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Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1.

ABSTRACT: A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1. A mutation in gckA leads to a specific C1-negative phenotype. The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis. Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and the tartrate utilization pathway share a series of reactions.

SUBMITTER: Chistoserdova L

PROVIDER: S-EPMC179346 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1.

Chistoserdova L L Lidstrom M E ME

Journal of bacteriology 19970801 15

A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1. A mutation in gckA leads to a specific C1-negative phenotype. The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis. Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and t ...[more]

PMID: 9244287

Similar Datasets

Poly-beta-hydroxybutyrate biosynthesis in the facultative methylotroph methylobacterium extorquens AM1: identification and mutation of gap11, gap20, and phaR.

Project description:Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C(1) and C(2) metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C(2) compounds, while they show wild-type growth characteristics on C(1) or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C(1) compounds and has defects in PHB accumulation but grows normally on C(3) and C(4) compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C(2) compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C(1), C(2), and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

| S-EPMC151960 | biostudies-literature

Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1.

Project description:Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.

| S-EPMC134890 | biostudies-literature

Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol.

Project description:Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

| S-EPMC303455 | biostudies-literature

Genetic and phenotypic comparison of facultative methylotrophy between Methylobacterium extorquens strains PA1 and AM1.

Project description:Methylobacterium extorquens AM1, a strain serendipitously isolated half a century ago, has become the best-characterized model system for the study of aerobic methylotrophy (the ability to grow on reduced single-carbon compounds). However, with 5 replicons and 174 insertion sequence (IS) elements in the genome as well as a long history of domestication in the laboratory, genetic and genomic analysis of M. extorquens AM1 face several challenges. On the contrary, a recently isolated strain - M. extorquens PA1- is closely related to M. extorquens AM1 (100% 16S rRNA identity) and contains a streamlined genome with a single replicon and only 20 IS elements. With the exception of the methylamine dehydrogenase encoding gene cluster (mau), genes known to be involved in methylotrophy are well conserved between M. extorquens AM1 and M. extorquens PA1. In this paper we report four primary findings regarding methylotrophy in PA1. First, with a few notable exceptions, the repertoire of methylotrophy genes between PA1 and AM1 is extremely similar. Second, PA1 grows faster with higher yields compared to AM1 on C1 and multi-C substrates in minimal media, but AM1 grows faster in rich medium. Third, deletion mutants in PA1 throughout methylotrophy modules have the same C1 growth phenotypes observed in AM1. Finally, the precision of our growth assays revealed several unexpected growth phenotypes for various knockout mutants that serve as leads for future work in understanding their basis and generality across Methylobacterium strains.

| S-EPMC4169470 | biostudies-literature

Complete Genome Sequence of the Facultative Methylotroph Methylobacterium extorquens TK 0001 Isolated from Soil in Poland.

Project description:Methylobacterium extorquens TK 0001 (DSM 1337, ATCC 43645) is an aerobic pink-pigmented facultative methylotrophic alphaproteobacterium isolated from soil in Poland. Here, we report the whole-genome sequence and annotation of this organism, which consists of a single 5.71-Mb chromosome.

| S-EPMC5824006 | biostudies-literature

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol.

Project description: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.

| S-EPMC111405 | biostudies-literature

Formaldehyde stress response in Methylobacterium extorquens AM1

Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1.

2015-01-01 | GSE15631 | GEO

Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(1) and C(2) compounds in the methylotroph Methylobacterium extorquens AM1.

Project description:Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.

| S-EPMC94972 | biostudies-literature

Co-consumption of methanol and succinate by Methylobacterium extorquens AM1.

Project description:Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. (13)C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO(2) for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.

| S-EPMC3486813 | biostudies-literature

Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production.

Project description:BackgroundButanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol.ResultsIn this work, we engineered a modified CoA-dependent pathway in Methylobacterium extorquens AM1 to produce 1-butanol. Engineered strains displayed different 1-butanol titers using ethylamine as a substrate. A strain overexpressing Treponema denticola trans-enoyl-CoA reductase, Clostridium acetobutylicum alcohol dehydrogenase, and native crotonase was able to generate the highest 1-butanol titer (15.2 mg l(-1)). In vitro isotopic tracing of metabolic flux and in vivo metabolite analysis showed the accumulation of butyryl-CoA, demonstrating the functionality of the synthetic pathway and identifying targets for future improvement.ConclusionsWe demonstrated the feasibility of using metabolic intermediates of the ethylmalonyl-CoA pathway in M. extorquens AM1 to generate value-added chemicals, with 1-butanol as the test case. This will not only establish the biotechnological potential of the ethylmalonyl-CoA pathway, but will also introduce M. extorquens AM1 as a potential platform to produce value-added chemicals.

| S-EPMC4207312 | biostudies-literature

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