isolated membrane fragments of Escherichia coli and Paracoccus denitrificans . Methylene blue reduction was also found to be inhibited . The oxidation of ascorbate was not inhibited in P . denitrificans . From these results it is suggested that MBT might interact with the respiratory chain at the level of flavoproteins or quinones and Fe-S clusters. J Biochem (Tokyo), 1994 Dec, 116(6), 1193 - 7 Structure of azurin from Achromobacter xylosoxidans NCIB11015 at 2.5 A resolution; Inoue T et al.; The crystal structure of azurin from a denitrifying bacterium, Achromobacter xylosoxidans NCIB11015, has been refined at 2.5 A resolution using diffraction data obtained by means of synchrotron radiation at KEK . Crystals suitable for X-ray experiment were obtained by the macro-seeding method and an intensity data were obtained on imaging plates mounted on a Weissenberg camera (Rmerge = 0.09) . The initial model was obtained by the molecular replacement method using the structure of azurin from Alcaligenes denitrificans NCTC8582 as a starting model . The structure was refined by molecular dynamics optimization and the restrained least-squares method to a crystallographic R-value of 0.205 . However, the current model gave an electron-density of the side-chain regions of several residues close to the N-terminus quite different from those expected from the amino acid sequences reported . Very recently, two kinds of azurins (Az-I and Az-II) were isolated from this bacterium by a slightly modified purification method and have been characterized and found to have different CD spectra . On analysis of amino acid sequences around the N-terminus, the second azurin (Az-II) was proved to be a new type of azurin in this bacterium . It was consequently revealed that the current model corresponds to a new type of azurin because of the complete agreement between the electron-density and the amino acid sequence of the newly determined 20 residues from the N-terminus . Determination of the whole amino acid sequence of this azurin and further refinement are in progress. Biodegradation, 1994 Dec, 5(3-4), 161 - 74 Genetics of alkane oxidation by Pseudomonas oleovorans; van Beilen JB et al.; Many Pseudomonads are able to use linear alkanes as sole carbon and energy source . The genetics and enzymology of alkane metabolism have been investigated in depth for Pseudomonas oleovorans, which is able to oxidize C5-C12 n-alkanes by virtue of two gene regions, localized on the OCT-plasmid . The so-called alk-genes have been cloned in pLAFR1, and were subsequent analyzed using minicell expression experiments, DNA sequencing and deletion analysis . This has led to the identification and characterization of of the alkBFGHJKL and alkST genes which encode all proteins necessary to convert alkanes to the corresponding acyl-CoA derivatives . These then enter the beta-oxidation-cycle, and can be utilized as carbon- and energy sources . Medium (C6-C12)- or long-chain (C13-C20) n-alkanes can be utilized by many strains, some of which have been partially characterized . The alkane-oxidizing enzymes used by some of these strains (e.g . two P . aeruginosa strains, a P . denitrificans strain and a marine Pseudomonas sp.) appear to be closely related to those encoded by the OCT-plasmid. Eur J Biochem, 1994 Nov 15, 226(1), 201 - 10 Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrificans is under control of a LysR-type transcriptional activator; Van Spanning RJ et al.; Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism . Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy . The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine . Repression is not observed when the growth medium contains methylamine and either choline or methanol . Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene . This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster . The mauR gene encodes a LysR-type transcriptional activator . Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine . The mutation is completely restored when the mauR gene is supplied in trans . The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein . Inactivation of the gene results in the inability of cells to grow on methylamine . Downstream from mauF and in the same transcription direction, mauB is located . This gene encodes the large subunit of methylamine dehydrogenase. FEBS Lett, 1994 Nov 7, 354(2), 160 - 4 Identification of amino acid residues associated with the {2Fe-2S} cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans; Yano T et al.; In order to identify the ligand residues of the {2Fe-2S} cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans, we mutated individually all seven cysteine residues (C61, C96, C101, C104, C113, C137, and C141) and one conserved histidine residue (H92) to Ser or Ala and expressed them in E . coli . After purification of the mutated 25 kDa subunits, the effect of mutations on the iron-sulfur cluster were characterized by chemical analyses and UV-visible and EPR spectroscopy . All mutated subunits, especially mutants of conserved cysteines, contained lower amounts of non-heme iron than wild-type . The subunits of three non-conserved cysteine residues (C61, C104, and C113) mutated to Ser and a histidine residue (H92) mutated to Ala exhibited essentially the same spectroscopic properties as those of the wild-type subunit . In contrast, mutation of the four conserved