Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and ...methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.
Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools. We describe the development of a broad-host-range cre-lox system that allows antibiotic ...marker recycling in a variety of gram-negative bacteria. This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase. We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400. This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria. Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.
Methods have been explored for detection of methylotrophs in natural samples, using environmental primers based on genes involved in the tetrahydromethanopterin (H₄MPT)-linked C₁ transfer pathway. ...The underlying hypotheses were that the H₄MPT-linked pathway is an ancient methylotrophy pathway, based on gene divergence, and that primers targeting more divergent genes will detect a broader variety of methylotrophs compared to the variety uncovered using probes and primers targeting highly conserved genes. Three groups of novel primer sets were developed targeting mch, mtdB, and fae, key genes in the H₄MPT-linked pathway, and these were used to assess the variety of microorganisms possessing these genes in sediments from Lake Washington in Seattle, WA. Environmental clone libraries were constructed for each of the genes and were analyzed by RFLP, and representatives of different RFLP groups were sequenced and subjected to phylogenetic analysis. A combination of all three sets of novel primers allowed detection of the two previously characterized groups of methylotrophs in the site: methanotrophs of the (α- and the γ-proteobacterial groups, belonghg to genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus. In addition to the genes belonging to known methanotroph populations, novel genes were identified, suggesting existence of previously undetected microbial groups possessing C₁ transfer functions in this site. These included sequences clustering with the well-characterized methylotrophic phyla, Methylobacterium, Hyphomicrobium, and Xanthobacter. In addition, sequences divergent from those known for any groups of methylotrophs or methanogens were obtained, suggesting the presence of a yet unidentified microbial group possessing this H₄MPT-linked C₁ transfer pathway.
Methanogenic and sulfate-reducing Archaea are considered to have an energy metabolism involving C1 transfer coenzymes and enzymes unique for this group of strictly anaerobic microorganisms. An ...aerobic methylotrophic bacterium, Methylobacterium extorquens AM1, was found to contain a cluster of genes that are predicted to encode some of these enzymes and was shown to contain two of the enzyme activities and one of the methanogenic coenzymes. Insertion mutants were all unable to grow on C1 compounds, suggesting that the archaeal enzymes function in aerobic C1 metabolism. Thus, methylotrophy and methanogenesis involve common genes that cross the bacterial/archaeal boundaries.
Life-on-a-chip Lidstrom, Mary E; Meldrum, Deirdre R
Nature reviews. Microbiology,
11/2003, Letnik:
1, Številka:
2
Journal Article
Recenzirano
Mechanistic studies of cellular processes are usually carried out with large populations of cells. However, parameters that are measured as averages of large populations can be misleading. For ...instance, an apparently linear response to a signal could, in fact, reflect an increasing number of cells in the population that have switched from 'off' to 'on', rather than a graded increase in response by all the cells. At present, the study of single cells is challenging, but new technologies mean it might soon be a reality.
Methylamine dehydrogenase (MADH), an $\alpha_2\beta_2$ enzyme from numerous methylotrophic soil bacteria, contains a novel quinonoid redox prosthetic group that is covalently bound to its small β ...subunit through two amino acyl residues. A comparison of the amino acid sequence deduced from the gene sequence of the small subunit for the enzyme from Methylobacterium extorquens AM1 with the published amino acid sequence obtained by the Edman degradation method, allowed the identification of the amino acyl constituents of the cofactor as two tryptophyl residues. This information was crucial for interpreting $^1$H and $^{13}$C nuclear magnetic reasonance, and mass spectral data collected for the semicarbazide- and carboxymethyl-derivatized bis(tripeptidyl)-cofactor of MADH from bacterium W3A1. The cofactor is composed of two cross-linked tryptophyl residues. Although there are many possible isomers, only one is consistent with all the data: The first tryptophyl residue in the peptide sequence exists as an indole-6,7-dione, and is attached at its 4 position to the 2 position of the second, otherwise unmodified, indole side group. Contrary to earlier reports, the cofactor of MADH is not 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), a derivative thereof, or pro-PQQ. This appears to be the only example of two cross-linked, modified amino acyl residues having a functional role in the active site of an enzyme, in the absence of other cofactors or metal ions.
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