Dataset Information

Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C.

ABSTRACT: The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1. The M. buryatense 5GB1 genome contains homologs for all enzymes necessary for a fermentative metabolism, and we hypothesize that a metabolic switch to fermentation can be induced by low-O2 conditions. Under prolonged O2-starvation in a closed vial, this methanotroph increases the amount of acetate excreted about 10-fold, but the formate, lactate, and succinate excreted do not respond to this culture condition. In bioreactor cultures, the amount of each excreted product is similar across a range of growth rates and limiting substrates, including O2-limitation. A set of mutants were generated in genes predicted to be involved in generating or regulating excretion of these compounds and tested for growth defects, and changes in excretion products. The phenotypes and associated metabolic flux modeling suggested that in M. buryatense 5GB1, formate and acetate are excreted in response to redox imbalance. Our results indicate that even under O2-starvation conditions, M. buryatense 5GB1 maintains a metabolic state representing a combination of fermentation and respiration metabolism.

SUBMITTER: Gilman A

PROVIDER: S-EPMC5652258 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C.

Gilman Alexey A Fu Yanfen Y Hendershott Melissa M Chu Frances F Puri Aaron W AW Smith Amanda Lee AL Pesesky Mitchell M Lieberman Rose R Beck David A C DAC Lidstrom Mary E ME

PeerJ 20171020

The bacteria that grow on methane aerobically (methanotrophs) support populations of non-methanotrophs in the natural environment by excreting methane-derived carbon. One group of excreted compounds are short-chain organic acids, generated in highest abundance when cultures are grown under O2-starvation. We examined this O2-starvation condition in the methanotroph Methylomicrobium buryatense 5GB1. The M. buryatense 5GB1 genome contains homologs for all enzymes ...[more]

PMID: 29062611

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Project description:Methylomicrobium buryatense 5GB1 is an obligate methylotroph which grows on methane or methanol with similar growth rates. It has long been assumed that the core metabolic pathways must be similar on the two substrates, but recent studies of methane metabolism in this bacterium suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach and a 13C tracer approach were taken to understand core carbon metabolism in M. buryatense 5GB1 during growth on methanol and to determine whether such differences occur. Our results suggest a systematic shift of active core metabolism in which increased flux occurred through both the Entner-Doudoroff (ED) pathway and the partial serine cycle, while the tricarboxylic acid (TCA) cycle was incomplete, in contrast to growth on methane. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol, and the results are consistent with predictions based on ATP and NADH changes. Transcriptomics analysis suggested that the changes in fluxes and metabolite levels represented results of posttranscriptional regulation. The combination of flux balance analysis of the genome-scale model and the flux ratio from 13C data changed the solution space for a better prediction of cell behavior and demonstrated the significant differences in physiology between growth on methane and growth on methanol.IMPORTANCE One-carbon compounds such as methane and methanol are of increasing interest as sustainable substrates for biological production of fuels and industrial chemicals. The bacteria that carry out these conversions have been studied for many decades, but gaps exist in our knowledge of their metabolic pathways. One such gap is the difference between growth on methane and growth on methanol. Understanding such metabolism is important, since each has advantages and disadvantages as a feedstock for production of chemicals and fuels. The significance of our research is in the demonstration that the metabolic network is substantially altered in each case and in the delineation of these changes. The resulting new insights into the core metabolism of this bacterium now provide an improved basis for future strain design.

Project description:UnlabelledMany methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive.ImportanceAerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.

Project description:Several of the metabolic enzymes in methanotrophic bacteria rely on metals for both their expression and their catalysis. The MxaFI methanol dehydrogenase enzyme complex uses calcium as a cofactor to oxidize methanol, while the alternative methanol dehydrogenase XoxF uses lanthanide metals such as lanthanum and cerium for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF This regulatory program, called the "lanthanide switch," is central to methylotrophic metabolism, but only some of its components are known. To uncover additional components of the lanthanide switch, we developed a chemical mutagenesis system in the type I gammaproteobacterial methanotroph "Methylotuvimicrobium buryatense" 5GB1C and designed a selection system for mutants unable to repress the mxaF promoter in the presence of lanthanum. Whole-genome resequencing for multiple lanthanide switch mutants identified several unique point mutations in a single gene encoding a TonB-dependent receptor, which we have named LanA. The LanA TonB-dependent receptor is absolutely required for the lanthanide switch and controls the expression of a small set of genes. While mutation of the lanA gene does not affect the amount of cell-associated lanthanum, it is essential for growth in the absence of the MxaF methanol dehydrogenase, suggesting that LanA is involved in lanthanum uptake to supply the XoxF methanol dehydrogenase with its critical metal ion cofactor. The discovery of this novel component of the lanthanide regulatory system highlights the complexity of this circuit and suggests that further components are likely involved.IMPORTANCE Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

Project description:Methylotuvimicrobium buryatense 5GB1C, a fast-growing gammaproteobacterial methanotroph, is equipped with two glycolytic pathways, the Entner-Doudoroff (ED) pathway and the Embden-Meyerhof-Parnas (EMP) pathway. Metabolic flux analysis and 13C-labeling experiments have shown the EMP pathway is the principal glycolytic route in M. buryatense 5GB1C, while the ED pathway appears to be metabolically and energetically insignificant. However, it has not been possible to obtain a null mutant in the edd-eda genes encoding the two unique enzymatic reactions in the ED pathway, suggesting the ED pathway may be essential for M. buryatense 5GB1C growth. In this study, the inducible P BAD promoter was used to manipulate gene expression of edd-eda, and in addition, the expression of these two genes was separated from that of a downstream gltA gene. The resulting strain shows arabinose-dependent growth that correlates with ED pathway activity, with normal growth achieved in the presence of ∼0.1 g/liter arabinose. Flux balance analysis shows that M. buryatense 5GB1C with a strong ED pathway has a reduced energy budget, thereby limiting the mutant growth at a high concentration of arabinose. Collectively, our study demonstrates that the ED pathway is essential for M. buryatense 5GB1C. However, no known mechanism can directly explain the essentiality of the ED pathway, and thus, it may have a yet unknown regulatory role required for sustaining a healthy and functional metabolism in this bacterium.IMPORTANCE The gammaproteobacterial methanotrophs possess a unique central metabolic architecture where methane and other reduced C1 carbon sources are assimilated through the ribulose monophosphate cycle. Although efforts have been made to better understand methanotrophic metabolism in these bacteria via experimental and computational approaches, many questions remain unanswered. One of these is the essentiality of the ED pathway for M. buryatense 5GB1C, as current results appear contradictory. By creating a construct with edd-eda and gltA genes controlled by P BAD and P J23101 , respectively, we demonstrated the essentiality of the ED pathway for this obligate methanotroph. It is also demonstrated that these genetic tools are applicable to M. buryatense 5GB1C and that expression of the target genes can be tightly controlled across an extensive range. Our study adds to the expanding knowledge of methanotrophic metabolism and practical approaches to genetic manipulation for obligate methanotrophs.

Dataset Information

Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C.

Publications

Oxygen-limited metabolism in the methanotroph <i>Methylomicrobium buryatense</i> 5GB1C.

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