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.

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.

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.

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.

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.

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

In Paracoccus denitrificans 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 ccoNOOP, 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 ccoNOOP 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 oxidase under certain conditions. Sequence alignment suggests that many residues that have been proposed to constitute the chemical and pumped proton channels in cytochrome aa3 (and probably also in cytochrome bb3) are not conserved in cytochrome cbb3. It is concluded that the design of the proton pump in cytochrome cbb3 differs significantly from that in the other oxidase types.

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 tensions P. 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 of P. 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 in P. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery.

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event.

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.

Isolation and characterization of a novel insertion sequence element, IS1248, in Paracoccus denitrificans.

A new suicide vector, pRVS3, was constructed to facilitate gene replacements in the genome of Paracoccus denitrificans. In control experiments, incorporation of this suicide vector into the genome did not depend on the presence of homologous DNA. Using appropriate restriction enzymes, the suicide vector and flanking DNA were recovered from the genomic DNA. Sequence analysis demonstrated that both up- and downstream of the ex-integrant vector there was an element that showed high homology with bacterial insertion sequences (IS). Southern blot analysis of wild-type and integrant strains revealed that at least four copies of this IS element reside in the P. denitrificans genome, one of which, designated IS1248, had been involved in the transpositional event described here. IS1248 is 830 bp long, has 13-bp imperfect inverted repeats at the borders, and contains five open reading frames. With respect to the organization and primary sequences of the open reading frames, IS1248 closely resembles IS869 and IS427 of Agrobacterium tumefaciens, IS402 of Pseudomonas cepacia, and ISmyco found in Mycobacterium tuberculosis.

Mutational analysis of mau genes involved in methylamine metabolism in Paracoccus denitrificans.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

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 cbb3-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.

Isolation, sequencing, and mutagenesis of the gene encoding NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) from Paracoccus denitrificans, in which GD-FALDH is essential for methylotrophic growth.

NAD- and glutathione-dependent formaldehyde dehydrogenase (GD-FALDH) of Paracoccus denitrificans has been purified as a tetramer with a relative molecular mass of 150 kDa. The gene encoding GD-FALDH (flhA) has been isolated, sequenced, and mutated by insertion of a kanamycin resistance gene. The mutant strain is not able to grow on methanol, methylamine, or choline, while heterotrophic growth is not influenced by the mutation. This finding indicates that GD-FALDH of P. denitrificans is essential for the oxidation of formaldehyde produced during methylotrophic growth.

Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrificans is under control of a LysR-type transcriptional activator.

Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR-type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.