cysteine residues (C96, C101, C137, and C141) to Ser or Ala considerably altered the UV-visible and EPR spectra from the wild-type subunit . These results indicate that the four conserved cysteine residues coordinate the {2Fe-2S} cluster in the P . denitrificans 25 kDa subunit. Appl Environ Microbiol, 1994 Nov, 60(11), 4047 - 52 Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria; Seyfried B et al.; Pseudomonas sp . strain T and Pseudomonas sp . strain K172 grow with toluene under denitrifying conditions . We demonstrated that anaerobic degradation of toluene was initiated by direct oxidation of the methyl group . Benzaldehyde and benzoate accumulated sequentially after toluene was added when cell suspensions were incubated at 5 degrees C . Strain T also grows anaerobically with m-xylene, and we demonstrated that degradation was initiated by oxidation of one methyl group . In cell suspensions incubated at 5 degrees C 3-methylbenzaldehyde and 3-methylbenzoate accumulated after m-xylene was added . Toluene- or m-xylene-grown strain T cells were induced to the same extent for oxidation of both hydrocarbons . In addition, the methyl group-oxidizing enzyme system of strain T also catalyzed the oxidation of each isomer of the chloro- and fluorotoluenes to the corresponding halogenated benzoate derivatives . In contrast, strain K172 only oxidized 4-fluorotoluene to 4-fluorobenzoate, probably because of the narrow substrate specificity of the methyl group-oxidizing enzymatic system . During anaerobic growth with toluene strains T and K172 produced two transformation products, benzylsuccinate and benzylfumarate . About 0.5% of the toluene carbon was converted to these products. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 482 - 6 Precipitation potential as a major factor in the formation of granular sludge in an upflow sludge-blanket reactor for denitrification of drinking water; Tarre S et al.; The effects of the chemical composition of water on granular sludge formation and characteristics in a denitrifying upflow sludge-blanket (USB) reactor were studied . Denitrification of drinking water showed different biomass sludge characteristics when the reactor was fed with groundwater as opposed to surface water USB reactors fed with groundwater produced granules with good settling characteristics, SVI (sludge volume index) values lower than 30 ml/g, and high reactor biomass concentrations (20-25 g/l), while surface-water-fed reactors exhibited lower biomass concentrations (10-15 g/l) due to poor settling characteristics (SVI values of 50-90 ml/g) . Sludge granules from the reactor fed with surface water had a low mineral content of between 10% and 20% as compared to a mineral content of 25%-50% in the groundwater reactor . The larger mineral content in the groundwater-fed reactor was due to a greater precipitation potential, i.e . higher concentrations of calcium and alkalinity present in groundwater combined with the release of alkalinity and subsequent increase in pH caused by biological denitrification . Verification for this phenomenon was established by enriching surface water with calcium and alkalinity, which increased the reactor's precipitation potential from 15 mg/l to 40 mg/l (as CaCO3) . The granules obtained from the reactor fed with enriched surface water had a high mineral content of between 40% and 50% and very low SVI values, contributing to improved granule-settling characteristics and reactor stability. Biochem Biophys Res Commun, 1994 Oct 14, 204(1), 120 - 8 Replacement of methionine as the axial ligand of Achromobacter cycloclastes cytochrome c554 at high pH values revealed by absorption, EPR and MCD spectroscopy; Saraiva LM et al.; Cytochrome c554 from the denitrifying bacterium Achromobacter cycloclastes is a monoheme class II c-type cytochrome with a His-Met axial coordination at neutral pH . The amino acid composition and the N-terminal sequence of the cytochrome have been determined . Subsequent determination of the pH-dependence of the redox potential and examination of the EPR and MCD spectra of ferricytochrome c554 revealed a new form at high pH values made apparent with both spectroscopies . These observations are consistent with the presence of lysine as the axial ligand for which methionine substitutes at high pH values. FEMS Microbiol Rev, 1994 Oct, 15(2-3), 137 - 53 Biology of polyphosphate-accumulating bacteria involved in enhanced biological phosphorus removal; Kortstee GJ et al.; Recent research on the process of biological phosphorus removal in lab-scale treatment systems has indicated that: (i) the development of an actively polyP-accumulating bacterial community after the introduction of an anaerobic period may take at least 4 months; (ii) up to 80% of all aerobic bacteria isolated from these communities are able to accumulate polyP; (iii) polyP synthesized by the bacterial communities of lab-scale treatment systems is probably mainly low polymeric, not exceeding 20 P-residues, and this polyP is rapidly degraded during the anaerobic period; (iv) the enzymatic hydrolysis of polyP under anaerobic conditions is accompanied by PHB formation from exogenous acetate, reducing equivalents are provided by the degradation of carbohydrates; and (v) nitric oxide inhibits the release of phosphate under anaerobic conditions in Renpho and F&D sludges . Bacteria belonging to the genus Acinetobacter occur in a