Relationship between nitrite reduction and active phosphate uptake in the phosphate-accumulating denitrifier Pseudomonas sp. strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Light-Mediated Nitrite Accumulation during Denitrification by Pseudomonas sp. Strain JR12

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier, Pseudomonas sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome c, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.     

Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c

Anammox is a bacterial energy metabolic process that forms N₂ gas from nitrite and ammonium ions. The enzymatic mechanisms of anammox have been gradually revealed; however, the electron transport chain in anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the anammox bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an anammox bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the anammox process.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Periplasmic Cytochrome c3 of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

Kinetic parameters and the role of cytochrome c₃ in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K_m = 220 μM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H₂ and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H₂ and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H₂, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H₂-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H₂ was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H₂ was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c₃ is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

Electron Transport Coupled to Nitrate Reduction in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

The electron transport system involved in nitrate reductionand its relationship to photosynthetic cyclic electron transportin a photodenitrifier, Rhodopseudomonas sphaeroides forma sp.denitrificans, were studied. Nitrate oxidized only b-type cytochromein the presence of cyanide, which inhibits nitrite reductase.Heptylhydroxyquinoline-N-oxide (HOQNO) inhibited the oxidationof b-type cytochrome by nitrate, but not the oxidation of b-and c-type cytochrome by nitrite. The inhibition by HOQNO wasovercome by phenazine methosulfate (PMS). Absorption changesof b-type cytochrome induced by illumination were in just theopposite directions for oxygen- and nitrate-oxidized cells;the cytochrome was reduced in oxygen-oxidized cells and oxidizedin nitrate-oxidized cells. Antimycin enhanced the reductionand inhibited the oxidation, but had no inhibitory effect onthe oxidation of b-type cytochrome by nitrate. Dithionite-reducedminus ferricyanide-oxidized difference spectra of cells at 77?Kshowed two b-type cytochrome components with a bands at 556.5and 562 nm. The proportion of the b-562 component decreasedin cells grown under denitrifying conditions. It was concludedthat a b-type cytochrome is involved in the nitrate reduction.The b-type cytochrome was presumed to be an alternative to thecytochrome b in the photosynthetic cyclic electron transport. ¹ Present address: Japanese Red Cross Tokyo-to Komagome BloodCenter, Komagome 2-2-2, Toshima-ku, Tokyo 170, Japan. (Received August 13, 1981; Accepted December 5, 1981)     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Relationship between amino acid transport and electron transport by membrane vesicles of Micrococcus denitrificans

Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.     

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme c-type cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ∼35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the γ-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

Influence of ammonium,nitrate and nitrite on the performance of the pure culture of <Emphasis Type="Italic">Acinetobacter junii</Emphasis>

The influence of different concentration ranges (0–500 mg/L) of ammonium, nitrate and nitrite presence in the wastewater, on the performance of the pure culture of phosphate-accumulating bacterium Acinetobacter junii in the anaerobic and aerobic conditions, was investigated. A. junii was able to use ammonium and nitrate salts as the source of nitrogen, unlike in the case of nitrite salt. Comparing to the control reactors with the peptone and yeast extract as the sources of nitrogen, at the lowest tested concentration of ammonium and nitrate the performance of the system was inhibited due to the nitrogen deficit in the wastewater, while at the highest concentration it was positively influenced. Nitrite in all concentrations detrimentally affected the phosphate release and uptake rates, chemical oxygen demand uptake rates, nitrogen uptake rates, as well as multiplication of A. junii. The higher the nitrite concentration, the more pronounced was the effect. At the highest nitrite concentration tested a complete failure of the system was observed.     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.     

The electron transport chain ofStreptomyces erythreus: Possible functions of cytochromeaa 3 and a novel green pigment

The cytochromes of the bacteriumStreptomyces erythreus have been investigated. Membrane-bounda-, b-, andc-type cytochromes were found together with a green pigment, which was found in both a soluble and membrane-bound form. Cells containing the green pigment exhibited cyanide-insensitive oxygen uptake. The CO-binding pigments included cytochromea ₃, ab-type cytochrome, cytochrome P450, and the green pigment. Photodissociation spectra at various low temperatures, in the presence or absence of oxygen, revealed cytochromeaa ₃ to be the predominant cytochrome terminal oxidase. The green pigment was capable of electron transport; the relationship of the pigment to the remainder of the electron transport chain remains to be ascertained.     

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c₅₅₁ in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c₅₅₁, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.     

Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.     

Genetic regulation of phosphate transport system II in neurospora

Phosphate transport system II, previously shown to be responsible for highaffinity phosphate uptake under condition of phosphorus stavation, is regulated by at least three genes: pcon-nuc-2, preg, and nuc-1. nuc-1 and nuc-2 mutants cannot be depressed for phosphate transport system II, while pcon^c and preg^c mutants are partially constitutive.