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

Abstract

Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual primary production, yet defining how substrate utilization preferences and resource partitioning structure microbial communities remains a challenge. In this study, proteomic stable isotope probing (proteomic SIP) was used to characterize population-specific assimilation of dissolved free amino acids (DFAAs), a major source of dissolved organic carbon for bacterial secondary production in aquatic environments. Microcosms of seawater collected from Newport, Oregon, and Monterey Bay, California, were incubated with 1 µM ¹³C-labeled amino acids for 15 and 32 h. The taxonomic compositions of microcosm metaproteomes were highly similar to those of the sampled natural communities, with Rhodobacteriales, SAR11, and Flavobacteriales representing the dominant taxa. Analysis of ¹³C incorporation into protein biomass allowed for quantification of the isotopic enrichment of identified proteins and subsequent determination of differential amino acid assimilation patterns between specific bacterioplankton populations. Proteins associated with Rhodobacterales tended to have a significantly high frequency of ¹³C-enriched peptides, opposite the trend for Flavobacteriales and SAR11 proteins. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of ¹³C-enriched spectra at time point 2. Alteromonadales proteins also had a significantly high frequency of ¹³C-enriched peptides, particularly within ribosomal proteins, demonstrating their rapid growth during incubations. Overall, proteomic SIP facilitated quantitative comparisons of DFAA assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population level metabolic responses to resource acquisition in complex microbial communities. IMPORTANCE 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.

Keywords: environmental microbiology; marine microbiology; microbial communities; microbial ecology; proteomics.

FIG 1

Overview of proteomic SIP experiment. GFF, glass fiber filters; PES, polyether sulfone.

FIG 2

Peptide identifications by sequence source. “Metagenome” refers to peptides identified in predicted protein coding sequences (CDS) from an assembled metagenome from the Monterey Bay (MB) samples. “Isolate” refers to peptides identified in CDS from reference genomes. “Shared” refers to peptides found in CDS from both sources.

FIG 3

Relative abundance of protein identifications by taxonomy and COG functional category. (A) Stacked bar chart of proportion of total protein identifications in each sample for abundant orders of Bacteria. “Other” refers to low abundance and unclassified proteins, including identified eukaryote and Archaea proteins. (B) Heat map of proportion of protein identifications in each sample for general COG categories represented in the metaproteomes.

FIG 4

Histogram of highly labeled peptide spectral matches (PSM). The histogram depicts the proportion of labeled spectra (¹³C enrichment of ≥2%) from 4% to 99% ¹³C enrichment within 3% enrichment bins. Each distribution depicts bars on a relative scale of 0 to 10% of total labeled spectra. Label frequency and average enrichment values for the samples are presented in Table 1.

FIG 5

Label frequency and average enrichment of taxa. (A and B) more-represented (A) and less-represented (B) orders in the metaproteomes. The z-scores of label frequency (x axis) and average enrichment (y axis) based on comparisons of observed values with distributions under the null model are shown. The shaded area represents the inner 95% of values under a standard normal distribution. Significance testing is described in Materials and Methods.