Genes of theN-Methylglutamate Pathway Are Essential for Growth of Methylobacterium extorquens DM4 with Monomethylamine

Abstract

ABSTRACTMonomethylamine (MMA, CH3NH2) can be used as a carbon and nitrogen source by many methylotrophic bacteria.Methylobacterium extorquensDM4 lacks the MMA dehydrogenase encoded bymaugenes, which inM. extorquensAM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by theN-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genesgmaS,mgsABC, andmgdABCDfor the three enzymes of the pathway, γ-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clustersmgsABC-gmaSandmgdABCDand their induction during growth with MMA. The genesmgdABCDandmgsABCwere found to be essential for utilization of MMA as a carbon and nitrogen source. The genegmaSwas essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaSgrowing with succinate and MMA as a nitrogen source was observed. Plasmid copies ofgmaSand thegmaShomolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaSand DM4gmaSΔmetdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaSand DM4gmaSΔmetdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source.