Biochim Biophys Acta, 1982 Sep 15, 681(3), 459 - 68 Discrimination of ascorbate-dependent nonenzymatic and enzymatic, membrane-bound reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus; Zumft WG et al.; The marine nitrite-respiring (denitrifying) bacterium, Pseudomonas perfectomarinus, catalyzes by a membrane-bound enzyme the reduction of nitric oxide to nitrous oxide with ascorbic-reduced phenazine methosulfate as electron donor . The entire nitric oxide-reducing capability of a cell-free system was membrane bound and this process was studied with respect to pH and substrate dependency . The enzymatic process was perturbed by an identical nonenzymatic reduction by iron(II) ascorbate in neutral to alkaline aqueous solution . 2 mol nitric oxide and 1 mol ascorbate were consumed per mol nitrous oxide formed . Enzymatic and nonenzymatic processes were discriminated by their differential behavior towards pH and metal-chelating agents . The pH optimum for the enzymatic and nonenzymatic reaction was 5.2 and greater than 7.0, respectively . EDTA (10 mM) inhibited the nonenzymatic reduction completely without interfering with the membrane-bound activity . The nonenzymatic system mimics the reaction of nitric oxide reductase and could serve as a model to study the formation of the N-N bond in denitrification . Enzymatic generation of nitric oxide by cytochrome cd and subsequent nonenzymatic reduction to nitrous oxide simulate an overall quasi-enzymatic nitrous oxide formation by cytochrome cd . The nonenzymatic reduction of nitric oxide might have occurred in previous work due to the ubiquitous use of ascorbate in studies on nitrite respiration and the likelihood of adventitious iron in biological samples. Farmaco {Sci}, 1982 Jul, 37(7), 463 - 74 The reductive metabolism of the nitroaromatic flukicidal agent nitroxinil by liver microsomal cytochrome P-450; Maffei Facino R et al.; Hepatic biotransformation of the flukicidal agent nitroxynil (3-iodo-4-hydroxy-5-nitrobenzonitrile) (I) has been studied with rat liver subcellular fractions as the source of enzymes: two metabolites, 3-iodo-4--hydroxy-5-aminobenzonitrile (II) and 3-iodo-4-hydroxy-5-nitrobenzamide (III) have been identified by standard analytical techniques (TLC, GLC and MS) . The nitroaromatic reduction product (II) is formed in the hepatocyte in a process in which cytosol and endoplasmic reticulum enzymes cooperate . This formation is maximal in anaerobic conditions, but takes also place aerobically and in the absence of electrogenic cofactors . Cytochrome P-450 plays a major role in the denitrification process, and consequently could be the cellular site most exposed to damage by the intermediate arylhydroxylamine formed by reduction. J Bacteriol, 1982 Jul, 151(1), 162 - 71 Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa; Carlson CA et al.; Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa . The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography . The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography . Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels . The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000 . The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight . Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5 . Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen . Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM . Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM) . Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation . The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization. Eur J Biochem, 1982 Jun 15, 125(1), 189 - 95 Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides; Gennis RB et al.; When grown aerobically in the dark, Rhodopseudomonas sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed . The aa3-type cytochrome c oxidase from Rps . sphaeroides has been purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c . The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000 . The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using either yeast or horse-heart cytochrome c . The Rps . sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae . A particularly close relationship is indicated in the case of P . denitrificans . The Rps . sphaeroides oxidase has been incorporated into phospholipid vesicles . The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5 . Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed . The data suggest that the Rps . sphaeroides oxidase does not function as a proton pump. J Biol Chem, 1982 May 25, 257(10), 5576 - 8 Solubilized cytochrome c oxidase from Paracoccus denitrificans is a monomer; Ludwig B et al.; Cytochrome c oxidase purified from the bacterium Paracoccus denitrificans was analyzed by analytical ultracentrifugation . In the detergent octyltetra/pentaoxyethylene (C8E45), the isolated enzyme exhibits a molecular weight of 79,000 to 84,000 . The detergent-solubilized enzyme is thus a monomer which contains one copy of each of the two subunits. Nucleic Acids Res, 1982 May 11, 10(9), 2963 - 70 The 5S ribosomal RNAs of Paracoccus denitrificans and Prochloron; MacKay RM et al.; The nucleotide sequences of the 5S rRNAs of Paracoccus denitrificans and Prochloron sp . are (formula: see text), respectively . Specific phylogenetic relationships of P . denitrificans with purple non-sulphur bacteria, and of Prochloron with cyanobacteria are demonstrated, and unique features of potential secondary structure are described. J Biol Chem, 1982 May 10, 257(9), 4705 - 8 Positional isotopic equivalence of nitrogen in N2O produced by the denitrifying bacterium Pseudomonas stutzeri . Indirect evidence for a nitroxyl pathway; Garber EA et al.; During the concomitant reduction of {15N}nitrite and 14NO, the denitrifying bacterium Pseudomonas stutzeri produced considerable amounts of isotopically mixed N2O (14,15N2O) but did not isotopically intermix the nitrite and NO pools (Garber, E . A . E., and Hollocher, T . C . (1981) J . Biol . Chem . 256, 5459-5465) . It was determined that the mass spectrometric abundance of 14N15NO was about equal to that of 15N14NO in 14,15N2O by examination of the abundances of 14NO+, 15NO+, and 15N2+ that arose from the fragmentation of N2O+ species in the mass spectrometer . This positional isotopic equivalence requires that N2O arose from {15N}nitrite and 14NO by a process in which loss of oxygen occurred with equal probability from 15N and 14N precursors and suggests that at some point the precursors were identical if monomeric or effectively symmetrical if dimeric . One pathway, which is consistent with the available data and for which there is chemical precedence, is the reduction of nitrite and NO to nitroxyl (NOH or HNO), dimerization of free nitroxyl to a dinitrogen intermediate of short half-life, and dehydration of this intermediate to form N2O. Ann Microbiol (Paris), 1982 May-Jun, 133(3), 471 - 88 {Numerical taxonomy of a thermophilic "Bacillus" species isolated from West African rice soils (author's transl)}; Garcia JL et al.; Fifty-seven strains of endospore-forming thermophilic bacteria, 37 of which were capable of denitrification, were isolated from rice soils of West Africa . They were compared with 17 strains of similar bacteria from culture collections, utilizing a total of 123 morphological, physiological and biochemical characteristics . A numerical analysis was performed using the complete linkage-clustering method and the Khi2 test . Seventy-five percent (55 strains) could be included in 12 groups at a taxonomic distance of 0.015 . Wild strains of denitrifiers issued in phenons 8 to 12 and strains of phenon 4 (not denitrifying) were related to the named strains of phenons 1 and 7 (Bacillus stearothermophilus) . Twenty-two wild strains, and 5 strains from culture collections, were only thermotolerating without growth at 65 degrees C . The strains of phenon 3 were related to the 3 named strains of B . coagulans . Phenons 5 and 6 were composed of strains related to B . circulans . The strains of phenon 2 denitrified and showed a swollen central endospore; they were closely related to B . brevis . The denitrifying thermophilic strains isolated from rice soils (phenons 8 to 12) were related to the first group (B . kaustophilus) of Walker and Wolf but their base compositions of DNA were significantly different from those found for the reference strains. Antonie Van Leeuwenhoek, 1982 May, 48(2), 131 - 43 Oxygen tolerance of strictly aerobic hydrogen-oxidizing bacteria; Wilde E et al.; Growth of various bacteria, especially aerobic hydrogen-oxidizing bacteria, in the presence of 2 to 100% (v/v) oxygen in the gas atmosphere was evaluated . The bacterial strains included Alcaligenes eutrophus, A . paradoxus, Aquaspirillum autotrophicum, Arthrobacter spec . strain 11 X, Escherichia coli, Arthrobacter globiformis, Nocardia opaca, N . autotrophica, Paracoccus denitrificans, Pseudomonas facilis, P . putida, and Xanthobacter autotrophicus . Under heterotrophic conditions with fructose or gluconate as substrates neither colony formation on solid medium nor the growth rates in liquid media were drastically impaired by up to 100% oxygen . In contrast, autotrophic growth--with hydrogen, carbon dioxide and up to 80% oxygen in the gas atmosphere--was strongly depressed by high oxygen concentrations . However, only the growth rate, not the viability of the cells, was decreased . Growth retardation was accompanied by a decrease of hydrogenase activity. J Bacteriol, 1982 Apr, 150(1), 100 - 4 Fixation of dinitrogen derived from denitrification of nitrate in a photosynthetic bacterium, Rhodopseudomonas sphaeroides forma sp . denitrificans; Dunstan RH et al.; Studies with 15N demonstrated that the phototrophic bacterium Rhodopseudomonas sphaeroides forma sp . denitrificans strain IL106 cannot assimilate NO-3 but rather denitrifies it to N2 . This strain also fixed N2 into cell protein, although nitrogenase activity was partially inhibited in the presence of NO-3 . Strain IL106 did not assimilate NO-3, but growing cultures and washed cell suspensions incorporated the tracer from 15NO-3 via denitrification to 15N2 and then via nitrogenase into cell nitrogen . This incorporation was inhibited in cells supplied with NH4+ or in the absence of light, thus confirming the participation of nitrogenase in the assimilation of nitrogen from nitrate . This represents a novel type of N2 recycling in a photodiazotrophic denitrifying bacterium. J Gen Microbiol, 1982 Apr, 128 (Pt 4), 731 - 45 Numerical classification of some Rhodococci, Corynebacteria and related organisms; Goodfellow M et al.; Nineteen strains of Corynebacterium sensu stricto, 23 received as Corynebacterium equi or Rhodococcus equi, marker cultures of Arthrobacter, Brevibacterium, Bacterionema matruchotii, Cellulomonas flavigena, Kurthia zopfii, Listeria denitrificans, Microbacterium lacticum, Rhodococcus rubropertinctus and 88 representatives of Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon were the subject of numerical phenetic analyses using 92 characters . The data were examined using the simple matching (SSM) and Jaccard (SJ) coefficients and clustering was achieved using the average linkage algorithm . With a single exception, strains containing meso-diaminopimelic acid, arabinose, galactose and mycolic acids were recovered in five aggregate clusters corresponding to Corynebacterium sensu stricto, Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon . Most of the Corynebacterium (Rhodococcus) equi strains formed a good taxospecies which included the type strain of Corynebacterium hoagii . The numerical data, and the results of earlier chemical and genetical studies, also provide sufficient evidence for the transfer of Bacterionema matruchotii to Corynebacterium sensu stricto as Corynebacterium matruchotii comb.nov . and for the recognition of Rhodococcus globerulus sp.nov . for some strains previously classified as Rhodococcus rubropertinctus (Hefferan) Goodfellow & Alderson . The classification of the remaining marker strains correlates well with other major developments in coryneform taxonomy. Biochim Biophys Acta, 1982 Mar 4, 701(3), 305 - 17 Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote, Paracoccus denitrificans; Vignais PM et al.; 1 . A protein with cyanide-sensitive superoxide dismutase activity was isolated from the prokaryote Paracoccus denitrificans . 