Search for: All records

Abstract Prochlorococcus cells are the numerically dominant phototrophs in the open ocean. Cyanophages that infect them are a notable fraction of the total viral population in the euphotic zone, and, as vehicles of horizontal gene transfer, appear to drive their evolution. Here we examine the propensity of three cyanophages—a podovirus, a siphovirus, and a myovirus—to mispackage host DNA in their capsids while infecting Prochlorococcus, the first step in phage-mediated horizontal gene transfer. We find the mispackaging frequencies are distinctly different among the three phages. Myoviruses mispackage host DNA at low and seemingly fixed frequencies, while podo- and siphoviruses vary in their mispackaging frequencies by orders of magnitude depending on growth light intensity. We link this difference to the concentration of intracellular reactive oxygen species and protein synthesis rates, both parameters increasing in response to higher light intensity. Based on our findings, we propose a model of mispackaging frequency determined by the imbalance between the production of capsids and the number of phage genome copies during infection: when protein synthesis rate increase to levels that the phage cannot regulate, they lead to an accumulation of empty capsids, in turn triggering more frequent host DNA mispackaging errors. 

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus. To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.