AUTHOR=Recalde Alejandra , Abdul-Nabi Jasmin , Junker Pierre , van der Does Chris , Elsässer Jana , van Wolferen Marleen , Albers Sonja-Verena TITLE=The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius JOURNAL=Frontiers in Microbiology VOLUME=15 YEAR=2024 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1445186 DOI=10.3389/fmicb.2024.1445186 ISSN=1664-302X ABSTRACT=Introduction

Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius.

Methods

We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy.

Results

Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments.

Discussion

These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.