2 . This enzyme, present in low amount in the cell, represented not more than 10% of the total cellular superoxide dismutase activity . It was obtained in a form which was 20-40-times less active than the main superoxide dismutase of P . denitrificans which is a manganese-containing enzyme . 3 . It was a soluble monomeric enzyme, highly negatively charged (pI = 4.8), with an apparent molecular weight of 33,000 . 4 . Cyanide sensitivity was observed by NMR assay, enzyme assay and by staining the protein for superoxide dismutase activity on polyacrylamide electrophoretogram . KCN was shown to be a competitive inhibitor of this dismutase, with an inhibitor constant of 0.15 mM . 5 . From the amino acid analysis, S delta Q values lower than 100 were obtained with copper-containing proteins such as the subunit II of cytochrome oxidase from P . denitrificans (69), the azurin from P . denitrificans (77), the bacteriocuprein from Photobacter leiognathi (71); with iron and manganese superoxide dismutases (40-88), and with some eukaryotic copper/zinc dismutases of fish origin (55-82). J Biol Chem, 1982 Feb 25, 257(4), 1579 - 82 The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system; Solioz M et al.; The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6 . With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium . When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed . This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin . Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c . We thus conclude that cytochrome c oxidase of P . denitrificans is a proton pump . Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers. Folia Microbiol (Praha), 1982, 27(6), 460 - 4 Tetraphenylborate-sensitive electrode for measuring membrane potential; Karlovsky P et al.; The paper describes the construction of a new type of ion-selective electrode sensitive to tetraphenylborate (TPB-) and its electric characteristics . The electrode responds to increasing concentrations of the TPB- anion in accordance with the Nernst equation and can be used down to 0.1 microM concentration . The applicability of the electrode for measuring the membrane potential (positive inside) was proved in inside-out oriented membrane vesicles derived from Paracoccus denitrificans . The calculated values were 175 +/- 12 mV with NADH and 180 +/- 6 mV with succinate. Antonie Van Leeuwenhoek, 1982, 48(6), 531 - 44 The pathway of nitrogen and reductive enzymes of denitrification; Hollocher TC; Some recent studies on the pathway of nitrogen and the reductases of denitrification are reviewed . The available evidence suggests that while the intermediates of denitrification can remain enzyme-bound (presumably to nitrite reductase) prior to formation of N2O, NO and nitroxyl (HNO) can be released in part by certain bacteria . Release of NO is recognized by a nitrite/NO-15N exchange reaction and isotopic scrambling in product N2O; release of nitroxyl by Pseudomonas stutzeri is recognized by isotopic scrambling of nitrite and NO in product N2O in absence of exchange and affords evidence that the first N-N bond forms in denitrification at the N1+ redox level . The recent purification and partial characterization of nitrous oxide reductase are described . The ability of the dissimilatory nitrite reductase to activate nitrite for nitrosyl transfer affords a new chemical probe into the mechanism of action of this central enzyme . It would appear that reduction of nitrite is subject to electrophilic catalysis . 18O studies show that dissociation of nitrite from nitrite reductase can be slow relative to competing reduction or nitrosyl transfer. Antonie Van Leeuwenhoek, 1982, 48(6), 585 - 607 Evolution of bacterial denitrification and denitrifier diversity; Betlach MR; Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms . Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria . Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true . Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group . However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification . Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms . Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety . Both types are distinct from other nitrate reductases . Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny . Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms . However, denitrification is restricted to genera within the purple photosynthetic group . Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes . Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today . Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time. Antonie Van Leeuwenhoek, 1982, 48(6), 569 - 83 Denitrification: ecological niches, competition and survival; Tiedje JM et al.; Organisms with the denitrification capacity are widely distributed and in high density in nature . It is not well understood why they are so successful . A survey of denitrifying enzyme content of various habitats is presented which indicates a role of carbon and oxygen, but not nitrate, in affecting denitrifier populations . It is suggested that organic carbon is more important than oxygen status in determining denitrifying enzyme content of habitats . In low oxygen environments, denitrifiers compete with organisms that dissimilate nitrate to ammonium, a process which conserves nitrogen . The energetic and kinetic parameters that affect this competition are evaluated . The latter is examined using Michaelis-Menten theoretical models by varying Vmax, Km, and So (substrate concentration) for the two competing populations . The outcome predicted by these models is presented and discussed in relation to previous data on population densities and Km values for representatives of these competing groups . These models suggest the conditions required to achieve changes in partitioning between the two fates of nitrate . These considerations are important if one is to be able to evaluate and successfully "manage" the fate of nitrate in any habitat. Antonie Van Leeuwenhoek, 1982, 48(6), 545 - 53 The bioenergetics of denitrification; Stouthamer AH et al.; In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b . Electron transport to nitrite and nitrous oxide involves c-type cytochromes . During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide . The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account . For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane . Evidence for two transport systems for nitrate was obtained . One is driven by the proton motive force; this system is used to initiate nitrate reduction . The second system is a nitrate-nitrite antiport system . A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented . The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o . These findings are in accordance with growth yield studies . YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport. Antonie Van Leeuwenhoek, 1982, 48(6), 555 - 67 The physiological genetics of denitrifying bacteria; Carlson CA; The genetics of denitrification is a relatively unexplored area that has great promise . Species of Pseudomonas are probably best suited for study because they are widely found among natural denitrifying populations and are quite readily amenable to genetic analysis . The techniques for mutagenesis and for the exchange of chromosomal genes to characterize mutant strains have been well-developed in P . aeruginosa and are being developed in P . stutzeri . Mutants defective in the denitrification of nitrate, nitrite, and nitrous oxide are now available and will aid in describing the catalytic and regulatory elements of the denitrification pathway. Folia Microbiol (Praha), 1982, 27(1), 11 - 8 Changes in the cytochrome c content during the aerobic adaptation of Paracoccus denitrificans; Stros M et al.; Concentration of cytochrome c decreases when shaking anaerobically grown cells in a non-growth medium down to 50% of the original amount in the cell, depending on the degree of aeration . Only 10-20% of this amount can be found in the adaptation medium . The main portion of cytochrome c is degraded during the adaptation . Inhibitors of proteinases do not influence the degradation . Addition of mammalian cytochrome c fully prevents the degradation of bacterial cytochrome c but not its release from cells . Potassium hexacyanoferrate(III) exhibits a similar effect as oxygen . The degradation system is probably localized in the periplasmic space of the cells. Biochemistry, 1981 Dec 22, 20(26), 7528 - 31 Resonance Raman spectra of cytochromes c and b in Paracoccus denitrificans membranes: evidence for heme--heme interactions; Adar F et al.; Resonance Raman (RR) scattering from cytochromes b and c in the bacterium Paracoccus denitrificans was recorded by exciting with the 568.2-, 530.9-, and 520.8-nm lines of a Kr+ laser . The main features of the spectra were similar to those of the analogous cytochrome b-c1 complex derived from pigeon breast mitochondria . Differences in the 1300-cm-1 region were interpreted in terms of marker bands for heme type and spectral coupling of the hemes on the membranes . It is difficult to explain the results without invoking sharing of electronic and vibrational wave functions among the hemes . This conclusion documents the potential to study the physics of electron transport in functioning membrane by monitoring the RR spectra. Biochim Biophys Acta, 1981 Dec 14, 638(2), 181 - 91 Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans; Boogerd FC et al.; (1)H+ leads to/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO2- or N2O . Under optimal H+-translocation conditions, the ratios leads to H+/O, H+ leads to/N2O, H+ leads to/NO2- for reduction to N2 and H+ leads to/NO2- for reduction to N2O were 6.0-6.3, 4.02, 5.79 and 3.37, respectively . (2) With ascorbate/N,N,N,'N'-tetramethyl-p-phenylene-diamine as exogenous substrate, addition of NO2- or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium . H+/N2O, H+/NO2- for reduction to N2 and H+/NO2- for reduction to N2O were -0.84, -2.33 and -1.90, respectively . (3) The H+/oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycin/K+ and the triphenylmethylphosphonium cation . (4) A simplified scheme of electron transport to O2, NO2- and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase . Electrons destined for NO2-, N2O or O2 pass two H+-trans-locating sites . The H+ leads to/electron acceptor ratios predicted by this scheme are in good agreement with the experimental values. Arch Microbiol, 1981 Dec, 130(4), 307 - 11 Removal of Paracoccus denitrificans outer membrane material by sodium chloride; Hindahl MS et al.; The effects of water washing and NaCl treatment on the cell surface of P . denitrificans were studied . Both treatments caused a release of material from cells . Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane . This treatment also increased the susceptibility of the organism to lysozyme . Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface . Both treatments were shown to alter the appearance of the cell surface . The disruptive effects of these procedures were found to be dependent upon the age of the culture. J Gen Microbiol, 1981 Dec, 127 (Pt 2), 261 - 8 The gradostat: a bidirectional compound chemostat and its application in microbiological research; Lovitt RW et al.; A multistage continuous culture system is described in which solutes are transferred between vessels in opposite directions simultaneously . The system, called a gradostat, produces opposing solute gradients and is a good laboratory model of many natural microbial ecosystems in which solute gradients are important . Theoretical predictions concerning solute transfer were confirmed under steady-state and non steady-state conditions, using a coloured dye . Paracoccus denitrificans grew anaerobically in the gradostat at the intersection between opposing gradients of succinate and nitrate . Opposing gradients of glucose and oxygen separated the growth of a Bacillus sp . (a facultative anaerobe) and Clostridium butyricum (an obligate anaerobe) . Viable counts for both species fell exponentially away from their growth positions at the ends of the gradostat . The potential value of the gradostat and possible alternative conformations are discussed. Antimicrob Agents Chemother, 1981 Dec, 20(6), 814 - 20 Antibiotic action of pyocyanin; Baron SS et al.; Biologically produced pyocyanin was purified, and the nature of its antibacterial action was determined for several bacteria . The pigment was shown to be bactericidal for all susceptible organisms . The bactericidal effect was dependent upon pyocyanin concentration and resulted in decreases in viability ranging from 1 to 8 log viable cells ml-1 . The gram-positive bacteria were more susceptible as a group to the antibiotic action than were the gram-negative bacteria . All apyocyanogenic pseudomonads tested were totally resistant to the pigment, suggesting that resistance may be a characteristic of the genus . Pseudomonas aeruginosa, the producer organism, was also essentially unaffected by high concentrations of pyocyanin . Facultative anaerobes were twofold or more times resistant to the action of the pigment under fermentative conditions; however, the antibiotic action did not require oxygen since denitrifying bacteria were more susceptible during anaerobic respiration than during aerobic respiration. Biochim Biophys Acta, 1981 Oct 12, 637(3), 504 - 11 Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations; Joshi S et al.; Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity . Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient sub-mitochondrial particles . This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations . The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor . Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F1 (0.3--0.6 nmol/mg) . Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3 . Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle. J Biol Chem, 1981 Oct 10, 256(19), 10092 - 8 Reaction of oxygen with cytochrome c oxidase from Paracoccus denitrificans; Ludwig B et al.; The reaction of reduced cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans (American Type Culture Collection 13543) with dioxygen has been followed by laser flash photolysis of the CO derivative . In detergent-stabilized solutions the reaction showed at least two distinct kinetic components, the faster of which was oxygen concentration dependent and had a rate of approximately 60 X 10(6) M-1 s-1 . The slower reaction was independent of oxygen concentration and had a rate of 9 X 10(2) s-1 . These rates are about 1.5 times greater than comparable rates for ox heart oxidase reported by C . Greenwood and Q . H . Gibson (J . Biol . Chem . (1967) 242, 1782-1787) . The kinetic components have markedly different optical spectra which agree precisely in form with those for ox heart enzyme (Greenwood, C., and Gibson, Q . H . (1967) J . Biol . Chem . 242, 1782-1787) but are shifted by 2 nm toward the red . In phospholipid vesicles, the spectral contribution of the faster component was augmented . The dissociation constant for CO at 20 degrees C is 1.6 microM, 6 times greater than for the ox heart enzyme . The bacterial enzyme binds one CO per 2 heme a . The enzyme has an absorption band at 830 nm in the oxidized form similar to that of the ox heart enzyme. Can J Microbiol, 1981 Sep, 27(9), 878 - 85 Denitrification and dissimilatory nitrate reduction to ammonium in digested sludge; Kaspar HF et al.; Acetylene inhibition and 13N methods were used to assay digested sludge for its potential to denitrify and to reduce nitrate to ammonium . At nitrate concentrations below 10 microM, the reduction of N2O to N2 was not inhibited by acetylene concentrations as high as 80 kPa, though at higher nitrate concentration acetylene was an effective inhibitor . NO, N2O, and N2 were produced immediately after addition of nitrate or nitrite, indicating that denitrifying enzymes were present . NO was maintained at a higher concentration of 2--5 nM, while nitrate or nitrite were being reduced, but this gas was depleted once the ionic N oxide substrates were exhausted . Acetylene had little effect on appearance and disappearance of NO . It was also noted that NO was readily consumed by chemical reactions in the anaerobic sludge . Added N2O was reduced without a lag, but pasteurized samples did not consume N2O although they produced it . Fresh digested sludge reduced 60--70% of the added 13NO3- to 13NH4+ with the rest of the NO3- -N presumably lost to denitrification . This agrees well with the nitrate partitioning observed by the acetylene inhibition method in which 30--40% of the NO3- -N was recovered as N2O . Denitrification capacity persisted in both digested sludge and a methanogenic enrichment culture which had been grown in a chemostat for 2.5 years with acetate and ammonium as the sole carbon and nitrogen source . This suggests that denitrifiers with capacities for alternative anaerobic energy metabolism may be more common than now known. Biochim Biophys Acta, 1981 Aug 24, 665(2), 270 - 82 Studies on cyclopropane fatty acid synthesis . Correlation between the state of reduction of respiratory components and the accumulation of methylene hexadecanoic acid by Pseudomonas denitrificans; Jacques NA; A delay in the onset of accumulation of methylene hexadecanoic acid could be engendered in Pseudomonas denitrificans growing under limited oxygen conditions when the concentration of citrate but not the concentration of succinate in the medium was increased from 0.1 to 0.5% . Ascorbate, which specifically reduced a cytochrome component possessing a maximum absorbance at 551 nm, partially inhibited the accumulation of methylene hexadecanoic acid under conditions which otherwise led to maximal production . Limiting terminal cytochrome oxidase activity by controlling the oxygen supply, or by the use of low concentrations of the oxidase inhibitors cyanide or azide also prevented the accumulation of the fatty acid regardless of the nature or concentration of carbon source in the medium . Measurement of the levels of ATP, NAD and NADH as well as the steady state of reduction of respiratory components in vivo showed that the onset of accumulation of methylene hexadecanoic acid could be specifically correlated with the state of reduction of respiratory components . The uniqueness of succinate respiration in promoting the synthesis of cyclopropane synthetase (unsaturated-phospholipid methyltransferase (EC 2.1.1.16) under limited oxygen conditions could therefore be assigned to the high degree of oxidation of respiratory components observed under this condition. Biochim Biophys Acta, 1981 Jul, 636(2), 162 - 7 Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans; Seelig A et al.; The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms . However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II) . The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P . denitrificans cells that had been grown in a Mn(II)-depleted medium . This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart . Manganese is apparently not an essential component of P . denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c . However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase . In contrast, manganese added to the yeast oxidase or to the manganese-free P . denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme . This suggests that the manganese normally associated with P . denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme. Biochim Biophys Acta, 1981 May 13, 635(3), 525 - 34 Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans; van Verseveld HW et al.; The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method . leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants . It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P . denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e- . The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P . denitrificans. Can J Microbiol, 1981 May, 27(5), 553 - 7 Ampicillin resistance in Neisseria denitrificans: studies on ampicillin-sensitive enzymes; MacKenzie CR et al.; The beta-lactam sensitivities of enzymes involved in peptidoglycan synthesis were examined in two strains of Neisseria denitrificans having widely disparate degrees of ampicillin sensitivity . One strain of n . denitrificans was 400 times more resistant to ampicillin than the other; the former is known to have altered penicillin-binding proteins . No differences in the levels of sensitivities of total peptidoglycan synthesis as measured by the incorporation of glutamate into peptidoglycan, or of D-alanine carboxypeptidase, were observed between the two strains . However, the rate of glutamate incorporation into peptidoglycan by logarithmic growth phase cells was somewhat less for the ampicillin-resistant cells than for the parent cells. Biochem J, 1981 Apr 15, 196(1), 311 - 21 Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration; McCarthy JE et al.; 1 . Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium . Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation . Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described . 2 . If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P . dentrificans respiring aerobically in 100 mM-Hepes {4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH . This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+ . 3 . Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media . The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration . These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force . 4 . No 204Tl+ uptake into respiring cells was detected . This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed . The absence of respiration-driven 204Tl+ uptake indicates that P . denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli. Appl Environ Microbiol, 1981 Mar, 41(3), 705 - 9 Dissimilatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene; Kaspar HF et al.; 15N tracer methods and gas chromatography coupled to an electron capture detector were used to investigate dissimilatory reduction of nitrate and nitrite by the rumen microbiota of a fistulated cow . Ammonium was the only 15N-labeled end product of quantitative significance . Only traces of nitrous oxide were detected as a product of nitrate reduction; but in experiments with nitrite, up to 0.3% of the added nitrogen accumulated as nitrous oxide, but it was not further reduced . Furthermore, when 13NO3- was incubated with rumen microbiota virtually no {13N}N2 was produced . Acetylene partially inhibited the reduction of nitrite to ammonium as well as the formation of nitrous oxide . It is suggested that in the rumen ecosystem nitrous oxide is a byproduct of dissimilatory nitrite reduction to ammonium rather than a product of denitrification and that the latter process is absent from the rumen habitat. Biochim Biophys Acta, 1981 Feb 13, 657(2), 411 - 24 A manganese-containing superoxide dismutase from Paracoccus denitrificans; Terech A et al.; A cyanide-insensitive superoxide dismutase (superoxide: superoxide dismutase EC 1.15.1.1) has been isolated from Paracoccus denitrificans, purified to homogeneity and characterized . It is a soluble, manganese-containing protein with an apparent molecular weight of 41 500 +/- 1000 . It is composed of two identical subunits (Mr 23 500) not bound by disulfide linkage . It's isoelectric point is 4.5 . The amino acid composition shows strong similarities with other dimeric procaryotic and with tetrameric mitochondrial Mn-superoxide dismutases . The fully active enzyme contained from 1.34 to 2 gatom Mn/mol enzyme. Biochim Biophys Acta, 1981 Feb 12, 634(2), 279 - 88 Midpoint potentials of cytochromes in vesicles of anaerobically-grown Paracoccus denitrificans determined by the indirect coulometric titration method; Kula T et al.; 1 . Multiplicity of redox components with spectral properties similar to b-type cytochromes was established in vesicles derived fro anaerobically-grown Paracoccus denitrificans . 2 . Multiplicity of c-type cytochromes was not apparent either from low temperature spectroscopy or potentiometric titrations . 3 . Cytochromes a + a3 and a component, only observable at liquid nitrogen temperature, with a spectral maximum at 582.5 nm were detected . 4 . Redox cycling of electron transport components using the indirect coulometric titration method was a convenient means of pairing redox potentials and was reproducible in total absorbance changes, midpoint potentials and spectral maxima. J Biol Chem, 1981 Jan 10, 256(1), 278 - 84 Energy coupling to K+ transport in Paracoccus denitrificans; Erecinska M et al.; Paracoccus denitrificans requires potassium for normal growth and transports this cation by at least two systems, one with low (Km approximately 1 to 2 mM) and another with high (Km approximately 0.1 microM) affinity . Neither of the two systems seems to be dependent on periplasmic components since each retains full activity in cells subjected to an osmotic shock . P . denitrificans accumulates potassium at high velocity (270 nmol of K+/min/mg dry weight of cells) and against a large concentration gradient . The intracellular concentration of K+ in media of high osmolarity (about 320 mosmol) is 0.4 M; this gives a concentration gradient {K+}i/{K+}e of greater than or equal to 2 X 10(4) . The uptake of potassium against its concentration gradient requires a source of energy and is eliminated by the addition of uncouplers . The increased rate of energy usage for potassium transport results in an increased rate of ATP synthesis by the respiratory chain and is expressed in enhanced rates of respiration and substrate utilization . The stimulation of respiration is accompanied by increased steady state reduction level of the components of the respiratory chain . The calculations show that two K+ are most likely to be transported per one ATP hydrolyzed. Folia Microbiol (Praha), 1981, 26(1), 29 - 36 Thiobacilli and sulphate production from inorganic sulphur compounds in upper horizons of spruce forest soils; Lettl A et al.; The species representation of Thiobacilli was investigated in horizons F, H and A of spruce forest at ten localities . Concentrations of Thiobacilli in the selected localities and ability of the soils to oxidize sulphur compounds to sulphate were determined . Horizons F exhibited a high oxidative activity, a lower activity was found in horizon H and the lowest one was detected in horizon A . The activities showed spring and autumn maxima . Horizons F, H and A contained 10(4)--10(5), 10(2)--10(3) and 10(1)--10(3), respectively, Thiobacilli in 1 g dry soil . Thiobacillus thioparus was detected in all three horizons from all localities, T.thiooxidans was found in all horizons F, only in some horizons H and was not detected in horizons A . T.novellus was found only in some samples without any relation to the horizons, T.denitrificans was not detected at all. Antonie Van Leeuwenhoek, 1981, 47(3), 231 - 43 Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans; Timer-ten Hoor A; From cell yields of Thiomicrospira denitrificans grown inthe chemostat at different growth rates under anaerobic conditions a value of 1.4 mM S2O3 = per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 dry wt per mole S2O3 = for the true growth yield . Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans . Though in Thiobacillus denitrificans at D = 0.03 h(-1) under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans . Under aerobic conditions at D = 0.03 h(-1) values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate . As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate . Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans . This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase. Ann N Y Acad Sci, 1981, 361, 330 - 40 The establishment of mitochondria: Paracoccus and Rhodopseudomonas; Whatley FR; Many aerobic bacteria (both facultative and obligate) possess a number of those biochemical features of mitochondria which are concerned with energy metabolism . However, only restricted number, notably Paracoccus denitrificans and Rhodopseudomonas spheroides, have the majority of these features . The theory of endosymbiosis proposes that a primitive eukaryote took up bacteria to yield mitochondria . The present-day Paracoccus then resembles the ancestral bacterium in many respects the primitive amoeba, Pelomyxa palustris, which lacks mitochondria but contains a permanent population of unique symbiotic bacteria, has many of the characteristics of a present-day transitional form . The evolution of mitochondria from endosymbiotic bacteria would involve their integration with the host cell both biochemically and structurally: a number of the intermediate steps are discussed . Attention is drawn to the existence in some ciliates of hydrogenosomes, which function as anaerobic mitochondria. Biochim Biophys Acta, 1980 Dec 3, 593(2), 224 - 9 A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans; Katre NV et al.; The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N3CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membrane of Paracoccus denitrificans and Tetrahymena pyriformis . The N3CCP uncouples respiration in P . denitrificans and T . pyriformis cells with U1/2 values of 1.05 microM and 0.24 microM, respectively . Binding studies show the presence of 0.65 +/- 0.05 high affinity sites per cytochrome alpha with Kd of 0.5 +/- 0.1 microM in P . denitrificans membranes and 1.4 +/- 0.2 sites per cytochrome alpha 2 with a Kd of 0.4 +/- 0.1 microM in T . pyriformis membranes . Irradiation of {3H}-N3CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10--15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis . It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V . and Wilson, D.F . (1978) Arch . Biochem . Biophys . 191, 647--656). Biochim Biophys Acta, 1980 Dec 3, 593(2), 173 - 86 A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy; Albracht SP et al.; A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart . All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane . Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems. J Bacteriol, 1980 Dec, 144(3), 975 - 82 Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite; Rake JB et al.; The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions . Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P . denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling . Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption . Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed . The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays . The H+/O ratio of P . aeruginosa increased when growth conditions were changed from aerobic to denitrifying . This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P . aeruginosa. Arch Microbiol, 1980 Nov, 128(1), 19 - 25 Substrate level versus oxidative phosphorylation in the generation of ATP in Thiobacillus denitrificans; Aminuddin M; Particulate fractions of Thiobacillus denitrificans catalyse that the phosphorylation of ADP to ATP during the oxidation of various inorganic sulphur compounds or NADH via an electron transport chain . On the other hand, a "soluble" cell-free fraction synthesized ATP from APS and inorganic phosphate . The production of ATP was verified either by the firefly luciferin-luciferase enzyme system or by the incorporation of 32Pi into ATP . During the oxidation of sulphide, sulphite and NADH the production of ATP from ADP by particulate fractions is inhibited by compounds that inhibit electron transfer and by uncouplers of oxidative phosphorylation . However, these compounds had little effect on the production of ATP from AMP during the oxidation of sulphite by the soluble fraction . NADH was the most effective electron donor for oxidative phosphorylation . The soluble fraction contained high activities of ATP sulphurylase, inorganic pyrophosphatase and adenylate kinase but ADP sulphurylase activity was relatively low . The effects of inhibitors on ATP production from APS and Pi are compared with those on adenylate kinase and ATP sulphurylase. Mikrobiologiia, 1980 Nov-Dec, 49(6), 990 - 4 {Change in the microbiol complexes of soddy podzol soil under the influence of long-term spring wheat monoculture}; Berestetskii OA et al.; The special composition and the properties of microorganisms predominant in the soil and in the rhizosphere of summer wheat were studied in the conditions of monoculture and corp rotation . Noticeable differences were found in the composition of microbial complexes: microorganisms belonging to the genus Arthrobacter prevailed in the conditions of corp rotation, whereas cultures of the genera Pseudomonas and Bacillus predominated in the conditions of monoculture . Many of the latter possessed weak catalase activity and drastically decreased the oxidation-reduction potential of the medium . Microbiol complexes in soddy-podzolic soil, when summer wheat was grown as a monoculture for a long period of time, were characterized by the following properties: most species were incapable of utilizing mineral nitrogen; the activity of proteolytic enzymes was low; the denitrifying activity was high. Biochem J, 1980 Oct 15, 192(1), 231 - 40 The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans; Alefounder PR et al.; 1 . A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity . Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome . The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts . In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate . It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously . 2 . Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate . These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells . The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase. J Gen Microbiol, 1980 Oct, 120(Pt 2), 439 - 46 A study of the Va-1 group of pseudomonads and its relationship to Pseudomonas pickettii; Pickett MJ et al.; The Va-1 group of denitrifying pseudomonads was characterized and compared with Pseudomonas pickettii (Va-2) . They share many features but differ in their production of acid from cellobiose, lactose and maltose and in their denitrification (gas from nitrate) at 35 degrees C . DNA-DNA hybridizations between a strain of Va-1 and the type strain of P . pickettii disclosed 84% homology and hence indicated that Va-1 is a biovar of this species . Since both are potential human pathogens, features are presented that distinguish these and other phenotypically similar species that are recovered from clinical specimens. Can J Biochem, 1980 Oct, 58(10), 996 - 1003 The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans; Pik JR et al.; There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria . It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity . We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans . The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived . Melittin-induced enhancement of TMPD:ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of nonpermeant substrate to the interior of the vesicle . The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control . Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography . These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell. Biochim Biophys Acta, 1980 Sep 8, 619(3), 453 - 70 Studies on cyclopropane fatty acid synthesis . Effect of carbon source and oxygen tension on cyclopropane fatty acid synthetase activity in Pseudomonas denitrificans; Jacques NA et al.; The cyclopropane fatty acid, methylene hexadecanoic acid, constituted from 1% to upwards of 30% of the total lipid fatty acids of the bacterium, Pseudomonas denitrificans . The amount of this component varied along with the levels of the enzyme, cyclopropane synthetase (unsaturated-phospholipid methyltransferase, EC 2.1.1.16) . When P . denitrificans was grown on succinate in a culture medium saturated with oxygen, cyclopropane synthetase remained repressed while cell densities were low . As cell densities increased, the enzyme was induced and the activity rose to a maximum over a period of 4-6 h . Cyclopropane synthetase could also be induced by rapidly limiting the oxygen supply to cells growing in conditions where oxygen was in excess . This phenomenon was independent of the phase of growth and could be prevented by addition of chloramphenicol to the medium . Growth on glucose was also shown to repress the synthesis of cyclopropane synthetase under similar conditions . However, once maximum levels of cyclopropane synthetase were reached, they remained constant for at least the following 15 h irrespective of the source of carbon in the medium . Methylene hexadecanoic acid accumulated in a linear manner throughout this period until a maximum level was achieved, the rate of accumulation being related to the activity of cyclopropane synthetase detected in vitro . The rate of conversion of total fatty acid to methylene hexadecanoic acid was approximately 1.3-1.5% per h, the methylene hexadecanoic acid being metabolically stable - the relative percentage of methylene hexadecanoic acid to total fatty acid in repressed cells, falling linearly with increase in cell number . Repression of enzyme synthesis was further investigated by growing cells on various sources of carbon other than glucose . The results indicated that succinate was unique amongst tricarboxylic acid cycle intermediates in depressing cyclopropane synthetase under limited oxygen conditions. Antimicrob Agents Chemother, 1980 Aug, 18(2), 249 - 56 Mode of action of copper complexes of some 2,2'-bipyridyl analogs on Paracoccus denitrificans; Smit H et al.; Copper complexes of 2,2'-bipyridyl and related compounds and CuSO4 inhibited the growth of paracoccus denitrificans . The copper(I) complex of 2,9-dimethyl-1,10-phenanthroline {Cu(DMP)2NO3} showed the highest activity, whereas the copper(II) complex of 1,10-phenanthroline and CuSO4 inhibited the growth to a lesser extent . The uncomplexed ligands (1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline) showed little activity, but in the presence of noninhibitory amounts of CuSO4 this activity increased markedly . Copper ions therefore proved to be essential for the growth-inhibitor effect . The extent of inhibition appeared to be strongly dependent on the initial cell density and on the growth medium . No selective inhibition of deoxyribonucleic acid, ribonucleic acid, or protein synthesis was observed with Cu(DMP)2NO3 . Respiratory electron transport of P . denitrificans appeared to be strongly inhibited by Cu(DMP)2NO3 and to a somewhat lesser extent by CuSO4 . Both aerobic and anaerobic respirations were inhibited to the same extent, and from the cytochrome redox kinetics it is concluded that the site of this inhibition in the respiratory electron transport chain must be located before cytochrome b . Cu(DMP)2NO3 did not significantly influence the H+/O ratio with whole cells of P . denitrificans, suggesting that the efficiency of oxidative phosphorylation is not affected by CU(DMP)2NO3 . Growing cultures of P . denitrificans showed a decrease in intracellular potassium ion content in the presence of increasing amounts of Cu(DMP)2NO3 . It is concluded that interference with the cytoplasmic membrane, resulting in inhibition of respiratory electron transport, probably constitutes the main mode of action of copper complexes of 2,2'-bipyridyl analogs on P . denitrificans. Ann Microbiol (Paris), 1980 Jul-Aug, 131B(1), 69 - 80 {Quantitative study of biological denitrification in soil with the aid of acetylene . I.--Application on different soils (author's transl)}; Germon JC; The use of acetylene for investigation about biological denitrification in soils requires study of intensity and persistence of N2O-reductase activity inhibition by this gas and the effects on the other stages of denitrification and on the general metabolism of microflora . The methodology used is described, taking into account N2O solubility . N2O-evolution rates from nitrite and nitrate were measured during long-term incubations under acetylene, and complete inhibition of N2O-reductase was observed . After 2 weeks, acetylene inhibition appears decreased . The nitrate reduction rate seems increased in the presence of acetylene. Appl Environ Microbiol, 1980 Jul, 40(1), 108 - 13 Microbial activity of trench leachates from shallow-land, low-level radioactive waste disposal sites; Francis AJ et al.; Trench leachate samples collected anoxically from shallow-land, low-level radioactive waste disposal sites were analyzed for total aerobic and anaerobic populations, sulfate reducers, denitrifiers, and methanogens . Among the several aerobic and anaerobic bacteria isolated, only Bacillus sp., Pseudomonas sp., Citrobacter sp., and Clostridium sp . were identified . Mixed bacterial cultures isolated from the trench leachates were able to grow anaerobically in trench leachates, which indicates that the radionuclides and organic chemicals present were not toxic to these bacteria . Changes in concentrations of several of the organic constituents of the waste leachate samples were observed due to anaerobic microbial activity . Growth of a mixed culture of trench-water bacteria in media containing a mixture of radionuclides, 60Co, 85Sr, and 134,137Cs, was not affected at total activity concentrations of 2.6 X 10(2) and 2.7 X 10(3) pCi/ml. Ann Microbiol (Paris), 1980 Jul-Aug, 131B(1), 81 - 90 {Quantitative study of biological denitrification in soils with the aid of acetylene . II.