Fluorescence-based analysis of the intracytoplasmic membranes of type I methanotrophs.

Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells. This fluorescence method is sensitive enough to show not only the characteristic shift in intracytoplasmic membrane formation that is present when methanotrophs are grown with or without copper, but also differences in intracytoplasmic membrane levels at intermediate copper concentrations. This technique can also be employed to monitor dynamic intracytoplasmic membrane changes in the same cell in real time under changing growth conditions. We anticipate that this approach will be of use to researchers wishing to visualize intracytoplasmic membranes who may not have access to electron microscopes. It will also have the capability to relate membrane changes in individual living cells to other measurements by fluorescence labelling or other single-cell analysis methods.