Assimilatory nitrate utilization by bacteria on the West Florida Shelf as determined by stable isotope probing and functional microarray analysis.

Dissolved inorganic nitrogen (DIN) uptake by marine heterotrophic bacteria has important implications for the global nitrogen (N) and carbon (C) cycles. Bacterial nitrate utilization is more prevalent in the marine environment than traditionally thought, but the taxonomic identity of bacteria that utilize nitrate is difficult to determine using traditional methodologies. (15) N-based DNA stable isotope probing was applied to document direct use of nitrate by heterotrophic bacteria on the West Florida Shelf. Seawater was incubated in the presence of 2 μM (15) N ammonium or (15) N nitrate. DNA was extracted, fractionated via CsCl ultracentrifugation, and each fraction was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis. TRFs that exhibited density shifts when compared to controls that had not received (15) N amendments were identified by comparison with 16S rRNA gene sequence libraries. Relevant marine proteobacterial lineages, notably Thalassobacter and Alteromonadales, displayed evidence of (15) N incorporation. RT-PCR and functional gene microarray analysis could not demonstrate the expression of the assimilatory nitrate reductase gene, nasA, but mRNA for dissimilatory pathways, i.e. nirS, nirK, narG, nosZ, napA, and nrfA was detected. These data directly implicate several bacterial populations in nitrate uptake, but suggest a more complex pattern for N flow than traditionally implied.