--Evolution of inhibitory effect of acetylene on N2O-reductase; influence of acetylene on denitrification rate and on nitrate immobilisation (author's transl)}; Germon JC; Study of kinetics of nitrate or nitrite disappearance in soils incubating with atmosphere with or without acetylene, shows that the presence of this gas increases the rate of nitrate or nitrite reduction, and therefore very probably the denitrification process . The decrease of inhibitory effect on N2O-reductase after incubation during 15 days appears to be due to a great extent at a biological transformation of this gas with a consecutive increase of CO2 production . On the other hand, acetylene does not seem to affect perceptibly nitrate immobilisation. J Inorg Biochem, 1980 Jul, 12(4), 343 - 51 Reduction of DL-selenocystine and isolation of L-seleoncysteine; Burnell JN et al.; Cystine, selenocytsine, and several analogs were reduced by dithiothreitol (DTT), beta-mercaptoethanol (ME) and sodium borohydride (NaBH4) . DTT was the most effective; DTT to cystine ratios from 10 to 80 were equally effective . With selenocysteine, however, absorption was considerably reduced at all ratios . Selenocysteine was identified as the reduction product by reaction with Gaitonde's reagent, comparison of absorption spectra, paper chromatograhy, utilization by cysteinyl-tRNA synthetase fro Paracoccus denitrificans and Vigna radiata, changes in solubility after DTT treatment, and comparison of infrared spectra . During the ATP-PPi exchange assay, DTT and ME convert cysteine and selenocysteine derivatives to cysteine and selenocysteine which serve as substrates for cysteinyl-tRNA synthetase. J Gen Microbiol, 1980 Jul, 119(1), 145 - 54 The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12; Poole RK et al.; Centrifugation of membrane vesicles, prepared from ultrasonically disrupted Escherichia coli K12, on to a planar surface followed by slow, partial dehydration results in a high degree of parallel orientation of the membrane planes with respect to each other and the supporting surface . Rotation of such membrane multilayers about a single axis parallel with the membrane planes within the magnetic field of an electron paramagnetic resonance (e.p.r.) spectrometer allows the orientation of anisotropic paramagnetic centres to be deduced . Computer simulations of the angular dependence of cytochrome e.p.r . spectra show two, or perhaps three, cytochromes, well-oriented with respect to the membrane plane . A low-spin cytochrome is oriented with the normal to its haem plane lying in the membrane plane . One (or perhaps two) high-spin cytochrome(s) lies with its haem plane making an angle of 45 degrees with the membrane plane . The orientation of the low-spin cytochrome haem is thus the same as that of haems in b-type cytochromes and cytochrome oxidases of the a type found in the mitochondria of higher animal and microbial cells and the bacterium Paracoccus denitrificans (Erecinska et al., 1979) . The possible identity of this low-spin component as the terminal oxidase, cytochrome o, is discussed. J Biochem (Tokyo), 1980 Jul, 88(1), 223 - 30 Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans; Sanemori H et al.; The existence of thiamine pyrophosphokinase {EC 2.7.6.2} in procaryotic cells was first demonstrated in Paracoccus denitrificans (J . Bacteriol, (1976) 126, 1030-1036) . The enzyme was therefore purified from this organism to determine its molecular structure and properties . Thiamine pyrophosphokinase which was purified 620-fold from P . denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000 . Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits . Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer . The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP . Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively . The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM . Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme . The activity was also inhibited by the product, TPP. J Biol Chem, 1980 Jun 10, 255(11), 5027 - 30 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification; Hollocher TC et al.; 13N- and 15N-labeling experiments were carried out with Paracoccus denitrificans, grown anaerobically on nitrate, to determine whether hyponitrite might be an obligatory intermediate in denitrification and a precursor of nitrous oxide . From experiments designed to trap {13N}- or {15N,15N}hyponitrite by dilution into authentic hyponitrite it was calculated that the intracellular concentration of a presumptive hyponitrite pool must be less than 0.4 mM . In order for a pool of this size to turn over rapidly enough to handle the flux of nitrogen during dentrifucation, the spontaneous rate of hyponitrite dehydration must be enhanced by a factor of several thousand through enzyme catalysis . Cell extracts failed to catalyze this reaction under a variety of conditions . It is concluded that hyponitrite cannot be an intermediate in dentrification . In addition, the assimilation of inorganic nitrogen was studied in P . denitrificans using 13N as tracer . At low concentrations (less than 10(-8) M) of labeled nitrate and nitrite 5 to 10% of the label was assimilated into non-volatile metabolites and 90 to 95% was reduced to N2 . Similarly, with 15 mM {13N}nitrate, 5% of the label went into metabolites and 95% to N2 . High pressure liquid chromatography analysis of the labeled metabolites indicated that the major pathway for assimilation of inorganic nitrogen in P . denitrificans under these conditions is through ammonia incorporation via the aspartase reaction. Arch Microbiol, 1980 Jun, 126(2), 155 - 9 Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans; Burke KA et al.; Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities . The wild type forms a membrane protein Mr150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme . Suspensions of mutuant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein Mr150,000 was formed under all conditions tested, including without inducers and without molybdenum . Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wild type . Autoradiography of preparations from cells incubated with 55Fe showed that the mutant and wild type proteins contained iron . However, in similar experiments with 99Mo, incorporation of molybdenum into the mutant protein was not detectable . We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes . This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers. Arch Microbiol, 1980 Jun, 126(2), 149 - 53 Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans; Calder K et al.; An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite . Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane . The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide Mr = 150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail . When incubated in the cell suspension system M-1 formed a membrance protein Mr = 150,000 similar to that attributed to nitrate reductase in the wild type . Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome . The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are co-regulated and that the active enzyme has a role in regulating its own synthesis. Mikrobiologiia, 1980 May-Jun, 49(3), 373 - 6 {Inhibition of the autotrophic growth of hydrogen bacteria by the autoregulation factor}; Savel'eva ND et al.; The purpose of this work was to study the action of the autoregulation factor isolated from the organotrophous culture of Pseudomonas carboxydoflava Z-1107 on autotrophous growth of this organism and some other cultures of hydrogen bacteria . If a sufficient dose of this metabolite was added to the culture growing lithoautotrophously, the growth of Ps . carboxydoflava Z-1107 stopped completely after 24 hours . A curve of false diauxia was observed if an increase in the concentration of the autoregulation factor in the culture was not great enough . When the factor acted on the autotrophously growing cultures of Alcaligenes eutrophus, Pseudomonas pantotropha and Paracoccus denitrificans, it was established that this endogenous metabolite possessed group specificity, and was capable of inhibiting, more or less, autotrophous growth of this hydrogen bacterium. Antimicrob Agents Chemother, 1980 May, 17(5), 789 - 97 Antibiotic resistance in Neisseria denitrificans; MacKenzie CR et al.; An ampicillin-resistant strain of Neisseria denitrificans was produced by serial passage of the organisms in media containing increased concentrations of antibiotic . The 400-fold increase in resistance obtained was a relatively stable characteristic . Ampicillin resistance in this organism was apparently related to a loss or modification of the penicillin-binding proteins associated with the cytoplasmic membranes . Membranes isolated from the ampicillin-resistant strain bound significantly less radioactive penicillin than those isolated from the parent strain and revealed one major and three minor penicillin-binding proteins . All four penicillin-binding proteins were present in reduced amounts or had a decreased capacity for penicillin binding in the ampicillin-resistant cells . The increased resistance did not involve enzymic degradation of the antibiotic or a general reduction in the permeability of the outer layers of the cell . No difference in the amount of peptidoglycan present in the parent and ampicillin-resistant cells or in the gross chemical structure of the peptidoglycans of the two strains was observed. Eur J Biochem, 1980 Apr, 105(2), 267 - 74 Isolation and characterization of an ornithine-containing lipid from Paracoccus denitrificans; Thiele OW et al.; The isolation and characterization of an ornithine-containing lipid from various strains of Paracoccus denitrificans (grown heteretrophically and autotrophically) is reported . The structure of this aminolipid was found to be H2N--CH2--(CH2)2--CH{NH--CO--CH2--CH(O--CO--R1)--R2}--CO2H, where R1 predominantly represents the residue of octadec-11-enoic acid, R2 the residue of a 3-hydroxyeicos-13-enoic acid . In addition, the major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol) were isolated and characterized. Mikrobiologiia, 1980 Mar-Apr, 49(2), 284 - 7 {Nitrogen transformation as a result of Pseudomonas denitrificans denitrification}; Il'ina TK et al.; The energy process of nitrate reduction was studied in the typical soil denitrifying cultures of Pseudomonas denitrificans by mass spectrometry . These microorganisms differed in certain characteristics of the process from the cultures of sporogenic soil denitrifying bacteria belonging to the Bacillus genus . The rate of denitrification by Ps . denitrificans was very high; as the result, losses of 15N from the medium exceeded 80--90% . Ammonia was not produced as the final product of nitrate reduction in the course of denitrification . Apparently, hydroxylamine, hyponitrite or an organic intermediate product of nitrate reduction is produced in the process of its assimilation by the cultures. Ann Microbiol (Paris), 1980 Mar-Apr, 131A(2), 197 - 207 {Distribution of heterotrophic aerobic microflora and specially denitrifying and free-living nitrogen-fixing bacteria in the rhizosphere of rice (author's transl)}; Roussos S et al.; The distribution of heterotrophic aerobic bacteria, actinomycetes and fungi was estimated in three samples (rhizospherical soil (SR), rhizoplane (R) and endorhizosphere (ER)) obtained from one rice seedling which had grown in pot during four mounths in a Casamance grey soil . The number of microbial populations were about the same from one sample to another: 1.1 to 2 X 10(8) bacteria, 3.3 to 8.6 X 10(6) actinomycetes and 0.2 to 8.9 X 10(4) fungi per gram of dry soil (SR) or dry roots (R and ER) . The denitrifying and free-living nitrogen fixing bacteria were numerous (10(7) bacteria/g) but lower for ER where the number of actinomycetes remained high . Thirty-six bacterial strains have been isolated from every sample with the use of a grid of isolation . The Gram-negative bacteria were dominant in SR and R where they represented respectively 70 and 94% of total count . The major groups were non-sporulated Gram-variable rods (SR) and