The LE1 Bacteriophage Replicates as a Plasmid within Leptospira biflexa: Construction of an L. biflexa-Escherichia coli Shuttle Vector
| Autor: | Catherine Ottone, Nyles W. Charon, Mathieu Picardeau, Isabelle Saint Girons, Pascale Bourhy, David B. Yelton, Philippe Glaser, Roger W. Hendrix |
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| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: |
Genetic Vectors
Molecular Sequence Data Replication Origin Origin of replication Virus Replication Microbiology Bacteriophage Plasmid Shuttle vector Leptospira Escherichia coli Bacteriophages Replicon Cloning Molecular Molecular Biology Prophage Southern blot Genetics biology Base Sequence biology.organism_classification Electrophoresis Gel Pulsed-Field DNA Viral Plasmids and Transposons Plasmids |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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