Genetic investigation of nitrate assimilation in marine microbes

Nitrogen is the macronutrient most likely to limit phytoplankton growth in the ocean, and its availability therefore is likely to influence the structure of marine communities and patterns of primary production. Microscopic phytoplankton dominate photosynthetic activity in the ocean, with diatoms contributing a quarter of global oceanic primary production. Diatoms characteristically form blooms in environments where nitrate is episodically supplied (e.g. subpolar spring blooms, coastal upwelling). We hypothesise that their success is due to their physiological ability to outcompete other phytoplankton for nitrate. This physiology is in turn encoded in their genes. Genetic studies of nitrate assimilation in marine microbes have been enabled only recently by molecular probes. The resolution and specificity that gene sequence information provides will allow us to link the genetic potential of phytoplankton groups to their ecological success. This dissertation investigates nitrate assimilation in marine microbes from a molecular perspective.Assimilatory nitrate reductase gene fragments were retrieved by polymerase chain reaction (PCR) from two microenvironments associated with seagrass blades. Phylogenetic groupings of nitrate reductase genes from diatoms (NR ) and heterotrophic bacteria (nasA) suggest that the differences among those genes represent functional differences in the corresponding enzymes. The new sequences significantly expanded the known database for microbial nitrate reductase genes.The short-term gene expression of assimilatory nitrate reductase and high affinity nitrate transporter (Nrt2) genes was quantified in the diatom Thalassiosira weissflogii in response to nitrate additions following nitrogen starvation. Significant Nrt2 and NR gene expression before exposure to NO-3 suggests that T. weissflogii maintains the ability to use NO-3 even in its absence. Rapid increase and decrease in gene expression occurred within 1.5 hr, suggesting that Nrt2 and NR are highly regulated to allow T. weissflogii to rapidly utilise nitrate.Targeting two major NR phylogenetic groups, identified in the genetic diversity study and which represent ecologically important species, we developed QPCR primers to detect group-specific dynamics in the ocean. T. weissflogii and S. costatum -specific primers correctly amplified the target gene from cultures. T. weissflogii -like sequences were more abundant in Western English Channel samples, and S. costatum -like sequences were more abundant in Monterey Bay samples. The differential abundance of these two groups suggests again that genetic differences may have ecological significance.