Alcaligenes-like bacteria (ER) . The pseudomonads represented quite 15% of total count in the three samples . On the other hand, the frequency of endospore-forming Gram-positive bacteria was high only in R where the Bacillus group was estimated to 45% of total count . Only 5 free-living nitrogen-fixing bacterial strains had shown a denitrifying ability. J Biol Chem, 1980 Jan 25, 255(2), 704 - 7 First practical assay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization; Kristjansson JK et al.; Lysis of spheroplasts made from denitrification-adapted Paracoccus denitrificans released a soluble nitrous oxide reductase which was assayed spectrophotometrically under anaerobic conditions by following the oxidation of methyl or benzyl viologen cation radical upon reduction of N2O to N2 . Other classes of reductants so far tested, including dithionite, could not substitute for viologen dyes . Viologen dyes, therefore, afford the first practical assay for this previously elusive enzyme of denitrifying bacteria . The assay is specifically an in vitro assay, because the dyes cannot couple with intracellular nitrous oxide reductase . The enzyme exhibited simple saturation kinetics with respect to both N2O and reduced viologen dye . The Km for N2O was about 5 microM at 22 degrees C and pH 7.1 and the apparent Km for reduced benzyl and methyl viologen was 0.9 and 0.5 microM, respectively . Both dyes afforded the same Vmax value . Oxidized viologen dyes were not inhibiting to 1 mM nor was N2 to 1 atm . In fresh lysates, Vmax was about 1.2 mumol of N2O X min-1 x mg of protein-1 or about twice that for intact cells or spheroplasts utilizing yeast extract or lactate . Enzyme activity was observed to be labile in crude preparations under anaerobic conditions . Nitrous oxide reductase was inhibited by acetylene, CO, azide, and cyanide with Ki values of 28, 3.5, 0.35, and 0.045 microM, respectively . All showed noncompetitive inhibition with respect to N2O. Ann Nutr Aliment, 1980, 34(5-6), 979 - 88 {Increase of nitrates in underground aquifers: study of a karstic basin in the Dijon area}; Simon JL et al.; The purpose of the study was to explain the enormous increase of nitrates in the waters of river Beze (Cote-d'Or) belonging to a well-defined karstic basin . This paper deals with surface and underground waters, pedologic formations and their rock base . It consisted in listing the oligoelements involved in the enzymic processes of nitrate reduction, the microflora and in determining the kinetics of denitrification by column perfusion . The presence of biodegradable carbon compounds is the governing factor of denitrification; their rapid lass related to new agricultural processes and the excessive use of nitrogene fertilizers are responsible for the enormous increase of nitrates observed. Antonie Van Leeuwenhoek, 1980, 46(2), 143 - 55 The regulation of hydrogenase formation as a differentiating character of strains of Paracoccus denitrificans; Nokhal TH et al.; Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation . In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides . The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero . In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions . Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions. Appl Environ Microbiol, 1980 Jan, 39(1), 105 - 8 Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens; Sorensen J et al.; The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied . Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV . The control without reductant had a redox potential near +200 mV . Production of 13NO, {13N}N2O, and {13N}N2 from 13NO3- and 13NO2- was followed . Total gas production was similar for all three treatments . The accumulation of 13NO was most significant in the presence of sulfide . A parallel control with autoclaved cells indicated that the 13NO production was largely biological . The sulfide inhibition was more dramatic at the level of N2O reduction; {13N}N2O became the major product instead of {13N}N2, the dominant product when either no reductant or Ti(III) was present . The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells. Proc Natl Acad Sci U S A, 1980 Jan, 77(1), 196 - 200 A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans; Ludwig B et al.; Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans . The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of apparent molecular weights 45,000 and 28,000; they appear to correspond to the two largest mitochondrially made subunits of the seven-subunit cytochrome c oxidase isolated from yeast mitochondria . Because of its structural simplicity . Paracoccus cytochrome c oxidase offers new possibilities for exploring the mechanism of cytochrome c oxidase function. Biochim Biophys Acta, 1979 Dec 6, 548(3), 484 - 99 Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus; Zumft WG et al.; A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate . The membrane system had a specific requirement for FMN with NAD(P)H as electron donors . Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate . The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell . As further terminal oxidase cytochrome o was identified . The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor. Z Naturforsch {C}, 1979 Dec, 34(12), 1272 - 4 Distribution of thioredoxins in Cyanobacteria; Schmidt A et al.; The presence of thioredoxin was demonstrated in 20 strains of cyanobacteria as well as in one phototrophic bacterium Rhodopseudomonas sulfidophila and in Thiobacillus denitrificans . Thioredoxin activity was not found in Cyanophora paradoxa and in Porphyridium cruentum using the thioredoxin-dependent PAPS-sulfotransferase activity from Synechococcus 6301 as assay system. Biochim Biophys Acta, 1979 Sep 12, 570(1), 43 - 55 Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans . Isolation of the hydrogenase; Sim E et al.; The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100 . The membrane-bound and detergent-solubilised forms of the enzyme have been compared . Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM . An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break' . The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme . The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane . Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme . The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite . The overall yield of activity is 23%, with a final purification of over 100-fold. Biochemistry, 1979 Sep 4, 18(18), 3921 - 6 Implications of the integrated rate law for the reactions of Paracoccus denitrificans nitrite reductase; Robinson MK et al.; The integrated rate law for the reaction of the nitrite reductase of Paracoccus denitrificans, a cytochrome cd, has been established for turnover assays using donor ferrocytochromes c and either nitrite or molecular oxygen as the ultimate acceptor . The time course for the concentration of ferrocytochrome follows the law: formula: (see text), where S is the concentration of donor ferrocytochrome c, So is the initial concentration, t is time, and u1, u2, and u3 are empirical parameters that are constant for a given experiment but depend upon the initial substrate concentration . In particular, all the u1 increase with decreasing initial ferrocytochrome concentration . Saturation of reaction rates at high donor ferrocytochrome concentrations was not observed . The parameter u1 was proportional to the enzyme concentration while u2 and u3 were not . The form of the integrated rate law and the behavior of the u1 impose severe restrictions on possible kinetic schemes for the activity of the enzyme . Contemporary mechanisms that have been proposed for mitochondrial oxidase aa3 are examined and found to be inadequate to explain the reactivity of cytochrome cd . The simplest interpretations of the cytochrome cd data suggest that the enzyme does not bind the ferri and ferro forms of donor cytochromes c with equal affinity and that the enzyme is subject to inhibition by a product of reaction . Eucaryotic horse cytochrome c reacts with the Paracoccus cytochrome cd with 77% of the activity when Paracoccus cytochrome c550 is used as the electron donor. J Bacteriol, 1979 Sep, 139(3), 1062 - 4 Transfer of kanamycin resistance mediated by plasmid R68.45 in Paracoccus denitrificans; Paraskeva C; Plasmid R68.45 mediates the transfer of kanamycin resistance from Pseudomonas aeruginosa to Paracoccus denitrificans . Kanamycin resistance could be transferred from one strain of P . denitrificans to another, thus opening up the possibility of using R68.45 as a sex factor in P . denitrificans. Biochem J, 1979 Sep 1, 181(3), 517 - 24 Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila; Bamforth CW et al.; 1 . A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol . 2 . The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme . 3 . The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel . 4 . The amino acid composition, ioselectric point, u.v . and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes . 5 . The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay . The iron could not be dissociated from the enzyme by dialysis against chelating agents . 6 . E.p.r . spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical . 8 . Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus . 9 . The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties. Arch Microbiol, 1979 Sep, 122(3), 263 - 70 Isolation and analysis of mutants of Pseudomonas aeruginosa unable to assimilate nitrate; Sias SR et al.; Pseudomonas aeruginosa can reduce nitrate to nitrite and evenutally to nitrogen gas by the denitrification pathway, thereby providing the organism with a mode of respiration and ATP generation in the absence of oxygen . P . aeruginosa can also reduce nitrate to nitrite through an assimilatory pathway that provides the cell with reduced nitrogen for biosyntheses . In order to establish whether this organism synthesizes a single nitrate reductase protein that functions in both pathways, or produces one for each pathway, we isolated mutants blocked in the assimilation of nitrate . These mutants are unaffected in the reduction of nitrate be the denitrification pathway, although they produce low or undectable levels of assimilatory nitrate reductase . On the basis of transductional analysis, the mutations were found to be distributed among four genes designated nasA, nasB, nasC, and nasD . Shifting a nasA mutant from anaerobic to aerobic growth eliminated the culture's ability to reduce nitrate, i.e . the anaerobic nitrate reductase cannot function in the presence of oxygen . Thus P . aeruginosa can synthesize two distinct proteins which reduce nitrate to nitrite: an assimilatory nitrate reductase and a dissimilatory nitrate reductase . If conditions of growth are fully aerobic, the latter is not synthesized and does not function . The former, synthesized under the control of at least four genes, is repressed by readily available nitrogen