Arch Biochem Biophys, 1992 Feb 1, 292(2), 576 - 82 Regulation of a cytochrome c2 isoform in wild-type and cytochrome c2 mutant strains of Rhodobacter sphaeroides; Rott MA et al.; In Rhodobacter sphaeroides, mutations that suppress the photosynthetic deficiency (spd mutations) of strains lacking cytochrome c2 (cyt c2) cause accumulation of a periplasmic cyt c2 isoform that has been designated isocytochrome c2 (isocyt c2) . In this study, a new method for purification of both cyt c2 and isocyt c2 is described that uses periplasmic fluid as a starting material . In addition, antiserum to isocyt c2 has been used to demonstrate that all suppressor mutants contain an isocyt c2 of approximately 15 kDa . Western blot analysis indicates that isocyt c2 was present at lower levels in both wild-type and cyt c2 mutants than in spd-containing mutants . Although isocyt c2 is detectable under all growth conditions in wild-type cells, the highest level of isocyt c2 is present under aerobic conditions . Our results demonstrate that spd mutations increase the steady state level of isocyt c2 under photosynthetic conditions . Although the physiological function of isocyt c2 in wild-type cells is not known, we show that a nitrate-regulated protein in Rhodobacter sphaeroides f . sp . denitrificans also reacts with the isocyt c2 antiserum. Biochim Biophys Acta, 1992 Jan 22, 1098(3), 343 - 50 Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans; Haltia T; Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al . (1981) Biochim . Biophys . Acta 636, 162-167) . In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein . A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA . The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme . The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane . One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well . Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O. Gene, 1992 Jan 2, 110(1), 105 - 8 Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region; Crouzet J et al.; Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290 . Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid. Appl Environ Microbiol, 1992 Jan, 58(1), 376 - 84 Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria; Smith GB et al.; The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced . In agreement with recent sequence information from another strain of P . stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene . P . stutzeri JM300 nir is 67% homologous to P . aeruginosa nir and 88% homologous to P . stutzeri ZoBell nir . Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P . aeruginosa . The denitrification genes in P . stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads . We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification . Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested . The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments . The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities. APMIS, 1992 Jan, 100(1), 48 - 56 Regulation of aspartate carbamoyltransferase in Neisseria and Branhamella species; Jyssum S; The regulatory characteristics of aspartate carbamoyltransferase (ACTase EC 2.1.3.2) from various species of Neisseria and Branhamella have been compared . Great differences in the regulatory nature of the enzymes were observed . ATP and GTP were positive effectors in Neisseria meningitidis, N . gonorrhoeae and nine other coccal "true neisseriae" species . In four "false neisseriae" species, including Branhamella catarrhalis, no stimulating effect of ATP or GTP was observed . The rod-shaped N . elongata behaved as the "false neisseriae" in these respects, despite its taxonomic affinity to the coccal "true neisseriae" species . Except in N . meningitidis and N . lactamica, CTP had no distinct stimulatory effect . CTP had a strong inhibitory effect on ACTases from N . elongata and the "false neisseriae" species N . caviae and B . catarrhalis . The inhibitory effect of CTP was weak in N . cinerea, N . denitrificans, and the "false neisseriae" species N . ovis and N . cuniculi . Thus, there was no sharp reflection of taxonomy in the regulation of ACTase by CTP in these groups of bacteria . The apparent {S}0.5 values for aspartate and carbamoyl phosphate, displayed for five of the eighteen species, showed great variability with {S}0.5 values for aspartate ranging from 6 to 34, and for carbamoyl phosphate from 2 to 9 . Treatment of the enzyme from the main test microbe N . meningitidis strain M1 by heat or para-chloromercuribenzoate (pCMB) showed that both the catalytic and the regulatory functions decreased in parallel as in the class A enzymes found in species of Pseudomonas . An estimation of the molecular weight (Mr) of the ACTase enzyme from N . meningitidis showed it to be about 295,000, which resembles the class B enzymes found in the Enterobacteriaceae. J Basic Microbiol, 1992, 32(2), 107 - 11 Microbial degradation of shrimp-shell waste; Sabry SA; A total of 40 strains of bacteria were isolated and tested for their potentiality to degrade chitin and utilize shrimp-shell waste for the production of chitinase . The activity ratio, percentage of weight loss and enzyme activity were determined for cultures exhibiting highest chitinolytic activities . The most active organisms were identified as Alcaligenes denitrificans, Bacillus amyloliquefaciens, B . megaterium and B . subtilis . The potentiality of the first two organisms to degrade chitin was reported for the first time. Arch Microbiol, 1992, 157(3), 218 - 22 Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri; Korner H et al.; Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N2O reductase and nitrite reductase (cytochrome cd1) in Pseudomonas stutzeri strain ZoBell . Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes . Immunocytochemical localization of N2O reductase in a mutant strain deficient in the chromophore of N2O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform. Arch Microbiol, 1992, 158(5), 335 - 43 Genes for a second terminal oxidase in Bradyrhizobium japonicum; Bott M et al.; Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa3-type cytochrome c oxidase . The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe . In the course of these studies, another B . japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa3 but also belonged to the superfamily of heme/copper oxidases . Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon . The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c . The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved . A remarkable feature of the DNA sequence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins . A B . japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean . This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid. Arch Microbiol, 1992, 158(1), 48 - 53 A strictly anaerobic nitrate-reducing bacterium growing with resorcinol and other aromatic compounds; Gorny N et al.; With resorcinol as sole source of energy and organic carbon, two stains of gram-negative, nitrate-reducing bacteria were isolated under strictly anaerobic conditions . Strain LuBRes1 was facultatively anaerobic and catalase- and superoxide dismutase-positive . This strain was affiliated with Alcaligenes denitrificans on the basis of substrate utilization spectrum and peritrichous flagellation . Strain LuFRes1 could grow only under anaerobic conditions with oxidized nitrogen compounds as electron acceptor . Cells were catalase-negative but superoxide dismutase-positive . Since this strain was apparently an obligate nitrate reducer, it could not be grouped with any existing genus . Resorcinol was completely oxidized to CO2 by both strains . Neither an enzyme activity reducing or hydrolyzing the resorcinol molecule, nor an acyl-CoA-synthetase activating resorcylic acids or benzoate was detected in cell-free extracts of cells grown with resorcinol . In dense cell suspensions, both strains produced a compound which was identified as 5-oxo-2-hexenoic acid by mass spectrometric analysis . This would indicate a direct, hydrolytic cleavage of the resorcinol nucleus without initial reduction. Int J Syst Bacteriol, 1992 Jan, 42(1), 19 - 26 Identification of xenobiotic-degrading isolates from the beta subclass of the Proteobacteria by a polyphasic approach including 16S rRNA partial sequencing; Busse HJ et al.; Nineteen gram-negative, aerobic, biodegradative isolates were identified by using a polyphasic taxonomic approach . The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) allowed allocation of these organisms to the beta subclass of the Proteobacteria . On the basis of the results of additional characterization experiments (i.e., API 20NE tests, determinations of soluble protein patterns, and DNA-DNA hybridization experiments), we classified six isolates as either Comamonas testosteroni, Comamonas acidovorans, or Alcaligenes xylosoxidans subsp . denitrificans . By using the same criteria we allocated two additional isolates to the genus Alcaligenes . A comparison of a 16S rRNA fragment (positions 1220 to 1377; Escherichia coli nomenclature) indicated that the remaining isolates should be allocated as follows: one is a member of C . testosteroni and one is a member of Acidovorax facilis, as confirmed by the results of additional DNA-DNA hybridizations; two others probably belong to the family Alcaligenaceae; six are related to "Alcaligenes eutrophus"; and one, strain NRRL 12228, occupies an isolated position. Acta Microbiol Pol, 1992, 41(3-4), 203 - 10 Comparison of dentrification by Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa; Blaszczyk M; The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied . These strains represent three different types of denitrification: 1 . reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P . denitrificans); 2 . partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P . aeruginosa) and 3 . two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P . stutzeri) . These observations differ from the results reported in the literature and possible reasons are discussed. J Biol Chem, 1991 Dec 5, 266(34), 22785 - 8 Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide; Braun C et al.; Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen . Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2 . Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance . Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation . NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification) . The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate . A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis . Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1. Biochem Biophys Res Commun, 1991 Nov 27, 181(1), 316 - 22 Molecular cloning of the cytochrome aa3 gene from the archaeon (Archaebacterium) Halobacterium halobium; Denda K et al.; A novel aa3-type cytochrome oxidase from the extremely halophilic archaeon, Halobacterium halobium, differs significantly from those of other prokaryotic and eukaryotic cytochrome oxidases (Fujiwara, T., Fukumori, Y., and Yamanaka, T . (1989) J . Biochem . 105, 287-292) . In the present study, we cloned and sequenced the gene which encodes the cytochrome aa3 by using the polymerase chain reaction methods . The deduced amino acid sequence of subunit I of H . halobium cytochrome aa3 was more similar to that of subunit I of the eukaryotic cytochrome (44%, maize mitochondria) than that of the cytochrome from other bacteria (36%, Paracoccus denitrificans) . The consensus sequence in putative metal binding residues is well-conserved also in H . halobium cytochrome aa3. J Bacteriol, 1991 Nov, 173(22), 7340 - 4 Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha; Mittenhuber G et al.; For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants . For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe . The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19 . An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker . Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants . Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion . To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E . coli . Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19 . A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13 . pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria . Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus . No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined. Biochem Soc Trans, 1991 Nov, 19(4), 976 - 81 Using the bacterium, Paracoccus denitrificans and other 'runaway mitochondria' as classroom models for respiratory electron transport studies; Bolgiano B et al.; Our suggestions for experiments demonstrating electron-transport-chain composition and reactions all exploit bacteria which can be prepared quickly, easily and cheaply from cells grown in Erlenmeyer flasks . While they have been designed from a cytochrome oxidase point of view using organisms of our own prejudice, strains containing mutations in other sites could be just as educational . Most bacteria that can grow aerobically have features in common with the mitochondrial respiratory chain . Because of the vital importance of oxygen utilization throughout most of evolution, and consequent conservation of electron-transport complexes and carriers, the teaching of bioenergetics, whether in the laboratory or lecture room, could benefit from the inclusion of micro-organisms in the curriculum. J Bacteriol, 1991 Nov, 173(21), 6971 - 9 Isolation, sequencing, and mutagenesis of the gene encoding cytochrome c553i of Paracoccus denitrificans and characterization of the mutant strain; Ras J et al.; The periplasmically located cytochrome c553i of Paracoccus denitrificans was purified from cells grown aerobically on choline as the carbon source . The purified protein was digested with trypsin to obtain several protein fragments . The N-terminal regions of these fragments were sequenced . On the basis of one of these sequences, a mix of 17-mer oligonucleotides was synthesized . By using this mix as a probe, the structural gene encoding cytochrome c553i (cycB) was isolated . The nucleotide sequence of this gene was determined from a genomic bank . The N-terminal region of the deduced amino acid sequence showed characteristics of a signal sequence . Based on the deduced amino acid sequence of the mature protein, the calculated molecular weight is 22,427 . The gene encoding cytochrome c553i was mutated by insertion of a kanamycin resistance gene . As a consequence of the mutation, cytochrome c553i was absent from the periplasmic protein fraction . The mutation in cycB resulted in a decreased maximum specific growth rate on methanol, while the molecular growth yield was not affected . Growth on methylamine or succinate was not affected at all . Upstream of cycB the 3' part of an open reading frame (ORF1) was identified . The deduced amino acid sequence of this part of ORF1 showed homology with methanol dehydrogenases from P . denitrificans and Methylobacterium extorquens AM1 . In addition, it showed homology with other quinoproteins like alcohol dehydrogenase from Acetobacter aceti and glucose dehydrogenase from both Acinetobacter calcoaceticus and Escherichia coli . Immediately downstream from cycB, the 5' part of another open reading frame (ORF2) was found . The deduced amino acid sequence of this part of ORF2 showed homology with the moxJ gene products from P . denitrificans and M . extorquens AM1. J Bacteriol, 1991 Nov, 173(21), 6962 - 70 A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i; Van Spanning RJ et al.; A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans . The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase . The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination . Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively . Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations . The wild type and mutant strains were all able to grow on succinate and choline chloride . In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities . cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate . The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities . moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities . The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain . The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes. J Bacteriol, 1991 Nov, 173(21), 6948 - 61 Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth; Van Spanning RJ et al.; By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans . The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one . The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1 . Directly downstream from these three genes, a new mox gene was identified . The gene is designated moxR . By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene . Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro . As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone . Growth on succinate and on methylamine was not affected . In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed . The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity . The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i . Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity . The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed . The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P . denitrificans . In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552 . In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase . It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase. J Bacteriol, 1991 Oct, 173(19), 6300 - 2 Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans; Debussche L et al.; Cob(I)alamin adenosyltransferase (EC 2.5.1.17) was purified to homogeneity from extracts of a Pseudomonas denitrificans recombinant strain and sequenced at its N terminus . It is a homodimer (each unit with an Mr of 28,000) encoded by cobO . The enzyme adenosylated all of the corrinoids isolated from this microorganism but did not adenosylate cobyrinic acid. J Bacteriol, 1991 Oct, 173(19), 6066 - 73 Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase; Cameron B et al.; Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes . Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus . Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P . denitrificans strain in which cobU and cobV were amplified . The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences . Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P . denitrificans . The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole . Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase. J Bacteriol, 1991 Oct, 173(19), 6058 - 65 Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes; Cameron B et al.; A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis . The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT) . Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertion-mediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid. J Bacteriol, 1991 Oct, 173(19), 6046 - 51 Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans; Blanche F et al.; The cobalamin biosynthetic pathway enzyme that catalyzes amidation of 5'-deoxy-5'-adenosyl-cobyrinic acid a,c-diamide was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by a four-column procedure . The purified protein had an isoelectric point of 5.6 and molecular weights of 97,300 as estimated by gel filtration and 57,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer . Stepwise Edman degradation provided the sequence of the first 16 amino acid residues at the N terminus . The enzyme catalyzed the four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of cobyrinic acid triamide, tetraamide, and pentaamide intermediates . The amidations are carried out in a specific order; this order was not determined . The enzyme was specific to coenzyme forms of substrates and did not carry out amidation of the carboxyl group at position f . The amidation reactions were ATP/Mg2+ dependent and exhibited a broad optimum around pH 7.5 . L-Glutamine was shown to be the preferred amide group donor (Km congruent to 45 microM) but could be replaced by ammonia (Km = 20 mM) . For all of the four partially amidated substrates, the Km values were in the micromolar range and the Vmax values were about 7,000 nmol h-1 mg-1. Antonie Van Leeuwenhoek, 1991 Oct-Nov, 60(3-4), 235 - 56 A new thermodynamically based correlation of chemotrophic biomass yields; Heijnen JJ; A new, generally applicable, thermodynamically based method is proposed to provide an estimation of the biomass yield on arbitrary organic and inorganic substrates . Aerobic, anaerobic, denitrifying growth systems with and without reversed electrontransport are covered . The biomass yield can be estimated with only 15% error in a very wide range of microbial growth systems and biomass yields (0.01-0.80 C-mol/(C)-mol) . This method is based on the use of 'Gibbs energy dissipated per C-mol produced biomass' (designated as Ds01/rAx) as the central parameter . Moreover the insufficiency of other methods based on YATP, YAve, eta o, YC and enthalpy or Gibbs energy efficiencies is shortly discussed . Also it appeared to be possible to understand the obtained correlation of Ds01/rAx in general biochemical terms. Appl Environ Microbiol, 1991 Oct, 57(10), 3043 - 5 Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil; Jones AM et al.; The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction . Most-probable-number estimates of naturally occurring F . canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F . canadensis (Is-11) . Our survey suggests that F . canadensis is common in the natural environment. FEMS Microbiol Lett, 1991 Oct 1, 67(2), 145 - 52 Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110; Rossbach S et al.; We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110 . The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B . japonicum strain CC705 . The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein . A B . japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation . Thus, the function of c552 remains unknown. J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2353 - 60 The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans; Long AR et al.; A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans . Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions-that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system . The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein . The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa . The M-protein is active in the 'physiological' assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde . It has its maximum effect when the ratio of M-protein:MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH. J Bacteriol, 1991 Oct, 173(19), 6074 - 87 Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase; Crouzet J et al.; A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced . The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW . Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase . CobN is proposed to play a role in cobalt insertion reactions . Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype. J Bacteriol, 1991 Oct, 173(19), 6052 - 7 A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities; Blanche F et al.; The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans . This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined . Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8 . Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase . However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme . Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction . The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM) . Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide . This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate. Biochemistry, 1991 Sep 24, 30(38), 9201 - 10 Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy; Backes G et al.; The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor . The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins . The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups . The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks . The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone . The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor . The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism . In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site . The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent . These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself. Biochemistry, 1991 Sep 3, 30(35), 8678 - 84 Characterization of the 25-kilodalton subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: sequence similarity to the 24-kilodalton subunit of the flavoprotein fraction of mammalian complex I; Xu XM et al.; The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides {Yagi, T . (1986) Arch . Biochem . Biophys . 250, 302-311} . Structural genes encoding the subunits of this enzyme complex constitute at least one gene cluster {Xu, X., Matsuno-Yagi, A., & Yagi, T . (1991) Biochemistry 30, 6422-6428} . The 25-kDa subunit (NQO2), which has been isolated from sodium dodecyl sulfate-polyacrylamide gels, is a polypeptide of this enzyme complex . The partial N-terminal amino acid sequence and amino acid composition of the NQO2 subunit have been determined . On the basis of the amino acid sequence, the NQO2 gene was found to be located 1.7 kilobase pairs upstream of the gene for NADH-binding subunit (NQO1) . The complete nucleotide sequence of the NQO2 gene was determined . It is composed of 717 base pairs and codes for 239 amino acid residues with a calculated molecular weight of 26,122 . The NQO2 subunit is homologous to the Mr 24,000 subunit of the mammalian mitochondrial NADH-ubiquinone oxidoreductase which bears an electron paramagnetic resonance-visible binuclear iron-sulfur cluster (probably cluster N1b) . Comparison of the predicted amino acid sequence of the Paracoccus NQO2 subunit with those of its mammalian counterparts suggests putative binding sites for the iron-sulfur cluster . In addition, nucleotide sequencing shows the presence of two unidentified reading frames between the NQO1 and NQO2 genes . These are designated URF1 and URF2 and are composed of 261 and 642 base pairs, respectively . The possible function of the protein coded for the URF2 is discussed. J Bacteriol, 1991 Sep, 173(17), 5494 - 501 Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp; Altenschmidt U et al.; The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp . anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources . Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate . ATP is cleaved into AMP and pyrophosphate . The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied . Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1) . It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km . Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids . The first one is identical with benzoate-CoA ligase (E1) . The second enzyme is a 2-aminobenzoate-CoA ligase (E2) . It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates . The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase . The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U . Altenschmidt, C . Eckerskorn, and G . Fuchs, Eur . J . Biochem . 