Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria
Open Access
- 6 November 2021
- journal article
- research article
- Published by Oxford University Press (OUP) in The ISME Journal
- Vol. 16 (4), 958-971
- https://doi.org/10.1038/s41396-021-01144-0
Abstract
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.Keywords
This publication has 140 references indexed in Scilit:
- MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and UsabilityMolecular Biology and Evolution, 2013
- Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell DivisionACS Chemical Biology, 2012
- Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamilyThe ISME Journal, 2011
- Fast and accurate long-read alignment with Burrows–Wheeler transformBioinformatics, 2010
- Thauera butanivorans sp. nov., a C2-C9 alkane-oxidizing bacterium previously referred to as 'Pseudomonas butanovora'International Journal of Systematic and Evolutionary Microbiology, 2009
- The Sequence Alignment/Map format and SAMtoolsBioinformatics, 2009
- The Ribosomal Database Project: improved alignments and new tools for rRNA analysisNucleic Acids Research, 2008
- Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In VivoCell Chemical Biology, 2007
- daime, a novel image analysis program for microbial ecology and biofilm researchEnvironmental Microbiology, 2005
- A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUESThe Journal of cell biology, 1973