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Journal of Bacteriology, October 2000, p. 5700-5705, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The LE1 Bacteriophage Replicates as a Plasmid within Leptospira biflexa: Construction of an L. biflexa-Escherichia coli Shuttle Vector

Isabelle Saint Girons,1,* Pascale Bourhy,1 Catherine Ottone,1 Mathieu Picardeau,1 David Yelton,2 Roger W. Hendrix,3 Philippe Glaser,4 and Nyles Charon2

Unité de Bactériologie Moléculaire et Médicale1 and Laboratoire de Génomique des Microorganismes Pathogènes,4 Institut Pasteur, 75724 Paris Cedex 15, France; Department of Microbiology and Immunology, Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506-91772; and Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 152603

Received 16 May 2000/Accepted 24 July 2000

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.

* Corresponding author. Mailing address: Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 25 rue du docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 1 45 68 83 66. Fax: 33 1 40 61 30 01. E-mail: isgirons{at}pasteur.fr.

Journal of Bacteriology, October 2000, p. 5700-5705, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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