Functional Analysis of Three Plasmids from Lactobacillus plantarum

Abstract

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.Lactic acid bacteria are used for the preservation of food and feed raw materials like milk, meat, and vegetables or other plant materials. Certain strains of lactic acid bacteria, in particular, strains from the genus Lactobacillus, have been attributed probiotic activities in humans and animals (30). Several lactic acid bacteria, including lactococci, streptococci, lactobacilli, and pediococci, are known to harbor plasmids. These may encode important traits like resistance to phages or antibiotics, lactose catabolism, and production of proteolytic enzymes or bacteriocins. Lactobacillus plantarum species often harbor several plasmids (49). Several of these have been sequenced (6,11,12,20,31,40,52,58). Although most of them are of unknown function, one plasmid encoding phage resistance (pMD5057, 10,877 bp) and another plasmid (pLKS, 2,025 bp) probably introduced from another source and coding for tetracycline resistance have been described previously (12,20). All are smaller than 11 kb and are predicted to replicate via the rolling-circle replication mechanism, except for the largest plasmid, pMD5057, which is predicted to replicate via the theta mechanism (12).The capacity for conjugal transfer is an important characteristic for plasmids. Self-transmissible conjugative plasmids have the ability to form effective cell-to-cell contact, while mobilizable plasmids are only able to prepare their DNA for transfer (38). Mobilization involves the action of a specific DNA-protein structure called the relaxosome to produce single-stranded cleavage at the nicking site (nic) within the origin of transfer (oriT) of the plasmid (38). To date, there is very little information on conjugation in lactobacilli. Sasaki et al. demonstrated conjugational transfer of the promiscuous thetareplicating plasmid pAM␤1 from Streptococcus faecalis to L. plantarum (51). Ahn et al. described an 8.5-kb chloramphenicol resistance plasmid which had been comobilized with pAM␤1 from L. plantarum to Carnobacterium maltaromaticum (3), previously known as Carnobacterium piscicola (43). To our knowledge, no conjugative L. plantarum plasmids have been reported...

Diversity of the genus Lactobacillus revealed by comparative genomics of five species

“…L. salivarius has the smallest chromosome, but the overall genome size is 2?06 Mb because of the presence of the megaplasmid pMP118. L. plantarum WCFS1 harbours three plasmids (1?9, 2?3 and 36 kb; van Kranenburg et al, 2005), and L. salivarius UCC118 harbours two plasmids of 20 kb and 44 kb (Flynn, 2001), while the other sequenced genomes lack plasmids, although extrachromosomal replicons are very common in lactobacilli (Wang & Lee, 1997). L. plantarum and L. sakei have a distinctively high GC content (above 40 mol%), whereas that of the other Lactobacillus genomes ranges from 32?9 to 34?7 % (Table 1).…”

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

“…Matings between L. salivarius LS201(pLS208) and L. fermentum DSM20055(pNZ8048), L. plantarum NCIMB8826(pLS208) and L. fermentum DSM20055 (pNZ8048), and L. plantarum NCIMB8826(pLS208) and L. salivarius JCM1045(pNZ8048) were tested for the mobilization of pLS208 into the recipient cells. L. plantarum NCIMB8826 harbors a conjugative plasmid, pWCFS103 (55), which might conceivably have facilitated the transfer of pLS208. For the donor L. salivarius LS201(pLS208), putative loci related to conjugation are present in pMP118 according to the annotation of the genome of L. salivarius UCC118 (5).…”

“…these plasmids may harbor genes encoding resistance to antibiotics (39,54) and metal ions (55), genes encoding bacteriocins (30,44), gene clusters for conjugation (55), genes involved in adherence and biotin metabolism (10), and genes encoding toxin-antitoxin (TA) proteins for plasmid maintenance (53). In addition to encoding such interesting traits, endogenous plasmids are the most commonly used systems to construct genetic tools especially for gene cloning and gene expression purposes (7,52) due to their ability to replicate in the original hosts.…”

Genetic tools for investigating the biology of commensal lactobacilli

“…Conjugation is an alternative approach for the introduction of novel DNA into poorlytransformable lactobacilli (79). Conjugal gene transfer can be achieved by conjugative plasmids (26), or comobilization of a nonconjugative plasmid with a conjugative plasmid (89). For instance, conjugal gene transfer was used to introduce a heterologous gene encoding beta-glucanase into a non-transformable strain of the species L. helveticus (90).…”

“…Some Lactobacillus species harbour transmissible or conjugative plasmids. For example, the sequenced L. plantarum strain WCFS1 harbours a conjugative plasmid pWCFS103 (26). The megaplasmid of L. salivarius UCC118 contains a tract of genes related to conjugation (36), though they appear to be genetically corrupt (28).…”