Using click chemistry to study microbial ecology and evolution

Abstract

Technological advances have largely driven the revolution in our understanding of the structure and function of microbial communities. Culturing, long the primary tool to probe microbial life, was supplanted by sequencing and other -omics approaches, which allowed detailed quantitative insights into species composition, metabolic potential, transcriptional activity, secretory responses and more. Although the ability to characterize “who’s there” has never been easier or cheaper, it remains technically challenging and expensive to understand what the diverse species and strains that comprise microbial communities are doing in situ, and how these behaviors change through time. Our aim in this brief review is to introduce a developing toolkit based on click chemistry that can accelerate and reduce the expense of functional analyses of the ecology and evolution of microbial communities. After first outlining the history of technological development in this field, we will discuss key applications to date using diverse labels, including BONCAT, and then end with a selective (biased) view of areas where click-chemistry and BONCAT-based approaches stand to have a significant impact on our understanding of microbial communities.

High amino acid osmotrophic incorporation by marine eukaryotic phytoplankton revealed by click chemistry

“…In any case, the fact that diatoms dominated the eukaryotic substrate incorporation, both in terms of BONCAT-positive cell abundance and area, supports that osmotrophy was the main pathway of HPG incorporation in the studied communities, as diatoms capable of phagotrophy are not known to date [ 5 , 16 ]. BONCAT may thus help complement the current omics efforts to better constrain the role of mixotrophy in organic matter flows in the ocean [ 16 , 68 ], and future efforts to synthetize BONCAT surrogate substrates other than HPG and AHA would widen its potential to explore eukaryotic osmotrophy [ 36 ].…”

“…However, the complexity, time-consuming and expensive nature of microautoradiography or nanoSIMS have discouraged an intense use of these techniques for the study of eukaryotic osmotrophy. Click chemistry-based approaches like biorthogonal noncanonical amino acid tagging (BONCAT) [ 32 , 33 ] have recently emerged as a promising alternative to visually identify translationally active microbial cells [ 34 - 36 ]. BONCAT uses synthetic amino acids (analogues for methionine) that when incorporated into cells can be fluorescently detected via copper-catalyzed alkyne–azide click chemistry.…”

Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation

“…Bio-orthogonal noncanonical amino acid tagging (BONCAT) was formally introduced by Dieterich and colleagues as a method for visualizing newly synthesized proteins in mammalian cells. It has since been used in bacteria, ,, in archaea, in microbial cultures, − in plant systems, and even for in vivo labeling in living organisms like fruit flies and mice. , BONCAT involves the incorporation of an azide or alkyne functionality into proteins that then can undergo click chemistry . Click chemistries refer to a set of chemical reactions that are readily catalyzed in aqueous solutions at atmospheric pressure and biologically compatible temperatures, with few toxic intermediates, and relatively fast reaction kinetics. ,, The azide–alkyne cycloaddition reaction is most widely used for BONCAT even though azide–phosphine reactions have also been demonstrated …”

Removal of soluble Se from mining influenced water by native mine site bacteria depends on the consortium composition

“…were dominant but did not correlate with e cient selenate reduction despite their genome encoding the SerABC enzyme. Future work using methods to track active organisms, for example using SIP 58 or BONCAT 59 , would be very useful in addition to consortia studies.…”