Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)

Bioorthogonal noncanonical amino acid tagging (BONCAT) is a recently developed method for studying microbial in situ activity. This technique is based on the in vivo incorporation of artificial amino acids that carry modifiable chemical tags into newly synthesized proteins. BONCAT has been demonstrated to be effective in labeling the proteomes of a wide range of taxonomically and physiologically distinct Archaea and bacteria without resulting in preferential synthesis or degradation of proteins. After chemical fixation of cells, surrogate-containing proteins can be detected by whole-cell fluorescence staining using azide-alkyne click chemistry. When used in conjunction with rRNA-targeted fluorescence in situ hybridization (FISH), BONCAT allows the simultaneous taxonomic identification of a microbial cell and its translational activity. Rather than studying the bulk proteome, BONCAT is able to specifically target proteins that have been expressed in reaction to an experimental condition. BONCAT-FISH thus provides researchers with a selective, sensitive, fast, and inexpensive fluorescence microscopy technique for studying microbial in situ activity on an individual cell level. This protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, l-azidohomoalanine (AHA) and l-homopropargylglycine (HPG), which are both surrogates of l-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT studies and how they can be overcome