Physiological Features during the Copper Switch in Methylococcus Capsulatus (Bath)

Abstract

forms (particulate or soluble) of methane monooxygenase enzyme in Methylococcus capsulatus (Bath). Under growth conditions of high copper-to-biomass ratio, the particulate enzyme is expressed while the soluble enzyme is up-regulated when this ratio is low. It is of interest to explore physiological features of cells in response to copper addition. To this end, M. capsulatus was grown on Nitrate Mineral Salts medium in a bioreactor and 30 µM copper was added at a dilution rate of 3.3 ml min -1 at steady state . Samples were collected at different time points and assayed for soluble methane monooxygenase (sMMO) expression using the naphthalene assay, SDS-PAGE of total cell protein and copper measurements. The results showed that the activity of sMMO was gradually decreased upon gradual copper addition until it became fully inactive after 4 hours of adding copper. Furthermore, the intracellular, extracellular and copper associated with biomass increased by adding copper over the time tested. Eleven bands from the SDS-PAGE gel of sMMO-expressing cells and pMMO-expressing cells, were analyzed by mass spectrometry. The results highlighted that 128 proteins were identified from 792 polypeptides detected from the eleven bands analysed. Typically, 506 polypeptides from sMMO-expressing cells (corresponding to 87 proteins) and 236 from pMMO-expressing cells (corresponding to 41) were identified. Collectively, the results confirmed that copper negatively regulates sMMO and induces pMMO in M. capsulatus.