Specificity of FNR-type regulators in Paracoccus denitrificans.

The three FNR (fumarate and nitrate reductase regulatory protein)-type transcription activators of Paracoccus denitrificans, NarR, NnrR and FnrP, appear to have specific tasks in gene regulation during the switch from aerobic growth to denitrification. We here set out a series of experiments to get a fundamental understanding of the mechanism underlying this specificity. In one of these, we changed the nucleotide sequence of an NnrR box, the binding site for NnrR, into one found in FnrP-regulated promoters. As a result, we observed a change in regulation of that promoter from NnrR to FnrP. In a second series, we constructed hybrid promoters of NnrR-, NarR- and FnrP-regulated promoters and analysed their expression profiles in cells grown under various growth conditions. Our results indicate that the specificity of the FNR-type regulators is determined in part by the quality of the FNR box and in part by the sequences downstream of the FNR box. The latter suggests that specific sigma factors are involved in binding any of the Fnr-type regulators in P. denitrificans.

Cytochromes c(550), c(552), and c(1) in the electron transport network of Paracoccus denitrificans: redundant or subtly different in function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

Regulation of expression of terminal oxidases in Paracoccus denitrificans.

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1-negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3-type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3-type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3-type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain.

Two-component system that regulates methanol and formaldehyde oxidation in Paracoccus denitrificans.

A chromosomal region encoding a two-component regulatory system, FlhRS, has been isolated from Paracoccus denitrificans. FlhRS-deficient mutants were unable to grow on methanol, methylamine, or choline as the carbon and energy source. Expression of the gene encoding glutathione-dependent formaldehyde dehydrogenase (fhlA) was undetectable in the mutant, and expression of the S-formylglutathione hydrolase gene (fghA) was reduced in the mutant background. In addition, methanol dehydrogenase was immunologically undetectable in cell extracts of FhlRS mutants. These results indicate that the FlhRS sensor-regulator pair is involved in the regulation of formaldehyde, methanol, and methylamine oxidation. The effect that the FlhRS proteins exert on the regulation of C(1) metabolism might be essential to maintain the internal concentration of formaldehyde below toxic levels.

Heterologous NNR-mediated nitric oxide signaling in Escherichia coli.

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

The NosX and NirX proteins of Paracoccus denitrificans are functional homologues: their role in maturation of nitrous oxide reductase.

The nos (nitrous oxide reductase) operon of Paracoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the Cu(A) center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon.

Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.

Anaerobic growth of Paracoccus denitrificans requires cobalamin: characterization of cobK and cobJ genes.

A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P. denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the cobK and cobJ genes with their orthologues suggests that P. denitrificans uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions, P. denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that P. denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans.

By using the 'lacZ gene, the activities of the nirI, nirS, and norC promoters were assayed in the wild type and in NNR-deficient mutants of Paracoccus denitrificans grown under various growth conditions. In addition, induction profiles of the three promoters in response to the presence of various nitrogenous oxides were determined. Transcription from the three promoters required the absence of oxygen and the presence both of the transcriptional activator NNR and of nitric oxide. The activity of the nnr promoter itself was halved after the cells had been switched from aerobic respiration to denitrification. This response was apparently not a result of autoregulation or of regulation by FnrP, since the nnr promoter was as active in the wild-type strain as it was in NNR- or FnrP-deficient mutants.

The reduction state of the Q-pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans.

In this work we demonstrate how the reduction state of the Q-pool determines the distribution of electron flow over the two quinol-oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases. The dependence of the electron-flow rate to oxygen on the fraction of quinol in the Q-pool was determined in membrane fractions and in intact cells of the wild-type strain, a bc1-negative mutant and a quinol oxidase-negative mutant. Membrane fractions of the bc1-negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild-type strain or the quinol oxidase-negative mutant. In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher. In intact cells, the dependence was weaker. In uncoupled cells the dependence of the oxygen-consumption rates on the fractions of quinol in the Q-pool in the wild-type strain and in the two mutants came closer to that found for the membrane fractions. We also determined the dependence for membrane fractions of the wild-type in the absence and presence of antimycin A, an inhibitor of the bc1 complex. The dependence in the presence of antimycin A resembled that of the bc1-negative mutant. These results indicate that electron-flow distribution between the two quinol-oxidising branches in P. denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q-pool.

Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c550.

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd1) has been purified from the anoxically grown cytochrome c550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N-terminal amino acid sequence). Under our assay conditions, the half-saturation of electron transport occurred at about 10 microM pseudoazurin; the reaction was retarded by increasing ionic strength.

Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility.

Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P. denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common. Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration. Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains. The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled. There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P. denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified. In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus. While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

MauE and MauD proteins are essential in methylamine metabolism of Paracoccus denitrificans.

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C1-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the beta-subunit and low levels of the alpha-subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the beta-subunit and that the absence of each of these proteins leads to production of a non-functional beta-subunit which becomes rapidly degraded.

FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation.

The Paracoccus denitrificans fnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high-affinity cbb3-type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox-sensitive [4Fe-4S] cluster in FNR. NNR, another FNR-like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane-bound nitrate reductase and the cbb3-type oxidase was significantly reduced, whilst the activity of the bb3-type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3-type oxidase, cytochrome C550, cytochrome C552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP- and NNR-regulated genes are highly similar to or even identical to each other, the absence of cross-talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.

The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli.

The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to approximately 80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.

Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria.

The taxonomy of Paracoccus denitrificans and related bacteria is discussed. Evidence is given which shows that the physiological differences between P. denitrificans and Thiosphaera pantotropha are less fundamental than previously thought. A proposal to consider a species P. pantotropha is mentioned. The properties of the denitrifying enzymes and the genes involved in their formation in P. denitrificans is discussed. The synthesis of the membrane-bound-nitrate reductase is regulated by FNR, that of the nitrite- and nitric oxide reductase by NNR. Evidence is given that FNR acts as a redox sensor rather than an oxygen sensor. The occurrence of aerobic denitrification and coupled heterotrophic nitrification-denitrification in the original strain of Thiosphaera pantotropha are explained by a limiting respiratory activity which activates FNR. Aerobic denitrification leads to a lower growth yield and an increase in mumax in batch culture when a limiting respiratory activity is assumed and when excess substrate is present. Coupled heterotrophic nitrification-denitrification gives a smaller increase in mumax and a more drastic reduction in yield. Both processes are thus advantageous to the organism. In a chemostat with limiting substrate these processes are disadvantageous. T. pantotropha has lost the ability for aerobic denitrification during extended cultivation. Possibly the substrate concentration was limiting during extended cultivation giving a selective advantage to variants which have lost these properties. The calculations predict that P. denitrificans should be able to grow chemolithotrophically with hydroxylamine.

Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

S-formylglutathione hydrolase of Paracoccus denitrificans is homologous to human esterase D: a universal pathway for formaldehyde detoxification?

Downstream of flhA, the Paracoccus denitrificans gene encoding glutathione-dependent formaldehyde dehydrogenase, an open reading frame was identified and called fghA. The gene product of fghA showed appreciable similarity with human esterase D and with the deduced amino acid sequences of open reading frames found in Escherichia coli, Haemophilus influenzae, and Saccharomyces cerevisiae. Mutating fghA strongly reduced S-formylglutathione hydrolase activity. The mutant was unable to grow on methanol and methylamine, indicating that the enzyme is essential for methylotrophic growth. S-Formylglutathione hydrolase appears to be part of a formaldehyde detoxification pathway that is universal in nature.