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Journal of Bacteriology, February 2009, p. 713-719, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01130-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli{triangledown} ,{dagger}

Dongchang Sun,1,2 Xuewu Zhang,3 Lingyu Wang,3 Marc Prudhomme,1,2 Zhixiong Xie,3 Bernard Martin,1,2 and Jean-Pierre Claverys1,2*

Centre National de la Recherche Scientifique, LMGM-UMR5100, F-31000 Toulouse, France,1 Université de Toulouse, UPS, Laboratoire de Microbiologie et Génétique Moléculaires, F-31000 Toulouse, France,2 State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China3

Received 12 August 2008/ Accepted 9 November 2008

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca2+, Fe2+, Mg2+, Mn2+, or Zn2+. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.

* Corresponding author. Mailing address: Université Paul Sabatier-CNRS, LMGM, Bât. IBCG, 118 Route de Narbonne, F-31062 Toulouse Cedex 9, France. Phone: (33) 561-33-59-11. Fax: (33) 561-33-58.86. E-mail: claverys{at}ibcg.biotoul.fr

{triangledown} Published ahead of print on 14 November 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, February 2009, p. 713-719, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01130-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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