NITRATE UPTAKE BY HETEROTROPHIC BACTERIA AND THE DIVERSITY OF BACTERIAL NITRATE ASSIMILATION GENES IN MARINE SYSTEMS

This study focused on the development of molecular methods to assay the diversity and abundance of groups of heterotrophic bacteria that are capable of aerobic NO3 assimilation. The variability of the abundance and diversity of populations of heterotrophic bacteria capable of NO3 utilization was studied in relation to patterns of bacterial NO3 uptake as indicated by 15N uptake experiments. A PCR primer set that could be used to selectively amplify a fragment of the nasA gene (assimilatory nitrate reductase) from heterotrophic bacteria was designed. Results suggest that nine groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments. 15N tracer experiments conducted in the Barents Sea and in the South Atlantic Bight indicate that bacteria assimilate, on average, between 15 and 40 % of the available NO3. These results suggest that bacteria play a larger role in NO3 utilization than previously hypothesized and that bacterial uptake of NO3 should not be ignored in estimates of new production. In the Barents Sea Marinobacter sp. nasA gene abundance, measured via a SYBR Green real-time PCR assay, was positively correlated with NO3, showing a two-fold increase in concentration relative to total bacteria at 80 m compared to 5 m. Compared to other variables tested, NO3 is the best predictor, by a factor of 10, of the variability associated with nasA community structure (assayed via T-RFLP) across the different water masses sampled in the Barents Sea. Studies conducted in the Skidaway River estuary in the South Atlantic Bight indicated a strong correlation, across seasons, between Marinobacter sp. nasA gene abundance and the magnitude of bacterial NO3 uptake. Of the different variables assayed, NO3 uptake rate was the best predictor, by a factor of 15, of the variability associated with nasA community structure. The finding that NO3 availability and patterns of NO3 utilization are positively correlated with nasA community structure variability and the abundance of particular groups of nasA genes, indicates that patterns of NO3 supply, in the marine environment, are sufficiently important to be a factor in regulating bacterial communities.
INDEX WORDS: Nitrate uptake, 15N, PCR, Q-PCR, T-RFLP, nasA, Heterotrophic bacteria, Marine bacteria, Nitrogen cycle, estuary