Award: OCE-1850692

Marine bacteria consume roughly half of all organic carbon produced by phytoplankton, yet the specific molecules they metabolize remain largely uncharacterized.  Our project sought to reveal these molecular diets by linking the messenger-RNA profiles (transcriptomes) of key bacterial taxa to the dissolved organic carbon (DOC) compounds available in their environment.  Achieving this goal required a two-stage strategy: (i) calibrating how laboratory cultures of the model bacterium Ruegeria pomeroyi DSS-3 alter gene transcription when supplied with defined carbon compounds, and (ii) applying those calibrations to complex estuarine communities where DOC sources and sinks fluctuate with light, season, and hydrology.

In continuous-culture experiments (chemostats), DSS-3 was grown under carbon limitation by twelve individual substrates spanning simple acids, aromatics, and environmentally relevant compounds such as glycine betaine.  Transcriptome sequencing of 42 chemostats revealed that genes encoding substrate-specific transporters and the first enzymatic steps of catabolic pathways serve as the most reliable indicators of compound availability, whereas constitutively expressed central-metabolism genes (e.g., the tricarboxylic-acid cycle) provide little diagnostic value.  Follow-up batch-culture studies using mixtures of seven substrates demonstrated that these indicator genes retain their specificity even in chemically complex media, providing a practical reference table for interpreting environmental transcriptomes without requiring exhaustive chemical analyses.

Because pandemic restrictions reduced our capability for field sampling, we established large-volume microcosms with water from North Carolina’s Pamlico Sound and manipulated light climate (photosynthetically active radiation (PAR), ultraviolet radiation (UV), and darkness) across summer and spring deployments.  Measurements of DOC quantity and quality, microbial respiration, and community composition showed that light regime and season jointly restructure both the carbon pool and its microbial consumers.  Metagenomic profiling indicated that Roseobacters and Flavobacteria dominated under light, whereas distinct heterotrophs flourished in the dark and ultraviolet treatments, particularly those specializing on C1 compounds.  Ongoing metatranscriptomic analyses are now mapping the laboratory-derived genetic markers onto these field communities, allowing us to infer which DOC fractions were preferentially utilized under each condition.

To test whether calibrated indicator genes can function as a biosensor we introduced DSS-3 and related isolates into filtered microcosm water over the course of the experiment.  Enhanced growth and elevated expression of substrate-specific genes late in incubations indicate that DOC became progressively more bioavailable, information that classical bulk measurements cannot provide.  Together, these results establish a mechanistic framework for reading community transcriptomes as real-time reporters of organic-carbon dynamics in coastal systems.

The intellectual merit of this work lies in delivering an experimentally validated gene-to-substrate map that can be applied directly to environmental RNA data, thereby transforming descriptive metatranscriptomics into a quantitative tool for carbon-cycle research.  By pinpointing the molecules fueling microbial metabolism, the approach improves our ability to model oceanic carbon fluxes and predict how they will respond to climate-driven changes in primary production and DOC composition.

Broader impacts were achieved through a multi-tiered education program.  We partnered with the Scientific Research and Education Network (SciREN) to create an inquiry-based lesson on aquatic metabolism aligned with North Carolina science standards.  This module, coupled with portable oxygen-optode instrumentation, was deployed in for two years in classrooms at Riverside High School, engaging roughly fifty students in measuring primary production and respiration in their local river.  Four undergraduates, two graduate students, and a high-school intern received training in high-throughput sequencing, bioinformatics, and quantitative ecology, cultivating a new cohort of scientists fluent in environmental ‘omics.  All sequence data have been deposited in the NCBI Sequence Read Archive. The results from this research have been presented at five international conferences, two Master’s students’ theses, and two doctoral student’s dissertations, all with manuscripts currently in preparation for submission.

By converting microbial genes into molecular signposts of carbon utilization, this project equips researchers with a powerful, culture-independent means to monitor the fate of organic matter from estuaries to the open ocean, providing insights essential for forecasting the ocean’s role in Earth’s carbon cycle.

Last Modified: 05/29/2025
Modified by: Scott Michael Gifford