Characterization of a Theta-Type Plasmid from Lactobacillus sakei : a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

Abstract

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 restrictionmodification 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.Lactobacilli belong to the lactic acid bacteria (LAB) group. The genus comprises several species important for the food industry, among which is Lactobacillus sakei, one of the most important bacterial species involved in meat fermentation and preservation. L. sakei belongs to the dominant microflora naturally occurring on vacuum-packed meat and is also used in Western Europe as starter cultures for fermented products (especially sausage) because of its ability to rapidly ferment carbohydrates in the meat environment (11,26).Most lactobacilli harbor one or more plasmids, the size of which can vary from 1.2 to 150 kb (for a review, see reference 50). Different functions have already been found on these plasmids, among which are genes for lactose metabolism, bacteriocin synthesis, exopolysaccharide production, or DNA restriction-modification (R-M). With the development of molecular biology and functional genomics of lactobacilli, interest is growing for the characterization of replicons themselves as potential useful (food-grade) vectors. Two modes of DNA replication are used by circular bacterial plasmids, namely, rolling circle (RC) and theta. RC plasmids have been assumed to be the most widespread in gram-positive bacteria. However, recent years have seen the characterization of a large number of theta-replicating plasmids from gram-positive, and especially from LAB, hosts. Several mechanisms are known for the initiation of theta replication. Theta replicons can be classified according to their dependence on three key components. These are plasmid-encoded initiator Rep proteins necessary for strand opening and/or interaction with other components of the initiation complex, origins of replication (generically termed ori) with specific DNA structural organization...

The SXT Conjugative Element and Linear Prophage N15 Encode Toxin-Antitoxin-Stabilizing Systems Homologous to the tad-ata Module of the Paracoccus aminophilus Plasmid pAMI2

“…AE003851], plasmids 1 and 3 of Nitrosospira multiformis [accession no. NC_007615 and NC_007617, respectively], pDC3000B of Pseudomonas syringae [18], pRV500 of Lactobacillus sakei [2], and pSRQ800 Lactococcus lactis [16]); (ii) linear bacteriophages (N15 of E. coli [accession no. NC_001901] and KO2 of Klebsiella oxytoca [22]); (iii) conjugative element SXT of Vibrio cholerae (12); and (iv) noncomposite transposon Tn5503 (Tn3 family) occurring in plasmid Rms149 of Pseudomonas aeruginosa (39) and a related unnamed transposon of plasmid pND6-1 from Pseudomonas sp.…”

Catabolism of N -Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins

“…PCR was carried out using Taq DNA polymerase purchased from Fermentas by using a conventional protocol comprising an initial denaturation step (4 min at 94°C), followed by 25 amplification cycles (30 s at 95°C; 1 min at 55°C; 2 min at 72°C) and a final elongation step (5 min at 72°C). Isolation of plasmid DNA from E. coli or L. sakei using commercially available reagents (Qiagen) and chro- mosomal DNA extraction from L. sakei were performed as previously described (40). Plasmid construction.…”

Intraspecies Genomic Diversity and Natural Population Structure of the Meat-Borne Lactic Acid Bacterium Lactobacillus sakei

“…For proteomic studies, bacterial strains were grown in a chemically defined medium (MCD) (28) supplemented with 0.5% glucose or MRS and incubated at 30°C. Strain 332F, cured of its endogenous plasmid pRV500 (2), was prepared as described earlier (4) by electroporating the parent strain L. sakei 332 with a pRV566 plasmid carrying resistance to erythromycin, which had been derived from a pRV500 replicon (2). One erythromycin-resistant clone was selected and cultivated for 200 generations in MRS broth without antibiotic at 30°C.…”