Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria.

Autor: Sakoula D; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands. dimitra.sakoula@univie.ac.at.; Division of Microbial Ecology, Center for Microbiology and Environmental Systems Science, University of Vienna, Althanstraße 14, 1090, Vienna, Austria. dimitra.sakoula@univie.ac.at., Smith GJ; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Frank J; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands.; Soehngen Institute of Anaerobic Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Mesman RJ; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Kop LFM; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Blom P; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Jetten MSM; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands.; Soehngen Institute of Anaerobic Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., van Kessel MAHJ; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands., Lücker S; Department of Microbiology, RIBES, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, the Netherlands. s.luecker@science.ru.nl.
Jazyk: angličtina
Zdroj: The ISME journal [ISME J] 2022 Apr; Vol. 16 (4), pp. 958-971. Date of Electronic Publication: 2021 Nov 06.
DOI: 10.1038/s41396-021-01144-0
Abstrakt: The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.
(© 2021. The Author(s).)
Databáze: MEDLINE
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