Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

Abstract

Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and Synthetic Biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed Super-Resolution Imaging techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have however not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform Single-Molecule Localization Microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photo-convertible fluorescent protein mEos3.2, enabling Photo-Activated Localization Microscopy (PALM) in most Mycoplasma species. We also describe the application of direct STochastic Optical Reconstruction Microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development SMLM strategies to study these organisms in the future.

Genome Engineering in Mycoplasma gallisepticum Using Exogenous Recombination Systems

“…Alternatively, the lack of transformants with the first set of plasmids may have resulted from poor expression of the antibiotic resistance marker with the p438 promoter. However, given that the p438 promoter is functional in Mgal , this second hypothesis appears to be less plausible. Finally, five transformants were randomly selected for each plasmid and further characterized by single-primer PCR and sequencing to determine the insertion sites of the transposon (Figure S3, Table S3).…”