Using molecular tools to identify unicellular cyanobacteria with the potential to utilize nitrate in oligotrophic oceans

The extent of the how genetic and physiological diversity is correlated in environmental populations of unicellular cyanobacteria is unknown. Genomic analysis of cultivated isolates of Synechococcus and Prochlorococcus has shown various gene deletion and rearrangement events between these cyanobacteria. In particular, the nitrate assimilation genes encoding for the assimilatory nitrate reductase (narB) and the bi-specific nitrate-nitrite permease (nrtP) have been lost in all current Prochlorococcus isolates yet are present in all current marine Synechococcus isolates. While the environmental diversity is unclear, the control of the nitrate assimilation pathway in cultivated representatives of Synechococcus is also widely unknown for the majority of Synechococcus strains. The goal of this dissertation was to elucidate the genetic diversity and control of the nitrate assimilation pathway in environmental and cultivated isolates of unicellular cyanobacteria. In order to achieve these goals, I used a multi-disciplinary approach in my work, incorporating oceanographic field work and sample collection, microbiological techniques such as aseptic culturing, molecular approaches including quantitative PCR, phylogenetic analysis using various software programs, and statistical analyses to study the role abiotic factors may play in the natural distributions of these organisms. This research revealed a greater diversity of the nitrate assimilation genes in natural populations of cyanobacteria than have been seen in previous studies. The field and laboratory work lead to the discovery of multiple novel narB and nrtP Prochlorococcus -like groups from samples taken in the oligotrophic environments of the Sargasso Sea and North Pacific Subtropical Ocean. These results were consistent with recent work by Martiny et al. (Martiny et al. 2009) where sequences from the Global Ocean Sampling (GOS) Dataset were analyzed and many were identified as Prochlorococcus based on GC content of their DNA. These results suggest that cultivated Prochlorococcus isolates represent a fraction of the natural diversity seen in these organisms. However, Prochlorococcus nrtP gene expression was not detected in these samples. We do detect nrtP expression from Synechococcusin our samples, despite the fact the majority of our DNA sequences correspond to Prochlorococcus. Therefore, these genes may be expressed at low levels in Prochlorococcus or regulated differently from Synechococcus. In metatranscriptomic libraries, Poretsky et al. (Poretsky et al. 2009) were able to detect environmental nrtP expression when 80L of water were filtered while Frias-Lopez et al. (Frias-Lopez et al. 2008) detected Prochlorococcus gene expression of various genes, including amt genes involved in ammonium metabolism, in 4L of water filtered, suggesting that the 1L volume filtered in our field samples may have been too low to detect these transcripts in the environment. This work also elucidated differences in narB and nrtP gene expression across five strains of marine Synechococcus , suggesting that our understanding of cultivated isolates is also minimal. The narB and nrtP genes in one representative of clade III and a strain from clade VI appear to be under a similar form of N control while it is unclear if another representative of clade III, and strains from clades II and V are under the same control. Synechococcus WH7805 (VI) and WH8102 (III) respond to nitrogen switching while WH7803 (V), WH8103 (III) and WH8109 (II) show no difference in nrtP and narB expression when grown in ammonium or nitrate. Synechococcus clades III and V also appear to cope with rapid nitrogen limitation better than strains from clades II and VI. However, there are discrepancies with some of our results and those obtained in previous studies for WH8103 (Wyman and Bird 2007) and WH8102 (Su et al. 2006), although those studies utilized different temperature and nitrogen concentrations. These results suggest that a culture condition standard, which would include specific light levels, temperature and a control media for growth rate, would be useful when working with these organisms in order to better compare data across studies. These results suggest that it could be difficult to make inferences regarding cellular nitrogen status from nitrogen assimilation gene expression patterns in the environment, unless each strain is truly representative of its phylogenetic clade. Future experiments assessing nitrogen assimilation physiology and gene expression at different temperatures and with manipulations of other relevant nutrients, such and phosphorus and iron, will also be helpful in discerning the potentially complex response of marine cyanobacteria to environmental variables