Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli.
The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.
Alpert
Carl-Alfred
CA
Flore Lactique et Environnement Carné, Institut National de la Recherche Agronomique, 78350 Jouy en Josas, France.
Crutz-Le Coq
Anne-Marie
AM
Malleret
Christine
C
Zagorec
Monique
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D016680:Genome, Bacterial; D019426:Integrases / Q000737:chemistry / Q000235:genetics; D007777:Lactobacillaceae / Q000145:classification; D007778:Lactobacillus / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D045522:Recombinases / Q000737:chemistry / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D045522:Recombinases; D019426:Integrases