Dataset Information

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath.

ABSTRACT: The increase in greenhouse gases with high global warming potential such as methane is a matter of concern and requires multifaceted efforts to reduce its emission and increase its mitigation from the environment. Microbes such as methanotrophs can assist in methane mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome-scale metabolic model (GSMM) of an obligate methanotroph, Methylococcus capsulatus str. Bath was reconstructed. The model contains 535 genes, 899 reactions and 865 metabolites and is named iMC535. The predictive potential of the model was validated using previously-reported experimental data. The model predicted the Entner-Duodoroff pathway to be essential for the growth of this bacterium, whereas the Embden-Meyerhof-Parnas pathway was found non-essential. The performance of the model was simulated on various carbon and nitrogen sources and found that M. capsulatus can grow on amino acids. The analysis of network topology of the model identified that six amino acids were in the top-ranked metabolic hubs. Using flux balance analysis, 29% of the metabolic genes were predicted to be essential, and 76 double knockout combinations involving 92 unique genes were predicted to be lethal. In conclusion, we have reconstructed a GSMM of a methanotroph Methylococcus capsulatus str. Bath. This is the first high quality GSMM of a Methylococcus strain which can serve as an important resource for further strain-specific models of the Methylococcus genus, as well as identifying the biotechnological potential of M. capsulatus Bath.

SUBMITTER: Gupta A

PROVIDER: S-EPMC6613435 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph <i>Methylococcus capsulatus</i> str. Bath.

Gupta Ankit A Ahmad Ahmad A Chothwe Dipesh D Madhu Midhun K MK Srivastava Shireesh S Sharma Vineet K VK

PeerJ 20190614

The increase in greenhouse gases with high global warming potential such as methane is a matter of concern and requires multifaceted efforts to reduce its emission and increase its mitigation from the environment. Microbes such as methanotrophs can assist in methane mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome-scale metabolic model (GSMM) of an obligate methanotroph, <i>Methylococcus capsulatus</i> str. Bath was reconstructed. The model contains 535 ge ...[more]

PMID: 31316867

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Project description:P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

Project description:The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Dataset Information

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath.

Publications

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph <i>Methylococcus capsulatus</i> str. Bath.

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