Quantitative Proteomic Analysis of Metabolic Regulation by Copper Ions in Methylococcus capsulatus (Bath)

Abstract

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C 1 metabolism pathways in this methanotroph and clarified the interrelationships between them.Methanotrophs are a unique group of Gram-negative bacteria that grow aerobically on methane and utilize methane as the sole source of carbon and energy. There has been considerable interest in methanotrophs over the past 30 years, since they can be used to produce single-cell bulk chemicals such as propylene oxide. The ability of these bacteria to co-oxidize a wide range of alkanes, alkenes, and substituted aliphatic compounds has been exploited in bioremediation processes, for example, in the degradation of key pollutants such as trichloroethylene in soil and groundwater. Methanotrophs also play an important role in the global methane cycle (1, 2).Methanotrophs are classified into three distinct types. Type I methanotrophs show disk-like intracellular membranes and use the RuMP (ribulose 5-phosphate) pathway for carbon assimilation. In contrast, Type II methanotrophs possess intracellular membranes in the cell periphery and use the serine pathway to degrade the formaldehyde produced. Methylococcus capsulatus (Bath) is classified as a Type X methanotroph (1), since it shows the physiological properties of both Type I and II methanotrophs, but it develops Type I intracytoplasmic membranes.Two methane monooxygenases (MMO) 1 catalyze the methane oxidation process in methanotrophs, converting methane to methanol. All methanotrophs express a membrane-bound copper-containing particulate MMO (pMMO) (1, 3), while Type X and a few Type II methanotrophic bacteria are capable of producing a second, soluble form (soluble methane monooxygenase, sMMO). Copper ions are known to switch the methane oxidation from sMMO to pMMO. At low copper-to-biomass ratios, the cytoplasmic sMMO is the dominant MMO. When the cells are grown at high copper-to-biomass ratios, pMMO is expressed and produced instead in the plasma membrane.Because of the unique role of copper ions in regulating the switch between sMMO and pMMO, we have applied cICAT (cleavable isot...

Quantitative proteomic profiling of the Escherichia coli response to metallic copper surfaces

“…However, quantitative proteomic analysis either by labeled or label-free methods have been used only rarely for the elucidation of cellular defenses and stress response mechanisms against copper (Bagwell et al 2010;Kao et al 2004). Similar studies with acute copper surface-exposed cells are lacking.…”

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

“…It has also been shown by proteomics that additional steps in the oxidation of methane to carbon dioxide are overexpressed with increasing availability of copper, such as that of proteins involved in lipid, cell wall, and membrane synthesis (66,115). Conversely, the ability of methanotrophs to direct carbon from methane to poly-3-hydroxybutyrate increases with decreasing copper availability (66,116), suggesting that to some extent energy metabolism of methanotrophs is controlled by copper.…”

Molecular Ecology Techniques for the Study of Aerobic Methanotrophs

“…Comparisons of the genome sequences of these representative obligate methanotrophs with the genome sequence of the facultative methanotroph Methylocella silvestris BL2 and with other obligate and facultative methylotrophs may well reveal the molecular basis for the obligate nature of most extant methanotrophs. Proteomic analyses of regulation of methanotrophy by copper ions (68) and of the outer membrane subproteome (6) in Methylococcus capsulatus Bath have also recently been published. These genomic and proteomic analyses provide a wealth of information for studying the biology of methane oxidation (24,69) and may provide insights into how methanotrophy is regulated under different environmental conditions.…”