Transcriptional Markers of Organic Substrate Utilization in a Marine Bacterium

Marine dissolved organic carbon is a chemically complex substrate pool that fuels heterotrophic bacteria, yet it remains difficult to determine which compounds are used by specific microbes. Bacterial transcriptomes offer a potential biosensor of substrate availability, but the reliability of this approach in chemically mixed substrates remains uncertain. Here, we evaluated the reliability of this transcriptional sensor approach using the model marine bacterium Ruegeria pomeroyi DSS-3 grown on either glucose or a defined mixture containing glycerol, benzoate, succinate, leucine, dimethylsulfoniopropionate, and trimethylamine N-oxide. Genome-wide transcription differed strongly between treatments, with the mixed-substrate treatment enriched in genes associated with C1 metabolism, sulfur oxidation, benzoate degradation, and motility. Across substrates, the most diagnostic transcriptional responses occurred at pathway entry points and first committed reactions, including glucose transport and Entner-Doudoroff metabolism, trimethylamine N-oxide transport and catabolism, and early steps of aerobic benzoate oxidation. In contrast, downstream metabolic genes were less substrate-specific, likely because multiple pathways converged on shared central metabolic intermediates. Transporter transcription was also less consistently diagnostic than expected, although substrate-binding subunits often showed the strongest responses within transporter complexes. Comparisons with previous single-substrate studies indicated that some transcriptional markers, particularly benzoate oxidation genes, remained detectable in the substrate mixture, whereas glycerol and succinate responses were weakened or lost. These findings show that transcriptomics can provide useful insight into bacterial substrate use, but interpretation is most robust when focused on experimentally validated transporters and early pathway genes, and when evaluated in the context of pathway connectivity, cellular physiology, and substrate mixture complexity.