The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.
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.
Girons
I S
IS
Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 75724 Paris Cedex 15, France. isgirons@pasteur.fr
Bourhy
P
P
Ottone
C
C
Picardeau
M
M
Yelton
D
D
Hendrix
R W
RW
Glaser
P
P
Charon
N
N
D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005822:Genetic Vectors; D007919:Leptospira / Q000235:genetics* / Q000821:virology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D004279:DNA, Viral