Abstract
The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB’. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB’, several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB’ and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB’. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per μg of DNA; in the new hosts, the plasmid was relatively stable as 29–53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.
Highlights
The aim of this study is the analysis and characterization of plasmids pRC12 and pRC18 detected in the draft genome sequence of L. curvatus CRL 705 [23]
While the nucleotide sequence of pRC12 was determined as a single contig (23-fold coverage) by using a wholegenome shotgun (WGS) strategy, the genome sequence of pRC18 was determined by a primer-walking strategy and confirmed by WGS [23]
Theta-type replicating plasmids are classified into six classes according to their dependency on three different components: a plasmid-encoded replication protein (Rep), an ori and a DNA polymerase encoded by the host [17]
Summary
The objective of this study was to determine and analyze the sequences of pRC12 and pRC18, the two plasmids harbored by L. curvatus CRL 705 that share regions with high identity, as the plasmidome of this species is not yet well understood. The aim of this study is the analysis and characterization of plasmids pRC12 and pRC18 detected in the draft genome sequence of L. curvatus CRL 705 [23]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have