The gene encoding the blue-copper protein amicyanin was isolated from a genomic bank of Paracoccus denitrificans by using a synthetic oligonucleotide. It is located directly downstream of the gene encoding the small subunit of methylamine dehydrogenase. Amicyanin is transcribed as a precursor 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.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus deniriJ'canj. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the S' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AMl. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPdl, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c5531 was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c_511 and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxj deletion mutant strain partly synthesized the latter two proteins, but methanol dehydrogenase activity could not be detected. The moxG deletion mutant strain lacks exclusively cytochrome c.,,,. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c_511 as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome cs_5l is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitifficans. In contrast to earlier suggestions, this cytochrome was found to be different from membranebound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.Paracoccus denitrificans is a gram-negative soil bacterium able to grow heterotrophically on a variety of multicarbon compounds, autotrophically on hydrogen and carbon dioxide, and methylotrophically on one-carbon compounds, such as methanol and methylamine. The physiology of P. denitrificans has recently been reviewed by Van Verseveld and Stouthamer (59) and by Harms and Van Spanning (19). During methyl...

Volume 172, no. 2, p. 990, Fig. 2: The amino acids under nucleotides 388 to 423 were transposed with those under nucleotides 424 to 474; the correct amino acid sequence of cytochrome c550 is shown below.

A new suicide vector, pRVSl, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denit4ficans. The vector was derived from suicide vector pGRPdl, which was equipped with the lacZ gene encoding 13-galactosidase. The reporter gene was found to be a successl screening marker for the discrimnation between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPdl and pRVSl were used in gene replacement tchniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the perAplasmic cytochromes c..., cssl1, and c5531, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the corrtnss of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type'levels of methylamine dehydrogenase activities. cycA mutant stan, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow, on met,hmol and showed wdid-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum spific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.Paracoccus denitrificans is a gram-negative soil bacterium, which is well adapted for aerobic and anaerobic growth on a variety of carbon sources. During aerobic heterotrophic growth, electrons can be passed from reducing compounds to oxygen via at least three different routes. One of these routes has typical mitochondrionlike characteristics, involving NADH dehydrogenase, the bc1 complex, and the aa3-type oxidase (1,5,6,12,19,43,44,46). Electron transport from the bc1 complex to the aa3-type oxidase is mediated by the constitutively formed cytochrome c550, which operates in the periplasm (32,41). An alternative route between the latter two proton-translocating complexes is suggested to involve membrane-bound cytochrome c552 (4,21). Besides the two routes to the aa3-type oxidase, P. denitrificans has the disposal of a third aerobic electron transport route in which oxygen reduction is mediated by a quinol oxidase (10,28).For growth on methanol or methylamine, additional periplasmically located dehydrogenases and electron carriers are induced to enable the bacterium to use the Cl compounds concerned as carbon and energy sources (2,6,(15)(16)(17)(18). Methylamine is oxidized by methylamine dehydrogenase, and electrons are transferred to their natural electron acceptor, amicyanin (16,18,42). This blue copper enzyme seems to be the branching point of tw...

To isolate genes from Escherichia coli which regulate the labile hydrogenase activity, a plasmid library was used to transform hydL mutants lacking the labile hydrogenase. A single type of gene, designated hydG, was isolated. This gene also partially restored the hydrogenase activity in hydF mutants (which are defective in all hydrogenase isoenzymes), although the low hydrogenase 1 and 2 levels were not induced. Therefore, hydG apparently regulates, specifically, the labile hydrogenase activity. Restoration of this latter activity in hydF mutants was accompanied by a proportional increase of the H2 uptake activity, suggesting a functional relationship. H2:fumarate oxidoreductase activity was not restored in complemented hydL mutants. These latter strains may therefore lack, in addition to the labile hydrogenase, a second component (provisionally designated component R), possibly an electron carrier coupling H2 oxidation to the anerobic respiratory chain. Sequence analysis showed an open reading frame of 1,314 base pairs for hydG. It was preceded by a ribosome-binding site but apparently lacked a promoter. Minicell experiments revealed a single polypeptide of approximately 50 kilodaltons. Comparison of the predicted amino acid sequence with a protein sequence data base revealed strong homology to NtrC from Klebsiella pneumoniae, a DNA-binding transcriptional activator. The 411 base pairs upstream from pHG40 contained a second open reading frame overlapping hydG by four bases. The deduced amino acid sequence showed considerable homology with the C-terminal part of NtrB. This sequence was therefore assumed to be part of a second gene, encoding the NtrB-like component, and was designated hydH. The labile hydrogenase activity in E. coli is apparently regulated by a multicomponent system analogous to the NtrB-NtrC system. This conclusion is in agreement with the results of Birkmann et al. (A. Birkmann, R. G. Sawers, and A. Böck, Mol. Gen. Genet. 210:535-542, 1987), who demonstrated ntrA dependence for the labile hydrogenase activity.