wide variety of activated sludges in which enhanced biological phosphate removal is observed . A . johnsonii 210A was studied in detail with respect to the elemental composition of polyP granules, the enzymatic synthesis and degradation of polyP, the regulation of polyP metabolism, and the transport of phosphate . A . johnsonii 210A reflects activated sludge in a number of ways as far as polyP metabolism is concerned but its polyP is highly polymeric and the phosphate efflux rate under anaerobic conditions is relatively low and not increased by exogenous acetate . In addition to Acinetobacter, other polyP-accumulating microorganisms may be involved in biological phosphorus removal . The isolation of polyP-accumulating denitrifying bacteria may well have interesting implications for a new process design in wastewater treatment systems . Further studies on the enzymes involved in polyP biosynthesis and on the uptake and efflux systems of phosphate, potassium, magnesium and lower fatty acids in pure cultures will enlarge our insight in the energetics of the metabolism of polyP . In addition, the regulation of the metabolism of polyP-accumulating organisms needs to be studied in more detail. Biochem J, 1994 Oct 1, 303 ( Pt 1), 141 - 5 Thermal stability of methanol dehydrogenase is altered by the replacement of enzyme-bound Ca2+ with Sr2+; Harris TK et al.; Methanol dehydrogenase (MEDH) possesses tightly bound Ca2+ in addition to its pyrroloquinoline quinone prosthetic group . Ca2+ was replaced with Sr2+ by growing the host bacterium, Paracoccus denitrificans, in media in which Ca2+ was replaced with Sr2+ . At temperatures in the transition region for stability, the rate constants for inactivation of MEDH purified from these cells (Sr-MEDH) were 2-fold lower than those for MEDH . However, Arrhenius plots yielded an activation energy (Ea) of 699 kJ (167 kcal)/mol for MEDH compared with 640 kJ (153 kcal)/mol for Sr-MEDH . Further analysis by transition-state theory yielded values for the activation enthalpy (delta H*) and activation entropy (delta S*) of 696 kJ (166 kcal)/mol and 1.73 kJ (414 cal)/mol per K for MEDH and 637 kJ (152 kcal)/mol and 1.55 kJ (371 cal)/mol per K for Sr-MEDH . The higher rate of inactivation of MEDH than Sr-MEDH at higher temperatures is a consequence of a more favourable net gain in entropy . This positive entropy contribution increases at high temperatures, and reduces the more favourable stability obtained from the enthalpy contribution for the free energy (delta G*) of inactivation . The differences in these thermodynamic data are discussed in relation to the recently determined crystal structure of MEDH as well as 1H electron-nuclear double resonance studies of the influence of Sr2+ substitution on the structure of the pyrroloquinoline quinone-derived radical in MEDH. J Bacteriol, 1994 Oct, 176(20), 6188 - 91 Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17; Wodara C et al.; The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19) . The corresponding wild-type region was cloned previously (G . Mittenhuber, K . Sonomoto, M . Egert, and C . G . Friedrich, J . Bacteriol . 173:7340-7344, 1991) . Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB . This polypeptide had an M(r) of 60.573, including a possible signal peptide . The function of the SoxB protein of P . denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus . The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P . denitrificans GB17. J Bacteriol, 1994 Oct, 176(19), 5919 - 28 Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity; Page MD et al.; A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis . P . denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes . In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type . The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104 . The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced . Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media . Disruption of the hemA gene generated a P . denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism . The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion. Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1859 - 65 Nitrile hydratase gene from Rhodococcus sp . N-774 requirement for its downstream region for efficient expression; Hashimoto Y et al.; For improvement of the production of nitrile hydratase (NHase) from Rhodococcus sp . N-774 by recombinant DNA techniques, several plasmids, each of which had a deletion of the upstream or downstream region of the genes encoding the alpha and beta subunits of NHase, were constructed . Enzyme assays of recombinant R . rhodochrous and Escherichia coli cells showed that a downstream region of the NHase genes was indispensable for the production of active NHase in both cells, but for the production of the active amidase, no genes other than the amidase structural gene were required . The nucleotide sequence of the downstream region contained a single open reading frame (Orf1188) with 396 amino acids . Orf1188 showed similarity in amino acid sequence to P47K, an open reading frame found downstream of the NHase genes from Pseudomonas chlororaphis B23, and also to the cobW gene product, which may be involved in cobalamin biosynthesis in Pseudomonas denitrificans . Because the distance between the TGA stop codon for the NHase beta-subunit and the ATG codon for Orf1188 is only 