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.
Highlights
Autotrophic ammonia- and methane-oxidizing bacteria (AOB and MOB, respectively) [1] are ubiquitous in the environment [2] and are of high biotechnological interest [3,4,5,6,7,8,9]
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. We show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkaneoxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes
activity-based protein profiling (ABPP)-based protocols have been successfully employed for the study of many microbial protein families, such as proteases, kinases, hydrolases, and glycosidases [20, 29]
Summary
Autotrophic ammonia- and methane-oxidizing bacteria (AOB and MOB, respectively) [1] are ubiquitous in the environment [2] and are of high biotechnological interest [3,4,5,6,7,8,9]. ABPP techniques employ bifunctional enzyme probes that feature (i) a reactive group, which covalently binds to the active site and thereby inhibits the enzyme, and (ii) an ethynyl or azide group that allows the attachment of a reporter group (e.g., fluorophores or biotin) to the enzyme via a Cu(I)-catalyzed alkyneazide cycloaddition (CuAAC) reaction [21] These protocols exhibit high specificity for the targeted enzymes as sufficient accessibility of the bifunctional enzyme probe to the active site and structural and/or chemical similarity with the substrate are necessary [20]. Enzymes in the copper-containing membrane monooxygenase (CuMMO) family catalyze diverse reactions, including the oxidation of ammonia, methane, and simple short-chain hydrocarbons [34]. These CuMMO enzymes generally exhibit high genetic, structural, and catalytic similarities [35,36,37].
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