Nucleotide sequence and analysis of pRC12 and pRC18, two theta-replicating plasmids harbored by Lactobacillus curvatus CRL 705

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.

Fig 1. Structure and organization of pRC12.

A) Physical and genetic map of plasmid pRC12 from L. curvatus CRL 705. Orientation of deduced CDSs are shown by purple arrows. B) Genetic map of plasmid pUCL287 (X75607.1) from Tetragenococcus halophilus ATCC33315. C) Structural organization of the replicative region of plasmids pRC12 and pUCL287. D) Comparison between plasmids pRC12 from L. curvatus CRL 705 and pUCL287 from Tetragenococcus halophilus ATCC33315; both plasmids share high identity of the replication region (ori-repA) and the integrase and toxin-antitoxin system.

Fig 2. Structure and organization of pRC18.

A) Physical and genetic map of the plasmid pRC18 from L. curvatus CRL 705. Deduced CDSs are shown by purple arrows indicating their orientation. B) Focus on an 11 kb insertion disrupting repA showing the presence of transposases, and the lactocin 705 operon.

Fig 3. Comparison between pRC12 and pRC18 plasmids.

The grey scale indicates the identity % between nucleotide sequences. Integrase, antitoxin/ toxin, and repA (pUCL287-like; disrupted in pRC18 by an 11 kb insertion DNA element) genes are common in plasmids pRC12 and pRC18.

Fig 4. Comparison of pRC18 structural organization with other plasmids.

GC skew (purple) and GC content (black) of pRC18 are shown in the inner circles. The pRC18 CDSs (red) are indicated and their paralogs in different plasmids are shown with a color code.

Fig 5. Genetic context of the replication region of the plasmid pRC18.

The coding sequence of repB’ gene (which encodes a 168 aa protein) is underlined. The sequences of directed repeats (DR) and inverted repeats (IR1 and IR2) are in bold type and indicated with arrows. The predicted position of the -35, -10 boxes and the ribosome binding sites are in bold type.

Fig 6. Map of plasmid p3B1.

It was constructed by ligating a 3.4 kb HpaI-EcoRI fragment of pRC18 (coordinates nt 10334–13806) into the HindIII (blunted)-EcoRI sites of a derivative-plasmid pBlueScript II SK (+) containing the chloramphenicol (cat) gene from plasmid pC194.