Proteomic Stable Isotope Probing Reveals Taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

Abstract

An estimated 50 gigatons of carbon is annually fixed within marine systems, of which heterotrophic microbial populations process nearly half. These communities vary in composition and activity across spatial and temporal scales, so understanding how these changes affect global processes requires the delineation of functional roles for individual members. In a step toward ascertaining these roles, we applied proteomic stable isotope probing to quantify the assimilation of organic carbon from DFAAs into microbial protein biomass, since the turnover of DFAAs accounts for a substantial fraction of marine microbial carbon metabolism that is directed into biomass production. We conducted experiments at two coastal North Pacific locations and found taxonomically distinct responses. This approach allowed us to compare amino acid assimilation by specific bacterioplankton populations and characterize their allocation of this substrate among cellular functions.

Microbial Community Structure–Function Relationships in Yaquina Bay Estuary Reveal Spatially Distinct Carbon and Nitrogen Cycling Capacities

“…Community DNA was extracted using the CTAB protocol described in ( Doherty et al, 2017 ). Total protein was extracted using SDS lysis method detailed in ( Bryson et al, 2016 , 2017 ). Extractions from 1 and 11 L of water yielded sufficient quantities of analytes for further analyses (1–5 μg DNA and 150–300 μg of protein, respectively).…”

“…Extracted proteins were digested with trypsin at room temperature (enzyme:substrate ratio of 1:100 w:w). Twenty-five micrograms of peptides were analyzed for each of the 12 metaproteomes using 2D-Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) with previously described conditions ( Bryson et al, 2016 ). LC-MS/MS measurements were performed on an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, MA, United States) ( Washburn et al, 2001 ).…”

“…Sipros Ensemble ( Guo et al, 2017 ) was used to search all MS spectra against a peptide database constructed from all metagenome CDS clustered at 100% identity using cd-hit ( Li and Godzik, 2006 ), and with reverse sequences of all peptides added as decoys to calculate false discovery rate (FDR) ( Bryson et al, 2016 ). Searches considered 7–60 residue peptides with a maximum of two missed tryptic cuts.…”

“…Searches considered 7–60 residue peptides with a maximum of two missed tryptic cuts. The two-peptide rule – one unique plus one shared peptide, or two unique peptides – was used to determine confident protein identifications, and a 1% FDR at the peptide level was implemented as in ( Bryson et al, 2016 ). Normalized balanced spectral counts (NBSC) of peptides for identified proteins were used as measures of protein relative abundances across all samples (Supplementary Table S2 ).…”

100 Days of marine Synechococcus–Ruegeria pomeroyi interaction: A detailed analysis of the exoproteome

“…Interestingly, it is between days 21 and 60 when R. pomeroyi shows an increased production of proteases (i.e., AAV95890 and AAV97448) and amidohydrolases (AAV97448 and AAV94260). Members of the Roseobacter group are known to preferentially target small nitrogen-rich DOM (Bryson et al, 2016;Teira et al, 2017), and hence, it is not surprising that these organisms will specialize in using these compounds when they are present. While the abundance of organic matter in ASW allows R. pomeroyi to shift its targeted polymeric DOM, in SW it shows a more sTable production of hydrolases throughout the 100-day co-culture, with only the increase of the pectate lyase AAV93776 (from 1.4% to 3.6%) and the protease AAV95890 (from < 0.01% to 0.13%) over time.…”