sources. Biochem J, 1979 Jul 1, 181(1), 159 - 69 Isolation and properties of cytochrome c peroxidase from Pseudomonas denitrificans; Coulson AF et al.; The isolation of cytochrome c peroxidase, cytochrome c4, cytochrome c-551 and azurin from Pseudomonas dentrificans is described . The peroxidase has a molecular weight of 63,000 and an isoelectric point of 5.6 . Its absorption spectrum suggests that it contains two haem c groups/molecule . Preliminary steady-state kinetic data are reported with cytochromes c-551 and c4 and azurin as the second substrate. Can J Microbiol, 1979 Jul, 25(7), 798 - 802 The effect of phenazine methosulfate-ascorbate on bacterial active transport and adenosine triphosphate formation: inhibition of Pseudomonas aeruginosa and stimulation of Escherichia coli; Eagon RG et al.; The artificial electron-donor system, phenazine methosulfate (PMS) ascorbate, inhibited active transport of glucose by Pseudomonas aeruginosa irrespective of whether the incubation systems were in air, flushed with oxygen, or gassed with nitrogen under anaerobic denitrifying conditions . Active transport of glucose by P . aeruginosa was also inhibited by reduced 5-N-methyl-phenazonium-3-sulfonate, a membrane-impermeable electron donor . PMS-ascorbate caused rapid depletion of intracellular adenosine triphosphate (ATP) when added to respiring cell suspensions of P . aeruginosa either in the presence or absence of glucose or succinate as oxidizable energy sources . In contrast, under identical conditions, Escherichia coli formed ATP with PMS-ascorbate as the sole oxidizable energy source and ATP formation continued when glucose or succinate was present in addition to PMS-ascorbate in the incubation system. Biol Bull Acad Sci USSR, 1979 Jul-Aug, 6(4), 450 - 9 Loss of nitrogen by denitrification; Smirnov PM et al.; A series of experiments showed the quantity and composition of nitrogen lost in gaseous form from fertilizers in soil is largely determined by the conditions of denitrification . Loss of nitrogen from ammonium sulfate or calcium nitrate was mainly through the release of nitrous oxide and molecular nitrogen, while nitrogen was released from sodium nitrite in the form of nitric oxide . Under anaerobic conditions and at neutral soil pH in the presence of glucose, the more reduced gaseous forms of nitrogen were released . However, the oxides of nitrogen predominated under conditions unfavorable for denitrification . The nitrogen oxides were not the terminal products of nitrogen conversion (nitrates and nitrites) . By a process of dissimilation, the nitrogen oxides acted as electron acceptors for microorganisms, being converted to N2O and N2 . The reduction of NO generally led to the formation of N2O as an intermediate, and depended on pH, aeration, and the presence of an energy source for the denitrifying organisms. Biochim Biophys Acta, 1979 Jun 6, 568(2), 454 - 66 Purification of Thiobacillus denitrificans siroheme sulfite reductase and investigation of some molecular and catalytic properties; Schedel M et al.; A siroheme-containing sulfite reductase was isolated from Thiobacillus denitrificans, purified to an electrophoretically homogenous state, and investigated with regard to some of its molecular and catalytic properties . The enzyme was a tetramer with a molecular weight of 160 000, consisting of two types of subunits arranged to an alpha 2 beta 2-structure . The molecular weight of the alpha-subunit was 38 000, that of the beta-subunit 43 000 . As prosthetic groups siroheme and Fe/S groupings could be detected . The absorption spectrum showed maxima at 273 nm, 393 nm, and 594 nm; the molar extinction coefficient at these wavelengths were 280, 181, and 60 . 10(3) cm2 . mmol-1, respectively . With reduced viologen dyes the enzyme reduced sulfite to sulfide, thiosulfate and trithionate . In many properties T . denitrificans sulfite reductase closely resembled desulfoviridin, the dissimilatory sulfite reductase of Dssulfovibrio species . It is proposed that the physiological function of this enzyme is not to reduce but rather to form sulfite from reduced sulfur compounds in the course of dissimilatory sulfur oxidation in T . denitrificans. Eur J Biochem, 1979 Jun, 97(1), 119 - 26 Hydrodynamic parameters of the detergent-solubilised hydrogenase from Paracoccus denitrificans; Sim E et al.; The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100 . The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O . The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100 . The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent . The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent . The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent . In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000 . The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100 . Removal of Triton X-100 results in association of tetramers to form octamers. Eur J Biochem, 1979 May 2, 96(1), 69 - 76 Anaerobic respiration and energy conservation in Paracoccus denitrificans . Functioning of iron-sulfur centers and the uncoupling effect of nitrite; Meijer EM et al.; 1 . Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain . In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase . 2 . Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase . Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced . These results contrast those observed for sulphate-limited growth of P . denitrificans under aerobic conditions {Eur . J . Biochem . (1977) 81, 267-275} . 3 . Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane . Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions . Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration {Arch . Microbiol . (1977) 112, 17-23} can be explained by the uncoupling action of nitrite. Z Naturforsch {C}, 1979 May-Jun, 34C(5-6), 346 - 9 Improved synthesis and rapid isolation millimole quantities of adenylysulfate; Cooper BP et al.; An improved enzymatic method for the synthesis of adenylysulfate (APS) from adenosine 5'-phosphate using APS-reductase from Thiobacillus denitrificans is described . Isolation of millimole quantitities of this sulfur nucleotide is achieved rapidly by means of ion exchange chromatography on a strongly basic ion exchange resin . A facile and reproducible desalting procedure is described. Biochem J, 1979 Apr 15, 180(1), 69 - 73 Interaction of lipophilic quinones with membrane fragments of Paracoccus denitrificans and Staphylococcus epidermidis; Mikes V et al.; In quinone-depleted mitochondrial and Paracoccus denitrificans membranes the quantum yield of fluorescence of ostruthin (6-geranyl-7-hydroxycoumarin) was maintained, whereas an increase in the quantum yield took place after extraction of Staphylococcus epidermidis membrane . A marked quenching effect of ubiquinone and menaquinone each with two isoprene units in the side chain on the ostruthin fluorescence was found with all types of quinone-depleted particles . When the homogues of menaquinone and ubiquinone with six isoprene units in the side chain were re-incorporated, a quenching of the ostruthin fluorescence was observed in the S . epidermidis membranes but not in those of P . denitrificans . The different behaviour of both bacterial preparations is attributable to the more specific finding of ubiquinone in the particles of P . denitrificans. J Bacteriol, 1979 Mar, 137(3), 1227 - 33 Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane; Kristjansson JK et al.; Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans . This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E . coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane . Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane . Nitrate pulse studies on E . coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses . By analogy with P . denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux . The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2. Biochim Biophys Acta, 1979 Feb 8, 545(2), 352 - 64 Studies of the orientation of the mitochondrial redox carriers . III . Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers; Erecinska M et al.; The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans . The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers . The directions of the g signals corresponding to the gx and gy axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase . It is suggested that this different orientation of gx and gy in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a3. Antonie Van Leeuwenhoek, 1979, 45(2), 225 - 40 Characterization and classification of fluorescent pseudomonads isolated from tap water and surface water; van der Kooij D; A total of 665 fluorescent pseudomonads, isolated from surface water and from different types of tap water, were classified into 22 groups defined by the results of the following 5 tests: hydrolysis of casein or gelatin, production of N2 from NO3-, and growth on sucrose, ethanol and D-sorbitol, respectively . Differences in colonial morphology and in the degree of proteolysis revealed that these groups were inhomogeneous . A more detailed subdivision was achieved by adding the following characters: growth on L-arabinose, D-mannitol, mesoinositol, and adonitol, respectively, and hydrolysis of Tween-80 . Repeating the tests for hydrolyzing enzymes, denitrification, and growth on the carbohydrates and alcohols with strains stored in the laboratory for 1 to 3 years revealed that most characters, except hydrolysis of Tween-80, denitrification, and growth on sucrose, were very stable . Forty-five biotypes of the fluorescent pseudomonads were defined, based on the results of the repeated tests and the results of tests on nine aromatic compounds . The observed changes of some characters in a number of isolates did not diminish the value of this classification, but indicated that close relationships exist between many biotypes . About half of the biotypes described in this paper are similar to those defined by Stanier, Palleroni and Doudoroff (1966) and by Doudoroff and Palleroni (1974a), confirming the wide-spread occurrence of well-definable biotypes of fluorescent pseudomonads . Representatives of some biotypes were most frequently isolated from surface water and from tap water prepared from surface water . Tap water prepared from anaerobic or aerobic ground water contained representatives of biotypes which were typical of these water types . Some pseudomonads were found to grow especially in filters . The observed relationships between origin of the fluorescent pseudomonads and their classification into biotypes as defined in this paper supported the presented classification. Antonie Van Leeuwenhoek, 1979, 45(2), 185 - 97 Structural analysis of four strains of Paracoccus denitrificans; Nokhal TH et al.; Two out of eleven newly isolated strains of Paracoccus denitrificans were investigated by light and electron microscopic methods and compared with two strains of P . denitrificans already kept in culture collections . Samples were taken from di