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.
van der Palen
C J
CJ
Department of Molecular and Cellular Biology, BioCentrum Amsterdam, Vrije Universiteit, The Netherlands.
Slotboom
D J
DJ
Jongejan
L
L
Reijnders
W N
WN
Harms
N
N
Duine
J A
JA
van Spanning
R J
RJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D005798:Genes, Bacterial; D008744:Methylamines / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D010231:Paracoccus denitrificans / Q000235:genetics* / Q000378:metabolism
D008744:Methylamines; C027451:methylamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors