Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.
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ABSTRACT:
SUBMITTER: Stolyar S
PROVIDER: S-EPMC95072 | biostudies-literature | 2001 Mar
REPOSITORIES: biostudies-literature
Publications
Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.
Stolyar S S Franke M M Lidstrom M E ME
Journal of bacteriology 20010301 5
The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth. ...[more]
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Project description:Background: Genome-scale metabolic models allow researchers to calculate yields, to predict consumption and production rates, and to study the effect of genetic modifications in silico, without running resource-intensive experiments. While these models have become an invaluable tool for optimizing industrial production hosts like Escherichia coli and S. cerevisiae, few such models exist for one-carbon (C1) metabolizers. Results: Here, we present a genome-scale metabolic model for Methylococcus capsulatus (Bath), a well-studied obligate methanotroph, which has been used as a production strain of single cell protein (SCP). The model was manually curated, and spans a total of 879 metabolites connected via 913 reactions. The inclusion of 730 genes and comprehensive annotations, make this model not only a useful tool for modeling metabolic physiology, but also a centralized knowledge base for M. capsulatus (Bath). With it, we determined that oxidation of methane by the particulate methane monooxygenase could be driven both through direct coupling or uphill electron transfer, both operating at reduced efficiency, as either scenario matches well with experimental data and observations from literature. Conclusion: The metabolic model will serve the ongoing fundamental research of C1 metabolism, and pave the way for rational strain design strategies toward improved SCP production processes in M. capsulatus.
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Project description:Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the well-characterized soluble MMO contains a bis-mu-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the alpha subunit (pmoB) of the alphabetagamma trimer and a site at the interface of the betagamma subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1-2 iron atoms per alphabetagamma show the highest catalytic activity. We have employed Mössbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per alphabetagamma of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the "Zn site" that accommodates the diiron center in active pMMO.
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Project description:The Gram-negative methanotroph Methylococcus capsulatus (Bath) was recently demonstrated to abrogate inflammation in a murine model of inflammatory bowel disease, suggesting interactions with cells involved in maintaining mucosal homeostasis and emphasizing the importance of understanding the many properties of M. capsulatus. Secreted proteins determine how bacteria may interact with their environment, and a comprehensive knowledge of such proteins is therefore vital to understand bacterial physiology and behavior. The aim of this study was to systematically analyze protein secretion in M. capsulatus (Bath) by identifying the secretion systems present and the respective secreted substrates. Computational analysis revealed that in addition to previously recognized type II secretion systems and a type VII secretion system, a type Vb (two-partner) secretion system and putative type I secretion systems are present in M. capsulatus (Bath). In silico analysis suggests that the diverse secretion systems in M.capsulatus transport proteins likely to be involved in adhesion, colonization, nutrient acquisition and homeostasis maintenance. Results of the computational analysis was verified and extended by an experimental approach showing that in addition an uncharacterized protein and putative moonlighting proteins are released to the medium during exponential growth of M. capsulatus (Bath).
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Project description:Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme.
| S-EPMC1163879 | biostudies-other