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions. J Reprod Med, 1991 Sep, 36(9), 685 - 7 Isolation of Kingella denitrificans from amniotic fluid in a woman with chorioamnionitis . A case report; Maccato M et al.; Kingella denitrificans and Streptococcus agalactiae were isolated from the amniotic fluid of a woman with chorioamnionitis . She was treated with intravenous ampicillin/sulbactam, with a good response. Zentralbl Hyg Umweltmed, 1991 Sep, 192(2), 134 - 45 Evaluation of an enzymatic method for fast identification of Listeria spp . in cheese and meat products; Comi G et al.; Listeria-Tek ELISA was compared with conventional cultural procedure of FIL/IDF (Federation International de Laiterie/International Dairy Federation) modified by us . The ELISA assay is able to detect Listeria innocua, Listeria monocytogenes, Listeria murrayi and Listeria welshimeri, after 48 h in artificially contaminated cheeses . No specific reactions, no false positive or negative were detected and the assay has a high correlation with the conventional method used in this research . We also analyzed meat products and cheeses and identified Listeria spp . and Listeria monocytogenes serotypes . In 313 food samples examined there were only 3 false positives . With ELISA Kit we obtained 0.9% more than with the cultural method and the percentage of false positives is 1.6% . So in commercial products the total absence of false negatives demonstrates the great sensitivity of the method in respect to investigated microorganisms . As determined by cultures 218 samples contained 47 Listeriae monocytogenes, 67 Listeriae innocua, 21 Listeriae welshimeri, 7 Listeriae ivanovii, 3 Listeriae murrayi and 3 Listeriae denitrificans . Listeria monocytogenens includes serotypes 1 (13) and serotypes 4 (14) . 20 Listeriae monocytogenes were serotyped by polyantisera Difco (Detroit, Michigan). Biochim Biophys Acta, 1991 Aug 23, 1059(2), 233 - 8 Cytochrome c' of Paracoccus denitrificans; Gilmour R et al.; Cytochrome c' was identified in periplasmic extracts of the Paracoccus denitrificans strains LMD 22.21 and LMD 52.44 . The cytochrome c' was purified from the latter using the device of sequential molecular exclusion chromatography in the dimeric and monomeric states . Although showing the overall spectroscopic features of the cytochrome c' family, the Paracoccus cytochrome c' is unusual in having a red-shifted oxidised Soret band at 407 nm . Also unusual is the midpoint potential of 202 mV, well above the known cytochrome c' range . The amino-acid composition of Pa . denitrificans cytochrome c' showed the high alanine and low proline content characteristic of the group and reflecting the predominantly alpha-helical character of the protein . Comparison of the amino-acid compositions suggests some similarity to the cytochromes c' of Chromatium vinosum and halotolerant Paracoccus. FEBS Lett, 1991 Aug 19, 288(1-2), 227 - 8 Nitrite activates the transcription of the Pseudomonas aeruginosa nitrite reductase and cytochrome c-551 operon under anaerobic conditions; Arai H et al.; The transcription of the Pseudomonas aeruginosa denAB operon, which consists of the nitrite reductase and cytochrome c-551 genes, is induced under anaerobic conditions . However, under anaerobic non-denitrifying conditions (anaerobic growth on arginine), the promoter activity of the operon was approximately one-fifth of that under anaerobic denitrifying conditions (anaerobic growth in the presence of nitrite or nitrate) . This result clearly demonstrates that the presence of nitrite or nitrate activates the transcription of P . aeruginosa denAB operon under anaerobic conditions. FEBS Lett, 1991 Aug 5, 287(1-2), 163 - 6 Crystallographic investigations of the tryptophan-derived cofactor in the quinoprotein methylamine dehydrogenase; Chen LY et al.; A model of tryptophan tryptophylquinone (TTQ), recently proposed by McIntire et al . (Science (1991) 252, 817-824) to be the prosthetic group of the quinoprotein methylamine dehydrogenase, has been compared with electron density maps of this dehydrogenase from Thiobacillus versutus and Paracoccus denitrificans . The comparison shows that the TTQ model can be neatly accommodated, providing strong supportive evidence that TTQ is indeed the cofactor for this group of quinoproteins. J Bacteriol, 1991 Aug, 173(15), 4893 - 6 Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium; Robin C et al.; A Bacillus megaterium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity was subcloned and sequenced . The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F . Blanche, L . Debussche, D . Thibaut, J . Crouzet, and B . Cameron, J . Bacteriol . 171:4222-4231, 1989) . The B . megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P . denitrificans SUMT, substrate inhibition at uroporphyrinogen III concentrations above 0.5 microM, suggesting a common regulation for aerobic cobalamin-producing organisms. J Bacteriol, 1991 Aug, 173(15), 4637 - 45 Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii; Blanche F et al.; An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield . The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions . The Km value for uroporphyrinogen III was 52 nM . The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2 . Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F . Blanche, L . Debussche, D . Thibaut, J . Crouzet and B . Cameron, J . Bacteriol . 171:4222-4231, 1989), M . ivanovii SUMT did not show substrate inhibition at uroporphyrinogen III concentrations of up to 20 microM . Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene . The encoded polypeptide showed more than 40% strict homology with P . denitrificans SUMT . The M . ivanovii SUMT structural gene is likely to be, as is P . denitrificans cobA, involved in corrinoid synthesis. Biophys J, 1991 Aug, 60(2), 415 - 23 Comparison of energy-transducing capabilities of the two- and three-subunit cytochromes aa3 from Paracoccus denitrificans and the 13-subunit beef heart enzyme; Hendler RW et al.; In the accompanying paper, we have shown that the two-subunit cytochrome aa3 isolated from Paracoccus denitrificans displays the same kind of complex and interactive redox behavior as the 13-subunit cytochrome aa3 from beef heart . Therefore, the redox characteristics are not dependent on the additional 11 subunits . In the current work, we have examined the energy-transducing capabilities of both the two- and three-subunit enzymes obtained from Paracoccus denitrificans in relation to that of the 13-unit mammalian enzyme . We have found that in all of the tested functions, which included the development of delta psi and delta pH, and the pumping of protons, that the two-subunit enzyme is at least as efficient as the structurally more complex mammalian enzyme . There is thus a correlation between the complex redox behavior and energy transducing capabilities of the two enzymes . There was also no difference in energy-transducing capabilities between the two- and three-subunit forms of the bacterial enzyme . It seems that only 2 subunits are required for an efficient energy-transducing cytochrome aa3 . The most likely role of the additional subunits in the mammalian enzyme, therefore, seems to be in regulation. Biophys J, 1991 Aug, 60(2), 408 - 14 Potentiometric and spectral studies with the two-subunit cytochrome aa3 from Paracoccus denitrificans . Comparison with the 13-subunit beef heart enzyme; Pardhasaradhi K et al.; Previous work from this laboratory has revealed a complex and interactive redox behavior for the active metal centers in beef heart cytochrome aa3 . All of these centers are contained in two of the 13 subunits which make up the enzyme . The isolated cytochrome aa3 of Paracoccus denitrificans contains only two subunits . The purpose of the current investigation was to see if the complex redox behavior is dependent on the presence of the additional 11 peptides that are present in the mammalian enzyme . In this paper we report that the structurally simpler bacterial enzyme displays a redox behavior which is very similar to that seen with the mammalian enzyme . Therefore, the observed redox behavior does not depend on interactions involving the additional peptides. Arch Biochem Biophys, 1991 Aug 1, 288(2), 380 - 5 Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans; Hochstein LI et al.; A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography . The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide . Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present . Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition . The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000) . Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt. Eur J Biochem, 1991 Aug 1, 199(3), 677 - 83 Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro . Correlation with photoinhibition studies; Richardson DJ et al.; 1 . Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm . The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen . The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived . Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex . 2 . Cells of R . capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction . A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant . The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131) . In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine {Ph(NMe2)2} were ineffective as mediators of electrons from isoascorbate . Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide . 