The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector

J Bacteriol. 2000 Oct;182(20):5700-5. doi: 10.1128/JB.182.20.5700-5705.2000.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophages / genetics*
  • Bacteriophages / physiology
  • Base Sequence
  • Cloning, Molecular
  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification
  • Electrophoresis, Gel, Pulsed-Field
  • Escherichia coli / genetics
  • Escherichia coli / virology
  • Genetic Vectors
  • Leptospira / genetics*
  • Leptospira / virology*
  • Molecular Sequence Data
  • Plasmids / genetics*
  • Replication Origin / genetics
  • Virus Replication / genetics*

Substances

  • DNA, Viral