Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants.

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RESUMO

The nucleotide sequence of the methylamine utilization (mau) gene region from Methylobacterium extorquens AM1 was determined. Open reading frames for 11 genes (mauFBEDACJGLMN) were found, all transcribed in the same orientation. The mauB, mauA, and mauC genes encode the periplasmic methylamine dehydrogenase (MADH) large and small subunit polypeptides and amicyanin, respectively. The products of mauD, mauG, mauL, and mauM were also predicted to be periplasmic. The products of mauF, mauE, and mauN were predicted to be membrane associated. The mauJ product is the only polypeptide encoded by the mau gene cluster which is predicted to be cytoplasmic. Computer analysis showed that the MauG polypeptide contains two putative heme binding sites and that the MauM and MauN polypeptides have four and two FeS cluster signatures, respectively. Mutants generated by insertions in mauF, mauB, mauE, mauD, mauA, mauG, and mauL were not able to grow on methylamine or any other primary amine as carbon sources, while a mutant generated from an insertion in mauC was not able to utilize methylamine as a source of carbon but utilized C2 to C4 n-alkylamines as carbon sources. Insertion mutations in mauJ, mauM, and mauN did not impair the ability of the mutants to utilize primary n-alkylamines as carbon sources. All mau mutants were able to utilize methylamine as a nitrogen source, implying the existence of an alternative (methyl)amine oxidation system, and a low activity of N-methylglutamate dehydrogenase was detected. The mauD, mauE, and mauF mutants were found to lack the MADH small subunit polypeptide and have a decreased amount of the MADH large subunit polypeptide. In the mauG and mauL mutants, the MADH large and small subunit polypeptides were present at wild-type levels, although the MADHs in these strains were not functional. In addition, MauG has sequence similarity to cytochrome c peroxidase from Pseudomonas sp. The mauA, mauD, and mauE genes from Paracoccus denitrificans and the mauD and mauG genes from Methylophilus methylotrophus W3A1 were able to complement corresponding mutants of M. extorquens AM1, confirming their functional equivalence. Comparison of amino acid sequences of polypeptides encoded by mau genes from M. extorquens AM1, P. denitrificans, and Thiobacillus versutus shows that they have considerable similarity.

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