5-6-7-8-tetrahydromethanopterin and methanopterin

In this study, the occurrence and chromosomal clustering of genes encoding C(1) transfer reactions linked to tetrahydromethanopterin (H(4)MPT) were analyzed in a variety of proteobacteria and in representatives of the Planctomycetes via genomic analysis or via partial sequencing by cosmid walking. Although a tendency for clustering was found common for the genes of interest, significant variations in gene order and the degree of clustering were uncovered both between and within different groups of Proteobacteria and between Proteobacteria and Planctomycetes. Phylogenetic analyses suggested that the evolution of genes encoding H(4)MPT-linked reactions in Proteobacteria involved lateral transfers within Proteobacteria and possibly between Proteobacteria and other phyla. Gene cluster comparisons revealed a number of novel genes potentially involved in the C(1) transfer reactions, and these were analyzed by mutation and expression analyses. Four genes, a homolog of pabB, and three genes conserved between methanogenic Archaea and Bacteria possessing H(4)MPT-linked functions, orfY, orf1, and afpA were shown to be involved in formaldehyde oxidation/detoxification, as judged by specific mutant phenotypes. In particular, pabB contributes to the biosynthesis of para-aminobenzoic acid, a precursor of both tetrahydrofolate and H(4)MPT, and afpA apparently encodes a novel dihydromethanopterin reductase, based on mutant complementation experiments.

Topics: Biological Evolution; Carbon; Formaldehyde; Methylobacterium extorquens; Multigene Family; Oxidation-Reduction; Pterins

In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.

Topics: Bacteria; Bacterial Proteins; DNA Primers; Fresh Water; Gene Library; Geologic Sediments; Phylogeny; Polymerase Chain Reaction; Pterins

The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1800 major phylotypes (the Sargasso Sea metagenome-SSM) presents a great source for expanding local databases of genes indicative of a specific function. In this article we analyse the SSM for the presence of methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment. The sequences representative of these major phylotypes do not appear to belong to any known microbial group capable of methanopterin-linked C1 transfer. Instead, these sequences separate from all known sequences on phylogenetic trees, pointing toward their affiliation with novel microbial phyla. These data imply a broader distribution of methanopterin-linked functions in the microbial world than has been previously known.

Topics: Biodiversity; Biological Transport; Carbon; Computational Biology; Formaldehyde; Genes, Archaeal; Genes, Bacterial; Marine Biology; Oceans and Seas; Oxidation-Reduction; Plankton; Pterins; Water Microbiology

An enzymatic assay was developed to measure tetrahydromethanopterin (H(4)MPT) levels in wild-type and mutant cells of Methylobacterium extorquens AM1. H(4)MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H(4)MPT biosynthesis. The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene 5'-phosphate synthase, the first committed step of H(4)MPT biosynthesis. These results provide the first biochemical evidence for H(4)MPT biosynthesis genes in bacteria.

Topics: Bacterial Proteins; Biological Assay; Methylobacterium extorquens; Mutation; Open Reading Frames; Oxidoreductases Acting on CH-NH Group Donors; Pterins

Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen. One of these genes encodes a product having identity with dihydrofolate reductase (DHFR). This mutant has a C(1)-defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H(4)MPT)-dependent formaldehyde oxidation. These results suggest that this gene, dmrA, may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H(4)MPT biosynthesis.

Topics: Amino Acid Sequence; Bacterial Proteins; DNA Transposable Elements; Genetic Complementation Test; Methanol; Methylobacterium extorquens; Molecular Sequence Data; Mutagenesis, Insertional; Oxidoreductases; Pterins; Sequence Alignment; Sequence Analysis, DNA; Tetrahydrofolate Dehydrogenase

Topics: Chromatography, High Pressure Liquid; Euryarchaeota; Pterins; Structure-Activity Relationship