Application of the Fluorescence-Activating and Absorption-Shifting Tag (FAST) for Flow Cytometry in Methanogenic Archaea

Norman Adlung, Silvan Scheller (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

1 Citation (Scopus)

Abstract

Methane-producing archaea play a crucial role in the global carbon cycle and are used for biotechnological fuel production. Methanogenic model organisms such as Methanococcus maripaludis and Methanosarcina acetivorans have been biochemically characterized and can be genetically engineered by using a variety of existing molecular tools. The anaerobic lifestyle and autofluorescence of methanogens, however, restrict the use of common fluorescent reporter proteins (e.g., GFP and derivatives), which require oxygen for chromophore maturation. Recently, the use of a novel oxygen-independent fluorescent activation and absorption-shifting tag (FAST) was demonstrated with M. maripaludis. Similarly, we now describe the use of the tandem activation and absorption-shifting tag protein 2 (tdFAST2), which fluoresces when the cell-permeable fluorescent ligand (fluorogen) 4-hydroxy-3, 5-dimethoxybenzylidene rhodanine (HBR-3, 5DOM) is present. Expression of tdFAST2 in M. acetivorans and M. maripaludis is noncytotoxic and tdFAST2:HBR-3, 5DOM fluorescence is clearly distinguishable from the autofluorescence. In flow cytometry experiments, mixed methanogen cultures can be distinguished, thereby allowing for the possibility of high-throughput investigations of the characteristic dynamics within single and mixed cultures.

Original languageEnglish
Article numbere0178622
JournalApplied and Environmental Microbiology
Volume89
Issue number4
DOIs
Publication statusPublished - Apr 2023
MoE publication typeA1 Journal article-refereed

Funding

This project was supported by grants from Novo Nordisk Foundation and the Academy of Finland (grant NNF19OC0055464 to N.A. and grants NNF19OC0054329 and 326020 to S.S.).

Keywords

  • Archaea/metabolism
  • Flow Cytometry
  • Methane/metabolism
  • Methanosarcina/metabolism
  • Methanosarcina
  • fluorescence
  • flow cytometry
  • Methanogenic archaea
  • FAST

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