UC San Diego

Recent research has shown the potential of bioorthogonal noncanonical amino acid tagging (BONCAT) methods in studying microbial activity in marine environments in situ without the use of a radioisotope label. Click chemistry with fluorescent dyes in addition to BONCAT allows for the identification of the marine microbial community that actively synthesizes proteins. When used in this way, community fluorescence intensity relates directly to total protein synthesis and is thus a reasonable proxy for bacterial production. Here, we present a BONCAT protocol with flow cytometry (FCM) tested on the analysis of bacterial production across a broad productivity gradient in the California Current ecosystem. Our method utilizes the non-canonical methionine substitute, L-azidohomoalanine (AHA) and strain-promoted click chemistry steps. The BONCAT-FCM method was first developed and tested on Escherichia coli cultures and coastal marine environmental samples. Samples processed using BONCAT-FCM were then directly compared to the 3H-Leucine assay commonly used to estimate bacterial production on the California Current Ecosystem (CCE) P2107 process cruise. 210 samples were collected from varying depths over sampling cycles of three discrete water parcels. Similar bacterial activity trends were observed between the sampling cycles though the overall agreement between both protocols was weak. The results show the potential of BONCAT to act as a proxy for bacterial production in environments were radioisotopic labelling isn’t feasible although more refinement of the BONCAT-FCM protocol is needed to reduce variability in measurements. Additionally, this method has greater potential for downstream analysis with fluorescence activated cell sorting (FACS) and subsequent genomic studies.