Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms

Mathilde Lindivat; Aud Larsen; Ole Kristian Hess-Erga; Gunnar Bratbak; Ingunn Alne Hoell

doi:10.3389/fmicb.2020.01929

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms

Front Microbiol. 2020 Aug 18:11:1929. doi: 10.3389/fmicb.2020.01929. eCollection 2020.

Authors

Mathilde Lindivat¹, Aud Larsen^{2

3}, Ole Kristian Hess-Erga⁴, Gunnar Bratbak³, Ingunn Alne Hoell¹

Affiliations

¹ Faculty of Engineering and Science, Institute of Safety Chemistry and Biomedical Laboratory Sciences, Western Norway University of Applied Sciences, Haugesund, Norway.
² NORCE Environment, NORCE Norwegian Research Centre AS, Bergen, Norway.
³ Department of Biological Sciences, University of Bergen, Bergen, Norway.
⁴ Norwegian Institute for Water Research, Bergen, Norway.

Abstract

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

Keywords: bioorthogonal non-canonical amino acid tagging; flow cytometry; marine microbes; protein synthesis; single cell activity.