Fluorescence‐based analysis of the intracytoplasmic membranes of type I methanotrophs

Abstract

Summary 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.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum

“…The direct coupling mechanism consists on the fact that the same redox cofactor co‐oxidized with methane, is responsible for the further oxidation of methanol to formaldehyde. Particulate methane monooxygenases (pMMOs), are located in so called intracytoplasmic membranes, recent fluorescence based experiments (Whiddon, Gudneppanavar, Hammer, West, & Konopka, 2019) have shown that these structures are connected to the cytoplasmic membrane, therefore redox carriers such as cytochromes, can be potentially exchanged between pMMOs and the respiratory chain. The direct coupling mechanism of methane oxidation allows M. alcaliphilum to obtain higher biomass yields on methane than those potentially achievable by type II methanotrophs such as bacteria from the genus Methylocystis (Bordel, Rodríguez, et al, 2019).…”

Intracytoplasmic membranes develop inGeobacter sulfurreducensunder thermodynamically limiting conditions

“…The ICM area as a fraction of total cell area had a median value of 5.7% in the electrode biofilm grown at -0.07 V and 4.2% in the fumarate cells, but there is a high variance in fractional area with some cells having over 30% of the area occupied by ICM (Figure S5 and Figure S6). This is lower than the cell area occupied by ICMs in methanotrophs 41 . By applying identical image analysis (ICM counting) to cells collected from different conditions, we found that a change in electron acceptor significantly affects the frequency of G. sulfurreducens cells displaying ICM (Figure 4a).…”

“…ICM abundance differences-We can also identify the presence of ICM in G. sulfurreducens with confocal microscopy. A similar technique has been used to identify ICM in methanotrophs 41 . We took confocal images of fixed G. sulfurreducens biofilm cells grown at 10 different anode potentials, or from cell suspensions grown with fumarate as electron acceptor, and each image had numerous individual cells (Table S1).…”

“…Producing an ICM must be a significant energy investment for a cell, and the structural organization of the ICM in G. sulfurreducens suggests a specialized function outside of lipid storage. The production of ICMs explains why G. sulfurreducens is higher in lipid content than other gram negative bacteria 46 , as the production of ICM in other bacteria causes elevated lipid fractions 41,47 .…”

Structure and activity of particulate methane monooxygenase arrays in methanotrophs

“…To accommodate such large quantities of pMMO, copper triggers the formation of intracytoplasmic membranes (ICMs) that fill the cell 11 . These methanotrophic ICMs, detected more than 50 years ago 12 , have been imaged using electron 7 , 11 , 13 , 14 and fluorescence 15 microscopies and cryo-electron tomography (cryoET) 16 , but their mechanism of biogenesis is not known. In addition, the arrangement of pMMO within the ICMs and the relationship between this environment and pMMO function remain unclear.…”