Compartmentalization and spatial organization play an important role in several metabolic processes; however bacteria lack organelles to accomplish this. In response, some bacteria produce intracytoplasmic membranes (ICMs) that allow them to pool and segregate metabolites, which in turn allow them to outcompete other bacteria for limited resources. Intracytoplasmic membranes may also house specialized enzymes that allow the bacteria to utilize unique metabolites. Among these are methanotrophic bacteria, which are able to use methane as their sole carbon and energy source. Methanotrophs house one of their primary methane oxidizing enzymes, particulate methane monooxygenase (pMMO), within their ICMs. These bacteria are of interest due to their ability to break down a potent greenhouse gas and use it build high amounts of potentially useful lipids. The ICMs of these methanotrops are of particular interest as they present a target for exploitation as a source of biofuels. ICMs can be visualized by techniques such as TEM or AFM, but these techniques cannot capture the dynamic properties of these membranes in vivo. Here we utilize fluorescence microscopy to investigate the in vivo dynamics of ICMs two methanotroph strains Methylomicrobium alcaliphilum 20z and Methylosinus trichosporium OB3b using fluorescent protein fusions and lipophilic dyes. We also use redox sensitive dyes to visualize cellular respiration, allowing us to relate ICM levels to cell vitality at the single cell level. Finally we demonstrate that these techniques can also be applied to other bacteria that produce ICMs such as photosynthetic or nitrogen fixing bacteria.

Single cell analysis of the intracytoplasmic membranes of methanotrophs via fluorescence microscopy

252nd National Meeting (2016)

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