3 . Purified ferroycytochrome c2 from R . capsulatus was oxidised by nitrous oxide in the presence of periplasm from R . capsulatus MTG4/S4 . The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates . The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl . 4 . Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase . However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase . The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction . 5 . Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS) FEMS Microbiol Lett, 1991 Jul 15, 66(1), 107 - 11 Metabolism of nitric oxide in denitrifying Pseudomonas aeruginosa and nitrate-respiring Bacillus cereus; Kalkowski I et al.; Suspensions of Pseudomonas aeruginosa and Bacillus cereus were continuously sparged with nitrogen to remove gaseous products of nitrate reduction . Under these conditions, P . aeruginosa denitrified nitrate to 4% NO, 47% N2O and 49% N2 . B . cereus reduced nitrate to 94% nitrite, 2% NO and 5% N2O . B . cereus was unable to reduce NO or N2O as sole electron acceptor, whereas P . aeruginosa reduced NO stoichiometrically to N2O when the N2O reductase was inhibited by acetylene or when the formed N2O was immediately flushed out of the incubation vessel . The mechanism and the reason for NO production in nitrate-respiring B . cereus are unknown, but the amounts of NO released were in a similar range as in the denitrifying P . aeruginosa and thus may be of similar environmental importance. J Biol Chem, 1991 Jul 15, 266(20), 12848 - 51 H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria . Evidence for an electrophilic nitrosyl during reduction of nitric oxide; Ye RW et al.; Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O . All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O . Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O . Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms . These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO . Trapping experiments with 15NO, 14N3-, and crude extracts of R . sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2- . The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A . cycloclastes and P . aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme. Biochemistry, 1991 Jul 2, 30(26), 6422 - 8 The NADH-binding subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans: gene cloning and deduced primary structure; Xu XM et al.; The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide {Yagi, T . (1986) Arch . Biochem . Biophys . 250, 302-311} . The NADH-binding subunit (Mr = 50,000) of this enzyme complex was identified by direct photoaffinity labeling with {32P}NADH {Yagi, T., & Dinh, T.M . (1990) Biochemistry 29, 5515-5520} . Primers were synthesized on the basis of the N-terminal amino acid sequence of this polypeptide, and these primers were used to synthesize an oligonucleotide probe by the polymerase chain reaction . This probe was utilized to isolate the gene encoding the NADH-binding subunit from a genomic library of P . denitrificans . The nucleotide sequence of the gene and the deduced amino acid sequence of the entire NADH-binding subunit were determined . The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191 . The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence . The deduced amino acid sequence of the Paracoccus NADH-binding subunit shows remarkable similarity to the alpha subunit of the NAD-linked hydrogenase of Alcaligenes eutrophus H16 . When partial DNA sequencing of the regions surrounding the gene encoding the NADH-binding subunit was carried out, sequences homologous to the 24-, 49-, and 75-kDa polypeptides of bovine complex I were detected, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex constitute a gene cluster. Biochim Biophys Acta, 1991 Jun 17, 1058(2), 256 - 60 Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans; Matchova I et al.; The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans . The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1) . In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c . The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin . In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate . All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells. J Biol Chem, 1991 Jun 15, 266(17), 11078 - 82 Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction; Shoun H et al.; From conditions for production in Fusarium oxysporum of the unique nitrate/nitrite-inducible cytochrome P-450, tentatively called P-450dNIR, it was expected that the fungus is capable of metabolizing nitrate dissimilatively . Here we report that F . oxysporum exhibits a distinct denitrifying ability which results in the anaerobic evolution of nitrous oxide (N2O) from nitrate or nitrite . Comparison of the cell growth during denitrification indicated that the dissimilatory reduction of nitrate to nitrite is an energetically favorable process in F . oxysporum; however, further reduction of nitrite to N2O might be energy-exhausting and may function as a detoxification mechanism . A potent nitrite reductase activity to form N2O could be reconstituted by combination of the cell-free extract prepared from the denitrifying cells and an NADH-phenadinemethosulfate-dependent reducing system . The activity was strongly inhibited by carbon monoxide, cyanide, oxygen (O2), and the antibody against P-450dNIR . The results, along with those concerning inducing conditions of P-450dNIR, were highly indicative that the cytochrome is involved in the denitrifying nitrite reduction . This work has thus presented not only the first demonstration that a eukaryote exhibits a marked denitrifying ability, but also the first instance of a cytochrome P-450 that is involved in a reducing reaction with a distinct physiological significance against a hydrophilic, inorganic substrate. J Biol Chem, 1991 Jun 15, 266(17), 10899 - 905 Nitric oxide reductase . Purification from Paracoccus denitrificans with use of a single column and some characteristics; Dermastia M et al.; Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column . The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH . These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values . The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method . The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit . The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000) . The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)) . Each subunit therefore contained only one heme group . The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A . The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen . It seemed not to be inactivated by aerobic solutions . These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J . (1990) Biochem . J . 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa . denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G . (1989) J . Bacteriol . 171, 3288-3297) . There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa . denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C . (1989) J . Biol . Chem . 264, 7556-7563). Appl Environ Microbiol, 1991 Jun, 57(6), 1783 - 9 Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp . denitrificans; Ingvorsen K et al.; A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp . denitrificans . Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate . The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media . Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A . xylosoxidans subsp . denitrificans DF3 . Strain DF3 hydrolyzed cyanide with great efficacy . Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h . Enzyme purification established that cyanide hydrolysis by A . xylosoxidans subsp . denitrificans DF3 was due to a single intracellular enzyme . The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation . The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da . The cyanide-hydrolyzing enzyme of A . xylosoxidans subsp . denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles. J Bacteriol, 1991 Jun, 173(11), 3277 - 81 Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f . sp . denitrificans; Yoshida Y et al.; Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f . sp . denitrificans, using immunoblotting analysis and radioactive labeling . A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO . An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with {35S}methionine . These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase . The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space . The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane . Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition . It was also inhibited by the addition of tungstate in the medium. Biochim Biophys Acta, 1991 May 23, 1058(1), 25 - 7 The cytochrome c peroxidase of Paracoccus denitrificans; Pettigrew GW; The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa . However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae . Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face' . Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall . This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles . A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa . denitrificans . Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism. Gene, 1991 May 15, 101(1), 133 - 7 The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species; Cao J et al.; The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans . A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined . The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon . The deduced amino acid (aa) sequence of subunit II of R . sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P . denitrificans oxidase (68% overall) . The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer . Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R . sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P . denitrificans. J Biol Chem, 1991 Apr 25, 266(12), 7676 - 81 Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I; Steinrucke P et al.; As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase . Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene . Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected . We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity . Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases . Wild-type phenotype, however, is only restored with the whole operon . Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors . Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins. Eur J Biochem, 1991 Apr 23, 197(2), 473 - 9 Catalytic properties of phenol carboxylase . In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction; Lack A et al.; Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis) . The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from {14C}phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent . The 14CO2 isotope exchange reaction was studied in vitro . The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+ . The actual substrate was CO2 rather than HCO3- . The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+ . The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+ . The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory . The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate . Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells . The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 262 - 8 Production of polyclonal and monoclonal antibodies for specific detection of nitrosation-proficient denitrifying bacteria in biological fluids; Venezia ND et al.; Polyclonal and monoclonal antibodies were raised against the nitrosating enzyme previously isolated and purified from a denitrifying bacteria Pseudomonas aeruginosa . Using the polyclonal antibodies, a preliminary ELISA test was set up which allowed the detection of the nitrosating enzyme in 2 ml urine samples with a minimal total bacteria count of greater than or equal to 10(5) cells/ml . The use of such a rapid immunological screening test in clinical settings should ascertain whether individual subjects at higher risk for cancer of the stomach or bladder harbour more nitrosation-proficient microorganisms in their microflora . The availability of monoclonal antibodies provides a tool for studying the mechanisms of bacteria-mediated nitrosamine formation from precursor amines. Appl Environ Microbiol, 1991 Apr, 57(4), 1139 - 45 Anaerobic degradation of toluene by a denitrifying bacterium; Evans PJ et al.; A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated . The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1 . Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass . The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification . Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase . The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension . Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation . In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1 . This transformation was dependent on the degradation of toluene. J Bioenerg Biomembr, 1991 Apr, 23(2), 187 - 210 C1 metabolism in Paracoccus denitrificans: genetics of Paracoccus denitrificans; Harms N et al.; Paracoccus denitrificans is able to grow on the C1 compounds methanol and methylamine . These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide . Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway . The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively . Both enzymes contain two different subunits in an alpha 2 beta 2 configuration . The genes encoding the subunits of methanol dehydrogenase (moxF and moxI) have been isolated and sequenced . They are located in one operon together with two other genes (moxJ and moxG) in the gene order moxFJGI . The function of the moxJ gene product is not yet known . MoxG codes for a cytochrome c551i, which functions as the electron acceptor of methanol dehydrogenase . Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor . These so-called quinoproteins are able to catalyze redox reactions by one-electron steps . The reaction mechanism of this oxidation will be described . Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochrome c . P . denitrificans is able to synthesize at least 10 different c-type cytochromes . Five could be detected in the periplasm and five have been found in the cytoplasmic membrane . The membrane-bound cytochrome c1 and cytochrome c552 and the periplasmic-located cytochrome c550 are present under all tested growth conditions . The cytochromes c551i and c553i, present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline . The other c-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions . An overview including the induction pattern of all P . denitrificans c-type cytochromes will be given . The genes encoding cytochrome c1, cytochrome c550, cytochrome c551i, and cytochrome c553i have been isolated and sequenced . By using site-directed mutagenesis these genes were mutated in the genome . The mutants thus obtained were used to study electron transport during growth on C1 compounds . This electron transport has also been studied by determining electron transfer rates in in vitro experiments . The exact pathways, however, are not yet fully understood . Electrons from methanol dehydrogenase are donated to cytochrome c551i . Further electron transport is either via cytochrome c550 or cytochrome c553i to cytochrome aa3 . However, direct electron transport from cytochrome c551i to the terminal oxidase might be possible as well.(ABSTRACT TRUNCATED AT 400 WORDS) J Bioenerg Biomembr, 1991 Apr, 23(2), 163 - 85 Metabolic regulation including anaerobic metabolism in Paracoccus denitrificans; Stouthamer AH; Under anaerobic circumstances in the presence of nitrate Paracoccus denitrificans is able to denitrify . The properties of the reductases involved in nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are described . For that purpose not only the properties of the enzymes of P . denitrificans are considered but also those from Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas stutzeri . Nitrate reductase consists of three subunits: the alpha subunit contains the molybdenum cofactor, the beta subunit contains the iron sulfur clusters, and the gamma subunit is a special cytochrome b . Nitrate is reduced at the cytoplasmic side of the membrane and evidence for the presence of a nitrate-nitrite antiporter is presented . Electron flow is from ubiquinol via the specific cytochrome b to the nitrate reductase . Nitrite reductase (which is identical to cytochrome cd1) and nitrous oxide reductase are periplasmic proteins . Nitric oxide reductase is a membrane-bound enzyme . The bc1 complex is involved in electron flow to these reductases and the whole reaction takes place at the periplasmic side of the membrane . It is now firmly established that NO is an obligatory intermediate between nitrite and nitrous oxide . Nitrous oxide reductase is a multi-copper protein . A large number of genes is involved in the acquisition of molybdenum and copper, the formation of the molybdenum cofactor, and the insertion of the metals . It is estimated that at least 40 genes are involved in the process of denitrification . The control of the expression of these genes in P . denitrificans is totally unknown . As an example of such complex regulatory systems the function of the fnr, narX, and narL gene products in the expression of nitrate reductase in E . coli is described . The control of the effects of oxygen on the reduction of nitrate, nitrite, and nitrous oxide are discussed . Oxygen inhibits reduction of nitrate by prevention of nitrate uptake in the cell . In the case of nitrite and nitrous oxide a competition between reductases and oxidases for a limited supply of electrons from primary dehydrogenases seems to play an important role . Under some circumstances NO formed from nitrite may inhibit oxidases, resulting in a redistribution of electron flow from oxygen to nitrite . P . denitrificans contains three main oxidases: cytochrome aa3, cytochrome o, and cytochrome co . Cytochrome o is proton translocating and receives its electrons from ubiquinol . Some properties of cytochrome co, which receives its electrons from cytochrome c, are reported.(ABSTRACT TRUNCATED AT 400 WORDS) Arch Biochem Biophys, 1991 Apr, 286(1), 159 - 63 Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa; Hernandez D et al.; Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification) . Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake . In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated . Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force . Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport . Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction . Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport . Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide. J Bacteriol, 1991 Apr, 173(7), 2238 - 43 Increase in spermine content coordinated with siderophore production in Paracoccus denitrificans; Bergeron RJ et al.; Spermine is present in relatively low amounts in Paracoccus denitrificans cultured aerobically in an ammonium succinate minimal salts medium supplemented with 50 microM iron(III) . However, in iron-deprived cultures {minimal salts medium containing 0.5 microM iron(III)}, spermine content increases by an order of magnitude in coordination with the well-known responses to iron derivation, e.g., derepression of siderophore synthesis and siderophore excretion . When iron-deprived cultures exhibiting both high spermine content and strong siderophore production are reseeded into fresh minimal salts medium containing 50 microM iron{III}, both siderophore production and spermine content fall rapidly . Five hours after iron supplementation, spermine is below limits of detection . These results suggest a specific role for spermine in the response of P . denitrificans to low-iron stress. J Bioenerg Biomembr, 1991 Apr, 23(2), 321 - 34 Evolutionary aspects of cytochrome c oxidase; Kadenbach B et al.; The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme . The number of these additional subunits increases with increasing evolutionary stage of the organism . Subunits I-III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mitochondrial DNA . The additional subunits are nuclear encoded . Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits . Dissociation of some of the nuclear-coded subunits (e.g . VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme . Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochrome c oxidation by the reconstituted enzyme from bovine heart but not from P . denitrificans . The regulatory function attributed to the nuclear-coded subunits of mammalian cytochrome c oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP . These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII . The results presented here are taken to indicate a regulation of cytochrome c oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes. J Bioenerg Biomembr, 1991 Apr, 23(2), 303 - 19 The reactions of the oxidase and reductases of Paracoccus denitrificans with cytochromes c; Smith L et al.; Electron transport in the Paracoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochrome c552 than with either soluble Paracoccus c550 or bovine cytochrome c; a pool function for cytochrome c is not necessary . Low concentrations of Paracoccus or bovine cytochrome c stimulate the oxidase activity . This observation could explain the multiphasic Scatchard plots which are obtained . A negatively charged area on the "back side" of Paracoccus c which is not present in mitochondrial c could be a control mechanism for Paracoccus reactions . Paracoccus oxidase and reductase reactions with bovine c show the same properties as mammalian systems; and this is true of Paracoccus oxidase reactions with its own soluble cytochrome c if added polycation masks the negatively charged area . Evidence for different oxidase and reductase reaction sites on cytochrome c include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies to Paracoccus cytochrome c; and (3) reaction of another bacterial cytochrome c with Paracoccus reductases but not oxidase . Rapid electron transport occurs in cytochrome c-less mutants of Paracoccus, suggesting that the reactions result from collision of diffusing complexes. J Bioenerg Biomembr, 1991 Apr, 23(2), 291 - 302 Cytochrome c oxidase metal centers: location and function; Muller M et al.; Cytochrome c oxidase of Paracoccus denitrificans is spectroscopically and functionally very similar to the mammalian enzyme . However, it has a very much simpler quaternary structure, consisting of only three subunits instead of the 13 of the bovine enzyme . The known primary structure of the Paracoccus denitrificans subunits, the knowledge of a large number of sequences from other species, and data on the controlled proteolytic digestion of the enzyme allow structural restrictions to be placed on the models describing the binding of the active metal centers to the polypeptide structure. J Bioenerg Biomembr, 1991 Apr, 23(2), 269 - 89 Cytochrome c oxidase in Paracoccus denitrificans . Protein, chemical, structural, and evolutionary aspects; Buse G et al.; Preparations and protein chemical characterizations performed with cytochrome c oxidase (E.C . 1.9.3.1) from the purple bacterium Paracoccus denitrificans are reviewed . The simplest catalytically competent complex of the enzyme consists of two subunits of 62012 and 2799 Da . The theoretical heme a/protein ratio of the purified enzyme is 22.0 nmol/mg . The amino acid sequences of both proteins are compared with examples of subunits I and II of mitochondrial terminal oxidases from the main kingdoms of eukaryotes . The significance of the emerging conserved features such as membrane penetration patterns, invariant residues, stoichiometry, and sites of prosthetic groups are discussed . The Paracoccus enzyme represents the only prokaryotic oxidase detailed so far, which is directly related to the mitochondrial oxidases by common ancestry in the growing O2 atmosphere. J Bioenerg Biomembr, 1991 Apr, 23(2), 257 - 67 Genes coding for cytochrome c oxidase in Paracoccus denitrificans; van der Oost J et al.; Several loci on the Paracoccus denitrificans chromosome are involved in the synthesis of cytochrome c oxidase . So far three genetic loci have been isolated . One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors . In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochrome c550 gene . An alignment of six promoter regions reveals only short common sequences. J Bioenerg Biomembr, 1991 Apr, 23(2), 241 - 55 The three-subunit cytochrome bc1 complex of Paracoccus denitrificans . Its physiological function, structure, and mechanism of electron transfer and energy transduction; Trumpower BL; The cytochrome bc1 complex purified from P . denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromes b, c1, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria . However, the bacterial bc1 complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary polypeptides, which have no obvious electron-transfer function . The P . denitrificans complex is a paradigm for the bc1 complexes of all gram-negative bacteria . In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, the P . denitrificans bc1 complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer. J Bioenerg Biomembr, 1991 Apr, 23(2), 227 - 39 The cytochrome c reductase/oxidase respiratory pathway of Paracoccus denitrificans: genetic and functional studies; Steinrucke P et al.; Data are presented on three components of the quinol oxidation branch of the Paracoccus respiratory chain: cytochrome c reductase, cytochrome c552, and the a-type terminal oxidase . Deletion mutants in the bc1 and the aa3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments . In addition, evidence for a role of cytochrome c552 in electron transport between complex III and IV is presented. Biochem Biophys Res Commun, 1991 Mar 29, 175(3), 901 - 5 Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide; Goretski J et al.; Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide . This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P . stutzeri and A . cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O . The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO . Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide . With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO. FEBS Lett, 1991 Mar 25, 280(2), 351 - 3 Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa; Arai H et al.; The nitrite reductase gene (denA) and the cytochrome c-551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome . We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions . The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E . coli. J Mol Biol, 1991 Mar 20, 218(2), 427 - 47 X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa; Nar H et al.; The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods . The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer . The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L . In each crystal form, there are four molecules in the asymmetric unit . They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L . The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L . The crystal structures reported here represent the first crystallographically refined structures for azurin from P . aeruginosa . The structure is very similar to that of azurin from Alcaligenes denitrificans . The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion . The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer. Biochem Pharmacol, 1991 Mar 1, 41(5), 677 - 84 Inhibitory effects of two structurally related carbocyanine laser dyes on the activity of bovine heart mitochondrial