Picocyanobacteria are important phytoplankton and primary producers in the ocean. Although extensive work has been conducted for picocyanobacteria (i.e. Synechococcus and Prochlorococcus) in coastal and oceanic waters, little is known about those found in estuaries like the Chesapeake Bay. Synechococcus CB0101, an estuarine isolate, is more tolerant to shifts in temperature, salinity, and metal toxicity than coastal and oceanic Synechococcus strains, WH7803 and WH7805. Further, CB0101 has a greater sensitivity to high light intensity, likely due to its adaptation to low light environments. A complete and annotated genome sequence of CB0101 was completed to explore its genetic capacity and to serve as a basis for further molecular analysis. Comparative genomics between CB0101, WH7803, and WH7805 show that CB0101 contains more genes involved in regulation, sensing, and stress response. At the transcript and protein level, CB0101 regulates its metabolic pathways, transport systems, and sensing mechanisms when nitrate and phosphate are limited. Zinc toxicity led to oxidative stress and a global down regulation of photosystems and the translation machinery. From the stress response studies seven chromosomal toxin-antitoxin (TA) genes, were identified in CB0101, which led to the discovery of TA genes in several marine Synechococcus strains. The activation of the relB2/relE1 TA system allows CB0101 to arrest its growth under stressful conditions, but the growth arrest is reversible, once the stressful environment dissipates. The genome of CB0101 contains a relatively large number of genomic island (GI) genes compared to known marine Synechococcus genomes. Interestingly, a massive shutdown (255 out of 343) of GI genes occurred after CB0101 was infected by a lytic phage. On the other hand, phage-encoded host-like proteins (hli, psbA, ThyX) were highly expressed upon phage infection. This research provides new evidence that estuarine Synechococcus like CB0101 have inherited unique genetic machinery, which allows them to be versatile in the estuarine environment.