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Applied and Environmental Microbiology, December 2006, p. 7607-7613, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.02034-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition{triangledown}

Andrew C. Tolonen,1* Gregory B. Liszt,2 and Wolfgang R. Hess3

Department of Biology, Massachusetts Institute of Technology-Woods Hole Oceanographic Institution Joint Program in Biological Oceanography, Cambridge, Massachusetts 02139,1 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,2 University of Freiburg, Institute for Biology II/Experimental Bioinformatics, D-79104 Freiburg, Germany3

Received 28 August 2006/ Accepted 5 October 2006

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.

* Corresponding author. Mailing address: Département de Microbiologie, Institut Pasteur, 25-28 Rue de Dr. Roux, Paris 75015, France. Phone: (33) 01 45 68 84 16. Fax: (33) 01 40 61 30 42. E-mail: tolonen{at}alum.mit.edu.

{triangledown} Published ahead of print on 13 October 2006.

Applied and Environmental Microbiology, December 2006, p. 7607-7613, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.02034-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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