and Paracoccus denitrificans NADH-ubiquinone reductase . Evidence for a rotenone-type mechanism; Anderson WM et al.; Two cationic, lipophilic laser dyes, 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (HIDC) and 1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide (HITC), inhibit bovine heart mitochondrial and Paracoccus denitrificans NADH oxidase activities . The mitochondrial I50 values were 0.5 microM (HIDC) and 1.2 microM (HITC), and the P . denitrificans I50 values 1.2 microM (HIDC) and 1.5 microM (HITC) . Neither succinate nor cytochrome oxidase (EC 1.9.3.1) activities were inhibited significantly by either compound, localizing the site of inhibition to the segment of each electron transport chain between NADH and ubiquinone . With submitochrondrial particles (SMP), NADH-dependent reduction of menadione, duroquinone and coenzyme Q1 was inhibited markedly (HIDC was the more potent inhibitor) . Using purified complex I, only NADH-dependent reduction of duroquinone and coenzyme Q1 was inhibited markedly (HIDC was the more potent inhibitor) and reduction of menadione was inhibited slightly . With P . denitrificans membrane vesicles, NADH-dependent reduction of menadione, juglone, and coenzyme Q1 was inhibited slightly and duroquinone reduction was inhibited markedly . Membrane-dependent interactions appear to be involved, since the compounds were more inhibitory with membrane preparations than with complex I . The mechanism of inhibition (except for the HIDC effect on coenzyme Q1 reduction with P . denitrificans) appeared to be through the interaction of dye with the rotenone site on NADH-ubiquinone reductase (EC 1.6.99.3), since rotenone-insensitive preparations of complex I and P . denitrificans membrane vesicles were also insensitive to HIDC and HITC inhibition. FEBS Lett, 1991 Feb 25, 279(2), 205 - 9 The nirSTBM region coding for cytochrome cd1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins; Jungst A et al.; Genes for respiratory nitrite reduction (denitrification) of Pseudomonas stutzeri are clustered within 7 kbp . A 4.6-kbp Hind III-Kpn I fragment carrying nirS, the structural gene for cytochrome cd1, was sequenced . An open reading frame immediately downstream of nirS codes for a 22.8-kDa protein with four heme c-binding motifs . Mutagenesis of this gene causes an apparent defect in electron donation to cytochrome cd1 . Following this ORF are the structural genes for cytochrome c552, cytochrome c551, and ORF5 that codes for a 11.9-kDa monoheme protein . All cytochromes have a signal sequence for protein export. Biochemistry, 1991 Feb 19, 30(7), 1924 - 8 Inhibition by cyclopropylamine of the quinoprotein methylamine dehydrogenase is mechanism-based and causes covalent cross-linking of alpha and beta subunits; Davidson VL et al.; Cyclopropylamine acted as a mechanism-based inhibitor of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans . The protein-bound quinone cofactor of this enzyme was rapidly reduced by addition of a stoichiometric amount of cyclopropylamine, but this compound did not serve as a substrate for the enzyme in the steady-state kinetic assay . Time-dependent inactivation of the enzyme by cyclopropylamine was observed only in the presence of a reoxidant . Saturation behavior was observed, and values of KI of 3.9 microM and K(inact) of 1.7 min-1 were determined . Enzyme inactivation was irreversible, as no restoration of activity was evident after gel filtration of methylamine dehydrogenase which had been incubated with cyclopropylamine in the presence of a reoxidant . The inactivated enzyme exhibited an altered absorption spectrum . Electrophoretic analysis of inactivated methylamine dehydrogenase indicated that covalent cross-linking of the alpha and beta subunits of this alpha 2 beta 2 oligomeric enzyme had occurred and that the quinone cofactor had been modified . A mechanism for this inhibition is proposed which is based upon the data presented and is consistent with the available structural information on methylamine dehydrogenase. Biotechnol Appl Biochem, 1991 Feb, 13(1), 112 - 9 Molecular cloning of the L-phenylalanine transaminase gene from Paracoccus denitrificans in Escherichia coli K-12; Takagi T et al.; The L-phenylalanine transaminase gene of Paracoccus denitrificans was cloned by a shotgun method using the Escherichia coli K-12 mutant DG30, which lacks three distinct transaminase genes . Plasmid pPAP142 was constructed by inserting a 2.2-kb fragment carrying the transaminase gene into pUC18 . Strain E . coli K-12 HB101 cells harboring the plasmid produced 20-fold to 30-fold more transaminase than wild type P . denitrificans cells . The nucleotide sequence of the 2.2-kb fragment was determined, revealing that the deduced amino acid sequence of the transaminase of P . denitrificans is similar to that of other transaminases. Appl Environ Microbiol, 1991 Feb, 57(2), 450 - 4 Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures; Evans PJ et al.; Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen . All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation . The depletion of o-xylene was correlated to and dependent on the metabolism of toluene . No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures . However, m-xylene was degraded by a subculture that was incubated on m-xylene alone . Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene . These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass . Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification . These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results. Mol Gen Genet, 1991 Feb, 225(2), 241 - 8 Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification; Jungst A et al.; The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone . A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS . Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS . Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments . Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked . The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb . Several genes for copper processing are located within the intervening region. J Bacteriol, 1991 Feb, 173(3), 1298 - 301 p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol; Hopper DJ et al.; A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp . The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified . The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome . It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500 . The midpoint redox potential of the cytochrome was 232 mV . The Km and kcat for p-cresol were 21 microM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used in the assays, was determined to be 1.7 mM . These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp. Eur J Biochem, 1991 Jan 30, 195(2), 517 - 25 The Bacillus subtilis cytochrome-c oxidase . Variations on a conserved protein theme; Saraste M et al.; The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced . Five genes, ctaB-F, are closely spaced . ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others . In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus . The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases . Subunit IVB, a small hydrophobic protein, is a novel subunit . These predictions suggest that the B . subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme . Cytochrome aa3, which was previously isolated from B . subtilis {de Vrij, W., Azzi, A . & Konings, W . N . (1983) Eur . J . Biochem . 131, 97-103} is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium. Biochim Biophys Acta, 1991 Jan 22, 1056(2), 133 - 8 Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli; Bonner FT et al.; The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon {HN2O3-} over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d . The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate . The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation . The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate . The kinetics show a biphasic dependence of initial rate upon nitrite concentration . The rate data at low {NO2-} are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM . The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems. Biochem J, 1991 Jan 15, 273(Pt 2), 423 - 7 Nitric and nitrous oxide reductases are active under aerobic conditions in cells of Thiosphaera pantotropha; Bell LC et al.; Use of Clark-type electrodes has shown that, in cells of Thiosphaera pantotropha, the nitrous oxide reductase is active in the presence of O2, and that the two gases involved (N2O, O2) are reduced simultaneously, but with mutual inhibition . Reduction of nitrate, or nitrite, to N2O under aerobic conditions involves NO as an intermediate, as judged by trapping experiments with the ferric form of extracellular horse heart cytochrome c and the demonstration that the cells possess a nitric oxide reductase activity . The overall conversion of nitrate to N2, the process of denitrification, under aerobic conditions, is thus not prevented by reaction of NO with O2 and depends upon a nitrous oxide reductase system which differs from that in other organisms by being neither directly inhibited nor inactivated by O2. J Biol Chem, 1991 Jan 5, 266(1), 45 - 51