Genetic manipulation of Prochlorococcus strain MIT9313: green fluorescent protein expression from an RSF1010 plasmid and Tn5 transposition.
Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria 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 strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.
Tolonen
Andrew C
AC
Department of Biology, Massachusetts Institute of Technology-Woods Hole Oceanographic Institution Joint Program in Biological Oceanography, Cambridge 02139, USA. tolonen@alum.mit.edu
Liszt
Gregory B
GB
Hess
Wolfgang R
WR
D017123:Bacteriophage T7; D001483:Base Sequence; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D048929:Prochlorococcus / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins