The unicellular planktonic non-nitrogen fixing cyanobacteria of the genera Synechococcus and Prochlorococcus are the dominant phytoplankters in the nitrogen poor, oligotrophic waters that cover vast areas of the world's oceans. The goals of this dissertation were to investigate the role of NtcA, a transcriptional activator, in the regulation of nitrogen metabolism in a model marine cyanobacterium, Synechococcus sp. strain WH7803, and to test the suitability of ntcA gene expression as a molecular indicator of the nitrogen status of picocyanobacterial field populations. Expression of the ntcA gene, found in a single copy, was basal in the presence of ammonium. Cells grown on nitrate or nitrite displayed enhanced ntcA expression whereas maximal transcript levels were found in cells that had been deprived of a combined nitrogen source. The addition of ammonium at concentrations in excess of 1 micromolar led to the rapid decline of ntcA transcript levels whereas the addition of l-methionine-sulfoximine (MSX), an inhibitor of ammonium assimilation, induced a nitrogen starvation response resulting in maximal ntcA expression. Cells deprived of phosphorus or iron did not display enhanced ntcA expression. Expression of ntcA in nitrate-grown cells fluctuated 5-10 fold over the course of a day with maximal expression around midday and a trough in expression at night. The persistence of this pattern under continuous light suggests that the differences in ntcA transcript levels were circadian controlled. Analysis of an ntcA-interruption mutant derived from Synechococcus sp. strain WH7803 showed that NtcA is essential for the transcriptional regulation of it own gene as well as of napA (encoding a nitrate transporter). Nitrogen uptake assays showed that cells grown in the presence of ammonium were incapable of nitrate or nitrite utilization, whereas those transferred to nitrate or nitrite developed maximal capacity for the uptake of both substrates. However cells deprived of a nitrogen source exhibited little, if any, capacity for nitrate or nitrite utilization. The addition of ammonium led to the immediate cessation of nitrate uptake, but only a gradual decline in nitrite uptake rates. Interstingly, the addition of nitrite inhibited nitrate utilization, whereas nitrite uptake was not affected by even high concentrations of nitrate.

A protocol was developed to differentiate between 3 different nitrogen states using ntcA gene expression. In this protocol, ntcA transcript levels in field populations of prokaryotic phytoplankton are compared to basal and maximal levels induced in the same populations by the addition of ammonium and MSX respectively. While basal ntcA transcript levels infers ammonium utilization, maximal ntcA expression is indicative of nitrogen deprivation. Intermediate ntcA transcript levels attests to the utilization of a nitrogen source, the identity of which cannot be fully resolved using this protocol. This protocol was tested on Synechococcus sp. strain WH7803 under controlled laboratory conditions and was shown to accurately depict its nitrogen status. Specific ntcA primers enabled the amplification of the ntcA gene from all marine cyanobacteria tested, but not from heterotrophic bacteria, anoxygenic photosynthetic bacteria nor eukaryotic algae. Furthermore, cyanobacteria-like ntcA fragments were specifically amplified from seawater samples irrespective of the presence of the myriad of other organisms found in the sea. A gene tree constructed from ntcA sequences amplified from cyanobacterial strains and field samples provided the basis for designing primers capable of specifically detecting ntcA from either Synechococcus or Prochlorococcus as well as from the distinct lineages found within each genus. These results pave the way for using ntcA gene expression to assess whether field populations of picocyanobacteria are utilizing regenerated nitrogen in the form of ammonium, are thriving on a nitrogen source other than ammonium, or are nitrogen deprived.