Rapid Assessment of Intracytoplasmic Membranes in Bacteria by Fluorescence Microscopy

Abstract

Intracytoplasmic membranes (ICMs) are known to play an important biological role in bacteria, but many details about these membrane systems remain a mystery. Previously, the primary tool for observing these ICMs was electron microscopy, which limited data collection due to long preparation times and inability to look at dynamic membrane changes. Here we describe a method to rapidly analyze intracytoplasmic membrane structures on a single cell level using fluorescence microscopy and lipophilic dyes on live cells. This allows us to rapidly determine whether or not individual cells possess ICMs. Furthermore we are able to track the growth of multiple single cells over time to elucidate the poorly understood mechanisms by which these membranes are gained or lost with time. Growth conditions also can be altered during imaging to observe how cells immediately react under varying conditions. This technique can be coupled to the use of other fluorescence imaging techniques, such as protein localization via fluorescent protein fusions, or quantitative methods to analyze relative abundance of lipids or proteins. These methods have primarily been applied to methanotropic bacteria, such as Methylomicrobium alcaliphilum 20z and Methylosinus trichosporium OB3b. Methanotrophs are a type of bacteria that can use single carbon molecules, such as methane, as their sole source of energy and carbon. These methanotrophs produce the membrane bound enzyme, particulate methane monooxygenase (pMMO) which is capable of oxidizing C-H bonds. Our goal is to understand the dynamic relationship between ICMs and pMMO.