W. N. M. Reijnders

Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor

Production of nitric oxide (NO) and nitrous oxide (N2O) by ammonia (NH3)‐oxidizing bacteria in natural and man‐made habitats is thought to contribute to the undesirable emission of NO and N2O into the earths atmosphere. The NH3‐oxidizing bacterium Nitrosomonas europaea expresses nitrite reductase (NirK), an enzyme that has so far been studied predominantly in heterotrophic denitrifying bacteria where it is involved in the production of these nitrogenous gases. The finding of nirK homologues in other NH3‐oxidizing bacteria suggests that NirK is widespread among this group; however, its role in these nitrifying bacteria remains unresolved. We identified a gene, nsrR, which encodes a novel nitrite (NO2–)‐sensitive transcription repressor that plays a pivotal role in the regulation of NirK expression in N. europaea. NsrR is a member of the Rrf2 family of putative transcription regulators. NirK was expressed aerobically in response to increasing concentrations of NO2– and decreasing pH. Disruption of nsrR resulted in the constitutive expression of NirK. NsrR repressed transcription from the nirK gene cluster promoter (Pnir), the activity of which correlated with NirK expression. Reconstruction of the NsrR‐Pnir system in Escherichia coli revealed that repression by NsrR was reversed by NO2– in a pH‐dependent manner. The findings are consistent with the hypothesis that N. europaea expresses NirK as a defence against the toxic NO2– that is produced during nitrification.

Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes

ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation.

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 denitrificansfnrP 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 terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa3‐type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa3‐type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem‐containing oxidase, called cytochrome bb3, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::KmR) an alternative cyto‐chrome c oxidase has been characterized; this cbb3‐type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox‐driven proton pumps. The proton‐pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N‐terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb 3‐type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation

The gene encoding the blue‐copper protein amicyanin was isolated from a genomic bank of Pracoccus denitrificans by using a synthetic oligonucleotide. It is located directly downstream of the gene encoding the small subunit of methylmine dehydrogenase. Amicyanin is transcribed as a presursor protein with a signal sequence, typical for periplasmic proteins. Specific inactivation of amicyanin by means of gene replacement techniques resulted in the complete loss of the ability to grow on methylamine.

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

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.

Regulation of oxidative phosphorylation: the flexible respiratory network of Paracoccus denitrificans

Paracoccus denitrificans is a facultative anaerobic bacterium that has the capacity to adjust its metabolic infrastructure, quantitatively and/or qualitatively, to the prevailing growth condition. In this bacterium the relative activity of distinct catabolic pathways is subject to a hierarchical control. In the presence of oxygen the aerobic respiration, the most efficient way of electron transfer-linked phosphorylation, has priority. At high oxygen tensionsP. denitrificans synthesizes an oxidase with a relatively low affinity for oxygen, whereas under oxygen limitation a high-affinity oxidase appears specifically induced. During anaerobiosis, the pathways with lower free energy-transducing efficiency are induced. In the presence of nitrate, the expression of a number of dehydrogenases ensures the continuation of oxidative phosphorylation via denitrification. After identification of the structural components that are involved in both the aerobic and the anaerobic respiratory networks ofP. denitrificans, the intriguing next challenge is to get insight in its regulation. Two transcription regulators have recently been demonstrated to be involved in the expression of a number of aerobic and/or anaerobic respiratory complexes inP. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery.

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 β-subunit and low levels of the α-subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the β-subunit and that the absence of each of these proteins leads to production of a non-functional β-subunit which becomes rapidly degraded.