The primary contribution of this paper is to describe the foundations of a genetic system for Prochlorococcus. We found conditions under which an interspecific conjugation system based on the RP4 plasmid family can be used to transfer DNA into Prochlorococcus MIT9313. pRL153, an RSF1010-derived plasmid, replicates autonomously in MIT9313, conferring resistance to kanamycin, and can be used to stably express foreign proteins, such as those for kanamycin resistance and for GFP. In addition, we found that Tn5 will transpose in vivo in Prochlorococcus. Once a liquid culture of kanamycin-resistant cells has been isolated, pour plating methods can be used to isolate individual colonies. These colonies can be transferred back to liquid medium for further characterization.
The methods described in this paper are highly repeatable; we isolated pure Prochlorococcus
cultures containing pRL153 in four successive conjugations. The isolation of transgenic Prochlorococcus
cultures does require a significant amount of time, however: 14 days to grow and mate the cells, 35 to 50 days to grow liquid transconjugant cultures after mating, 40 to 60 days to isolate transconjugant colonies on plates, 30 days to grow cultures isolated from colonies, and finally, 14 days for treatment with phage T7 and growth of pure cultures. Transgenic MIT9313 cultures can thus be isolated in 135 to 175 days (4.5 to 6 months). Experiments with Prochlorococcus
are inevitably time consuming due its low growth rate, but there are other Prochlorococcus
strains that grow faster than MIT9313. While MIT9313 grew with a doubling time of 3.3 days under the conditions in this study, Prochlorococcus
strain MED4, another axenic strain, grows with a maximum growth rate of one doubling every 1.10 days (14
). We avoided Prochlorococcus
strain MED4 in these experiments because it is naturally resistant to kanamycin, even at 100 μg ml−1
(data not shown). MED4 is highly sensitive to other antibiotics, however, such as 0.5 μg ml−1
chloramphenicol, suggesting plasmids carrying chloramphenicol resistance may be appropriate for this strain. Prochlorococcus
MIT9312, the third axenic strain, has a maximum growth rate of one doubling every 0.88 days (14
) and thus may also be a good candidate for genetic manipulation. Improvement of growth on plates is an area for potentially increasing the efficiency of the isolation of Prochlorococcus
mutants. MIT9313 colonies form on plates in 40 to 60 days with an efficiency of 1 colony per 100 to 10,000 cells. It is possible that yet-unidentified changes to the composition of the plating medium could improve the efficiency of MIT9313 colony formation. Further, future experiments to test the plating efficiencies of other Prochlorococcus
strains may reveal strains that grow more efficiently on plates.
This study is the first report of GFP expression in oceanic cyanobacteria, which has a number of potential applications. The division cycle of cells in Prochlorococcus
cultures synchronizes when entrained to a light/dark cycle (23
), and global gene expression is controlled by a central oscillator, similar to the case with other cyanobacteria (reviewed in reference 8
). Transcriptional fusions of Prochlorococcus
promoters to GFP could be used to study the diel cycling in the expression of different genes in Prochlorococcus
cultures. Although we were unable to quantify GFP fluorescence in individual cells, future studies using potentially stronger promoters or GFP variants with higher fluorescence (22
) may make this possible. Direct subcellular localization of GFP expression is likely not feasible for Prochlorococcus
, however, because the cell size (500 to 700 nm) approaches the lower limit of light microscopy as well as the wavelength of GFP fluorescence (maximum emission, ~510 nm). Relative to other cyanobacteria, Prochlorococcus
is a good candidate for studies to quantify GFP fluorescence on the whole-cell level. It does contain very low quantities of phycoerythrin, which in Prochlorococcus
has a fluorescence maximum of 549 nm (and in some low-light-adapted strains, an additional maximum of 495 nm) (24
). These maxima are close to that of GFP and could, in theory, be overlapping with it. However, the quantity of phycoerythrin in Prochlorococcus
is so low that it is undetectable by direct spectroscopic measurement and thus does not occlude GFP fluorescence. In addition, GFP expression could provide a means to sort transgenic from nontransgenic cells by flow cytometry. Faced with variable and overall low plating efficiencies, flow sorting of cells is an attractive alternative in order to isolate mutants following conjugation. Alternatively, RSF1010-derived plasmids could be modified to cause Prochlorococcus
to express other foreign proteins. For example, a tagged MIT9313 protein could be cloned into pRL153 and transferred into Prochlorococcus
by conjugation. The ectopically expressed, tagged protein could then be immunoprecipitated to determine which proteins interact with it in vivo.
The Tn5 transposon from pRL27 can be conjugally transferred to Prochlorococcus as a means of making tagged mutations in the chromosome. Our results suggest that Prochlorococcus transconjugants do not survive to form colonies if they are plated directly after mating. Consequently, transconjugants are first grown under kanamycin selection as a liquid culture containing a diversity of transposon mutants. Because the liquid transconjugant culture represented a mixed population of transposon mutants, some competitively dominant mutants likely increased in relative abundance and were among those that we identified. These mutants were likely relatively abundant in the culture because they had transposon insertions in selectively neutral sites in the chromosome. These sites may be ideal for future studies seeking to insert exogenous DNA into selectively neutral sites in the Prochlorococcus chromosome. Collectively, the methods described in this study show that genetic methods including transposon mutagenesis are tractable for Prochlorococcus, thus providing a foundation for future genetic studies with this ecologically important microbe.