98 bp, and because production of both Orf1188 and NHase is dependent on a promoter upstream of the amidase gene, these genes appear to be co-transcribed in a polycistronic manner, forming an operon . By optimization of the culture conditions of R . rhodochrous carrying pKRNH2, which contained the amidase, NHase, and Orf1188 genes, the transformant showed the NHase activity 6-fold higher than that of the original strain, Rhodococcus sp . N-774. J Biol Chem, 1994 Sep 16, 269(37), 23079 - 86 A cytochrome ba3 functions as a quinol oxidase in Paracoccus denitrificans . Purification, cloning, and sequence comparison; Richter OM et al.; A quinol oxidase has been purified from the cytoplasmic membrane of Paracoccus denitrificans; its heme composition and CO binding properties identify it as a cytochrome ba3 . On SDS gels, the purified enzyme complex is separated into five polypeptides . Using partial peptide sequence information for subunit II, the gene locus has been cloned and sequenced . In a typical operon pattern, four genes were identified: qoxA, -B, -C, and -D, coding for subunits II, I, III, and IV . DNA-derived amino acid sequence comparisons reveal extensive similarities to other members of the terminal oxidase superfamily. Eur J Biochem, 1994 Sep 15, 224(3), 1011 - 7 Physico-chemical and spectroscopic properties of the monohemic cytochrome C552 from Pseudomonas nautica 617; Saraiva LM et al.; A c-type monohemic ferricytochrome C552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor . The NH2-terminal sequence and the amino acid composition of the cytochrome were determined . The heme iron of the cytochrome C552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6 . The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies . The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments . The EPR spectrum of the oxidised form of the cytochrome C552 is typical of a low-spin ferric heme. Appl Environ Microbiol, 1994 Sep, 60(9), 3336 - 42 Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2; Hayase N et al.; We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230 . E . coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter . Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively . Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested . In these transconjugated hydrogen-utilizing bacteria, P . pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively. Biochim Biophys Acta, 1994 Aug 30, 1187(2), 260 - 3 Bacterial genes and proteins involved in the biogenesis of c-type cytochromes and terminal oxidases; Thony-Meyer L et al.; A total of nine genes potentially concerned with the biosynthesis of c-type cytochromes have been identified recently in the bacteria Bradyrhizobium japonicum and Rhodobacter capsulatus, and homologous counterparts appear to be present also in Escherichia coli . Most of the respective gene products are membrane-bound, while others are located in the periplasmic space . As inferred from sequence analyses, several of these proteins may play roles in membrane transport or redox processes, both functions being consistent with the required steps in cytochrome c formation (membrane translocation of heme; covalent linkage of protoheme IX to cysteine thiols) . Further genes of B . japonicum, E . coli, Bacillus subtilis and Paracoccus denitrificans have been studied whose products are necessary for the formation of intact heme/copper oxidases . Some of them are probably required in protein folding and assembly whereas others appear to be enzymes catalyzing steps in the biosynthesis of the heme cofactors. J Biol Chem, 1994 Aug 19, 269(33), 21037 - 42 Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I); Majander A et al.; Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters . In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of the protein-bound FMN can be visualized . It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region . DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme . Inhibition requires preincubation of enzyme in the presence of NADH and DPI . The lower the NADH/NAD+ ratio or the pH, or the higher the NAD+/DPI ratio, the more DPI is required for inhibition . NAD+ and DPI apparently compete for a common site . Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75% . Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction . DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli. J Biol Chem, 1994 Aug 19, 269(33), 21030 - 6 Properties of the iron-sulfur center in the 25-kilodalton subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans; Crouse BR et al.; The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity . EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single {2Fe-2S} cluster (Yano, T., Sled', V . D., Ohnishi, T., and Yagi, T . (1994) Biochemistry 33, 494-499) . In this report, the electronic, magnetic, and vibrational properties of the {2Fe-2S}2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple {2Fe-2S}-containing proteins . The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the {2Fe-2S} cluster in the P . denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type {2Fe-2S} centers typified by Clostridium pasteurianum 2Fe ferredoxin . The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations. Biochemistry, 1994 Aug 16, 33(32), 9731 - 40 Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans; Haltia T et al.; The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy . Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically . In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III . Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier . Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III . Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity . The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved . The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation . Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein . The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein . The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted. FEBS Lett, 1994 Aug 15, 350(1), 61 - 5 Sulfide dehydrogenase is identical with the SoxB protein of the thiosulfate-oxidizing enzyme system of Paracoccus denitrificans GB17; Schneider A et al.; Thiosulfate induced cells of Paracoccus denitrificans GB17 oxidize thiosulfate and sulfide to sulfate . A mutant carrying a Tn5-mob insertion in the soxB gene is unable to oxidize thiosulfate or sulfide suggesting a linkage of both activities . To test this assumption we have separated the components of the thiosulfate-oxidizing enzyme system of the wild-type by ion exchange chromatography . The SoxB protein coeluted with a highly active sulfide dehydrogenase . Analysis by polyacrylamide gel electrophoresis revealed one major protein of M(r) 32k . Thus, the SoxB protein appeared to be identical with sulfide dehydrogenase. Appl Environ Microbiol, 1994 Aug, 60(8), 2802 - 10 Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats; Fries MR et al.; Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world . Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm . From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained . Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different . Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate . All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch . All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes . All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes . Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe . Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp . Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1994 Aug 1, 121(1), 1 - 9 The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase; van der Oost J et al.; Among aerobic prokaryotes, many different terminal oxidase complexes have been described . Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase . A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase . Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well . Tentative structural, functional and evolutionary implications are discussed. Eur J Clin Microbiol Infect Dis, 1994 Aug, 13(8), 679 - 85 In vitro activity of FK-037, a novel parenteral cephalosporin, against bacterial isolates from neutropenic cancer patients; Dholakia N et al.; The in vitro activity of FK-037, a novel parenteral cephalosporin, was compared to that of ceftazidime, aztreonam and piperacillin (agents often used in empiric regimens in cancer patients) against recent bacterial isolates from patients with cancer . FK-037 was either equal to or 2 to 16-fold more active than the comparative agents against members of the Enterobacteriaceae . It was also active against Acinetobacter spp., Aeromonas spp., Pseudomonas aeruginosa, and other Pseudomonas spp . Xanthomonas maltophilia and Alcaligenes denitrificans were relatively resistant to all four agents . FK-037 was also 4 to 16-fold more active against most gram-positive organisms (including some methicillin-resistant staphylococci) than was ceftazidime . Enterococcus spp., Listeria monocytogenes and Staphylococcus haemolyticus were relatively resistant to FK-037 and ceftazidime . Overall, FK-037 has a broad antimicrobial spectrum that includes the majority of gram-positive and gram-negative isolates. Biochemistry, 1994 Jul 26, 33(29), 8673 - 7 Kinetics and thermodynamics of CO binding to cytochrome P450nor; Shiro Y et al.; The CO-binding reaction of cytochrome P450nor isolated from denitrifying fungus, Fusarium oxysporum, has been studied by using a flash photolysis method in the millisecond time domain . We obtained the CO on- and off-rate constants in the bimolecular reaction, and determined the activation free energy, enthalpy, and entropy from the temperature dependence of these rate constants . To discuss the structural characteristics of P450nor, these parameters were compared with those of other cytochrome P450s, such as cytochrome P450cam from Pseudomonas putida and myoglobin . The on-rate constant (k(on)) for P450nor is larger than those of camphor-bound P450cam {P450cam(+)}, suggesting that ligand entry to the heme pocket of P450nor is sterically less restricted than that of P450cam(+) . In the P450norCO complex, the IR stretching band of the iron-bound CO is observed at 1942 cm-1, which is the same position as in P450cam(+)CO . This result suggests that the heme pocket immediate to the ligand-binding site is the same size in the two enzymes, in good agreement with the observation that the equilibrium constant (K = k(on)/k(off)) is identical in P450nor and P450cam(+) . On the other hand, the entropy changes in the equilibrium and the off-activation processes are smaller in P450nor than in P450cam(+) . This feature could reflect the lack of a bound substrate at the active site of P450nor . These structural characteristics of P450nor are discussed in relation to its unique catalytic property, rapid NO reduction to yield N2O. Can J Microbiol, 1994 Jul, 40(7), 576 - 82 Cellular regulation of nitrate uptake in denitrifying Flexibacter canadensis; Wu Q et al.; Nitrate uptake and its regulation were investigated using an ion-specific nitrate electrode for denitrifying Flexibacter canadensis under anaerobic conditions . Glucose supported a greater rate of nitrate uptake than did glycerol, glutamate, lactose, cellobiose, or ethanol . Nitrate uptake closely approximated Michaelis--Menten kinetics; the estimated Ks(glucose) and apparent Km(nitrate) for nitrate uptake were 21 and 44 microM, respectively . Nitrate disappearance was correlated with nitrite accumulation, and nitrate had an inhibitory effect on nitrite reduction . Oxygen inhibition of nitrate uptake increased as the percent air saturation increased, and reversed readily as the percent air saturation decreased . The minimal air saturation showing inhibition of nitrate uptake was about 2-4% . Azide and cyanide completely inhibited nitrate uptake . No nitrate uptake was observed in cells grown in the presence of 1 or 5 mM tungstate (no added molybdate) . When molybdate (100-200 microM) was present in the medium, nitrate uptake was exhibited by organisms grown with 1 mM, but not with 5 mM, tungstate, indicating that nitrate uptake was dependent on the presence of an active nitrate reductase, and that competition between tungsten and molybdenum occurred during the formation of nitrate reductase . Nitrite production from nitrate by whole cells but not cell-free extracts was inhibited by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, indicating that nitrate and (or) nitrite transport depended upon the electrochemical proton gradient. J Bacteriol, 1994 Jul, 176(13), 4052 - 65 Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants; Chistoserdov AY et al.; The nucleotide sequence of the methylamine utilization (mau) gene region from Methylobacterium extorquens AM1 was determined . Open reading frames for 11 genes (mauFBEDACJGLMN) were found, all transcribed in the same orientation . The mauB, mauA, and mauC genes encode the periplasmic methylamine dehydrogenase (MADH) large and small subunit polypeptides and amicyanin, respectively . The products of mauD, mauG, mauL, and mauM were also predicted to be periplasmic . The products of mauF, mauE, and mauN were predicted to be membrane associated . The mauJ product is the only polypeptide encoded by the mau gene cluster which is predicted to be cytoplasmic . Computer analysis showed that the MauG polypeptide contains two putative heme binding sites and that the MauM and MauN polypeptides have four and two FeS cluster signatures, respectively . Mutants generated by insertions in mauF, mauB, mauE, mauD, mauA, mauG, and mauL were not able to grow on methylamine or any other primary amine as carbon sources, while a mutant generated from an insertion in mauC was not able to utilize methylamine as a source of carbon but utilized C2 to C4 n-alkylamines as carbon sources . Insertion mutations in mauJ, mauM, and mauN did not impair the ability of the mutants to utilize primary n-alkylamines as carbon sources . All mau mutants were able to utilize methylamine as a nitrogen source, implying the existence of an alternative (methyl)amine oxidation system, and a low activity of N-methylglutamate dehydrogenase was detected . The mauD, mauE, and mauF mutants were found to lack the MADH small subunit polypeptide and have a decreased amount of the MADH large subunit polypeptide . In the mauG and mauL mutants, the MADH large and small subunit polypeptides were present at wild-type levels, although the MADHs in these strains were not functional . In addition, MauG has sequence similarity to cytochrome c peroxidase from Pseudomonas sp . The mauA, mauD, and mauE genes from Paracoccus denitrificans and the mauD and mauG genes from Methylophilus methylotrophus W3A1 were able to complement corresponding mutants of M . extorquens AM1, confirming their functional equivalence . Comparison of amino acid sequences of polypeptides encoded by mau genes from M . extorquens AM1, P . denitrificans, and Thiobacillus versutus shows that they have considerable similarity. Mol Microbiol, 1994 Jul, 13(2), 183 - 96 The terminal oxidases of Paracoccus denitrificans; de Gier JW et al.; Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans . Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII . Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase . The genomic locus of a quinol oxidase has been isolated: cyoABC . This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used . In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified . Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps . The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed. J Biochem (Tokyo), 1994 Jul, 116(1), 88 - 94 Nitric oxide reductase cytochrome P-450 gene, CYP 55, of the fungus Fusarium oxysporum containing a potential binding-site for FNR, the transcription factor involved in the regulation of anaerobic growth of Escherichia coli; Tomura D et al.; A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase . The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest . The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced . The P-450nor gene contained 7 introns that consisted of 49 to 55 bp . The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution . Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region . One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria . The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system . Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level . These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite. Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1286 - 91 Structure and ANR-dependent transcription of the nir genes for denitrification from Pseudomonas aeruginosa; Arai H et al.; In the denitrification gene cluster from Pseudomonas aeruginosa, an operon encoding three open reading frames (nirQ, ORF2, ORF3) was upstream of the structural gene for nitrite reductase (nirS) as a divergent transcriptional organization . A nucleotide-binding protein encoded by nirQ was 76% identical to the Pseudomonas stutzeri nirQ gene product, which was shown to be necessary for activating nitrite and nitric oxide reductases . The gene product of ORF2 was homologous to subunit III of cytochrome oxidases . The nirQ gene was transcribed under denitrifying conditions . The intergenic region of nirS and nirQ has only one binding motif for ANR, a regulatory protein for anaerobic gene expression correspond to FNR in E . coli . Complementation analyses showed that the transcription of both nirS and nirQ completely depended on ANR. Int J Syst Bacteriol, 1994 Jul, 44(3), 461 - 73 Phenotypic and genotypic characterization of bradyrhizobia nodulating the leguminous tree Acacia albida; Dupuy N et al.; Rhizobial isolates that were obtained from both surface and deep soil samples in the Sahelian and Sudano-Guinean areas of Senegal (West Africa) under Acacia albida trees were compared with representative strains of known rhizobial species and genera . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was used to determine the taxonomic positions of these organisms and the relationships between isolates obtained from the surface and isolates obtained from deep soil . Most of the isolates belonged to eight electrophoretic clusters containing representative strains of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium sp . Isolates were also characterized by the Biolog system, and the results were compared with the results obtained by SDS-PAGE of total proteins; the level of correlation was very low . DNA-rRNA hybridizations with 16S or 23S rRNA from Bradyrhizobium japonicum LMG 6138T (T = type strain) confirmed that most of the protein electrophoretic clusters belong in the Bradyrhizobium-Rhodopseudomonas rRNA complex . Sequencing of 16S rRNA genes showed that some of the A . albida-nodulating isolates belong to a separate lineage together with representatives of other protein electrophoretic clusters . Other isolates that belong to the same electrophoretic cluster as the type strain of Bradyrhizobium japonicum are considered members of the lineage represented by this type strain . The first lineage is as far removed from Bradyrhizobium japonicum as it is from the genus Afipia, Blastobacter denitrificans, and the genus Rhodopseudomonas . The possible relationship among electrophoretic group, geographic origin, and depth of isolation at a particular site is discussed. FEBS Lett, 1994 Jun 20, 347(1), 22 - 6 Purification, and some molecular and enzymatic features of a novel ccb-type cytochrome c oxidase from a microaerobic denitrifier, Magnetospirillum magnetotacticum; Tamegai H et al.; A novel ccb-type cytochrome c oxidase was purified from the magnetic bacterium, Magnetospirillum magnetotacticum MS-1 . The enzyme was composed of three subunits with M(r)'s of 43,000, 34,000 and 28,000, respectively, and contained 0.91 mol of protoheme, 2.0 mol of heme c and 0.70 g atom of copper per mol of minimal structural unit . One mol of enzyme oxidized 187 mol of horse heart ferrocytochrome c and 34.4 mol of M . magnetotacticum ferrocytochrome c550/s . The cytochrome c oxidase activity of the enzyme was 50% inhibited by 12 microM KCN . The enzyme seems to function as the terminal oxidase in microaerobic respiration. Biochem J, 1994 Jun 15, 300 ( Pt 3), 907 - 14 The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase; Gilmour R et al.; In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans {Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem . J . 294, 745-752}, we have studied the kinetics of oxidation of cytochrome c by this enzyme . The enzyme, as isolated, is in the fully oxidized form and is relatively inactive . Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme . Full activation is achieved by addition of 1 mM CaCl2 . Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem . EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity . We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+ . Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution . Most of the activity lost upon dilution can be recovered after reconcentration . The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations . Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer . We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers . From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium . The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations . The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM . The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 mi