Methanol oxidation genes in the marine methanotroph Methylomonas sp. strain A4

Abstract

Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenasespecific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially.Gram-negative methylotrophic bacteria oxidize methanol via a periplasmic methanol dehydrogenase, which contains the quinone cofactor pyrrolo-quinoline quinone and utilizes a soluble cytochrome c (called cytochrome CL) as an electron acceptor (5). This methanol oxidation system is complex, and more than 20 genes (mox genes) that are required for methanol dehydrogenase regulation, synthesis, and assembly and for synthesis of the cofactor pyrrolo-quinoline quinone (19) have been identified in the facultative methanol utilizer Methylobacterium exworquens AML.The structural genes for the methanol dehydrogenase alpha and beta subunits (moxF and moxI, respectively) have been cloned from the facultative methanol utilizers M. extorquens AM1 (28, 29), Methylobactenum organophilum XX (24), and Paracoccus denitrificans (16). In all three cases, the genes have been found to reside in a gene cluster together with the gene for cytochrome CL (moxG) and another gene of unknown function (mcxJ) (19). The order of these genes in all three bacteria is moxFJGI. Transcriptional start sites for this gene cluster have been mapped for the two Methylobacterium strains (4, 23), and a comparison of upstream sequences has identified a putative promoter sequence for these genes as follows: AAAGACA-18 bp-TAGAAA-4 to 5 bp-+1 (19).Although most of the genetic studies of ...

Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA

“…This GAAA motif is also present in front of the moxF gene in methane utilizers Methylomonas albus BG8 and Methylomonas sp. strain A4, but in these bacteria, this motif apparently is not involved in promoter structures (11,59). A few copies of this motif were also found upstream of the moxWgene in M. organophilum XX (63) and upstream of pqqD and pqqC genes of M. extorquens AM1 (37), and in this case they also do not participate in promoter structures.…”

Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1

“…A total of 50 ml of cell culture was quickly chilled on dry ice and centrifuged (10,000 ϫ g) at 4ЊC, and the pellet of cells was resuspended in 15 ml of diethylpyrocarbonate-treated water. The remaining steps in the RNA isolation procedure were done as described by Waechter-Brulla et al (28).…”

“…For the chase phase, 1 l of 10 mM dCTP was then added, and the incubation was continued for an additional 40 min. The products of transcription were prepared as described previously (28) and subjected to electrophoresis on 6% (wt/vol) polyacrylamide gels simultaneously with a sequencing ladder generated with the same primer, with DNA from plasmid pCM187 as the template.…”

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

“…The methanol produced by MMO is oxidized to formaldehyde by a calcium or rare-earth-dependent pyrroloquinoline quinone (PQQ)-containing MeDH (87)(88)(89)(90)(91). Formaldehyde is an important branch point in the metabolism of methanotrophic metabolism and represents the point at which one-carbon (C 1 ) intermediates can be either oxidized to CO 2 to derive energy or assimilated into biomass.…”