Prochlorococcus genetic transformation and genomics of nitrogen metabolism

Abstract

Prochlorococcus, a unicellular cyanobacterium, is the most abundant phytoplankton in the oligotrophic, oceanic gyres where major plant nutrients such as nitrogen (N) and phosphorus (P) are at nanomolar concentrations. Nitrogen availability controls primary productivity in many of these regions. The cellular mechanisms that Prochlorococcus uses to acquire and metabolize nitrogen are thus central to its ecology. One of the goals of this thesis was to investigate how two Prochlorococcus strains responded on a physiological and genetic level to changes in ambient nitrogen. We characterized the N-starvation response of Prochlorococcus MED4 and MIT9313 by quantifying changes in global mRNA expression, chlorophyll fluorescence, and Fv/Fm along a time-series of increasing N starvation. In addition to efficiently scavenging ambient nitrogen, Prochlorococcus strains are hypothesized to niche-partition the water column by utilizing different N sources. We thus studied the global mRNA expression profiles of these two Prochlorococcus strains on different N sources. The recent sequencing of a number of Prochlorococcus genomes has revealed that nearly half of Prochlorococcus genes are of unknown function.

(cont.) Genetic methods such as reporter gene assays and tagged mutagenesis are critical tools for unveiling the function of these genes. As the basis for such approaches, another goal of this thesis was to find conditions by which interspecific conjugation with Escherichia coli could be used to transfer plasmid DNA into Prochlorococcus MIT9313. Following conjugation, E. coli were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus MIT9313. When this plasmid was modified to contain green fluorescent protein (GFP) we detected its expression in Prochlorococcus by Western blot and cellular fluorescence. Further, we applied these conjugation methods to show that Tn5 will transpose in vivo in Prochlorococcus. Collectively, these methods provide a means to experimentally alter the expression of genes in the Prochlorococcus cell.