Systemsbiology Studies of the Methanotrophic Methylococcus capsulatus (Bath)
Date Issued
2004
Date
2004
Author(s)
Kao, Wei-Chun
DOI
en-US
Abstract
Copper ions are known to switch the methane oxidation from the soluble methane monooxygenase (sMMO) to the particulate methane monooxygenase (pMMO) in certain strains of methanotrophic bacteria. Aside from being a transcriptional switch, copper is also a metabolic activator, stimulating the production of high concentrations of pMMO and lipid biosynthesis to form the extensive networks of intracellular membranes in which the membrane-bound pMMO resides. To elucidate the molecular mechanism associated with this copper ion regulation, we have applied genomic sequencing and large-scale comparative quantitative proteome analysis to the cytosolic and membrane-associated proteins in Methylococcus capsulatus (Bath) grown under different copper-to-biomass ratios. Proteins associated with the membrane and the cytosolic portions of the proteome under different copper ion concentrations were differentially labeled with an acid cleavable isotope-coded affinity tag (cICAT) reagent, and the labeled peptides were separated by two-dimensional chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The study confirmed the expression of 682 unique proteins, of which 623 have not been sequenced previously. In 682 proteins identified, 68 and 60 proteins were overexpressed at least two fold in the absence and presence of Cu, respectively. These 68 proteins are mainly responsible for cellular signaling process while the 60 proteins cover methane and carbohydrate metabolic enzymes, indicating a role of copper in expression of the genes associated with metabolism of the organism downstream of methane oxidation.
The metabolism route of M. capsulatus (Bath) was also clarified. Levels of the enzymes involved in the C1 metabolism (RuMP, RuBP, serine, and H4MPT pathway) of the organism were dramatically affected, most notably, the soluble and particulate forms of the methane monooxygenase that mediate the methane oxidation under copper-deficient and copper-rich medium, respectively. In addition, high expression levels were observed for a number of key enzymes involved in the C1 metabolism pathway, suggesting a regulatory role of copper ions in regulating the expression of genes involved in the metabolism of the organism downstream of methane oxidation as well.
Hemerythrin was observed to be overexpressed about three fold in high copper environment. In order to prove the existence and expression status of M. capsulatus (Bath) hemerythrin, purification and spectroscopic studies were undertaken. The hemerythrin was purified successfully by desalting, ion-exchange, and gel-filtration chromatography techniques, and UV-visible spectrum revealed that the absorbance of M. capsulatus (Bath) hemerythrin was in accord with those previously reported for hemerythrins from higher organisms. A molecular weight estimation indicated that the native hemerythrin in M. capsulatus (Bath) is most likely a monomer, in contrast to hemerythrins from other organisms, where they usually exist as a tetramer or higher oligomers.
In summary, we have demonstrated in this work the first applications of the genomic sequencing of M. capsulatus (Bath): the quantitative proteomic analysis of copper regulation, and the discovery and purification of a native hemerythrin in bacteria. The present Systemsbiology study of M. capsulatus (Bath) has not only provided a genomic, proteomic and metabolic overview of this organism, but also clarified a number of long standing evolutionary issues, including elucidation of the C1 metabolic pathways and oxygen transporting mechanisms in this interesting methanotroph.
Subjects
系統生物學
微生物代謝
蛋白質體學
嗜甲烷菌
proteomics
ICAT
methanotrophic bacteria
microbiol metabolism
systemsbiology
bioinformatics
Type
thesis
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