Distribution of Megaplasmids in Lactobacillus salivarius and Other Lactobacilli

Abstract

The genome of Lactobacillus salivarius UCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains of L. salivarius isolated from humans and animals all harbor a megaplasmid, which hybridized with the repA and repE replication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118 repA probe were also found in some strains of L. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of the repE and groEL sequences of 28 L. salivarius strains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids in L. salivarius was highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six other Lactobacillus species from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids in L. salivarius, and restricted carriage by other Lactobacillus species, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology of Lactobacillus.Lactic acid bacteria (LAB) are a diverse group of gram-positive bacteria that are found in a wide variety of nutrient-rich environments (29). LAB have been extensively exploited because of their ability to preserve food, beverages, and forage (43). Many LAB have been subjected to comprehensive genetic and genomic analyses (33,40,42) to provide a molecular basis for understanding their distinctive properties, some of which are determined extrachromosomally. Plasmids are commonly found in many members of the lactic acid bacteria (25). Many of these plasmids are cryptic (61), and their contribution to the biology of the strain harboring them is unclear. However, it is recognized that plasmid-borne traits are major accessories that are key to the phenotypes of industrially important groups such as the lactococci (reviewed in reference 45). Noteworthy lactococcal properties that are plasmid encoded include the production of the PrtP protease (12), abortive infection mechanisms to prevent bacteriophage attack (6, 49), exopolysaccharide biosynthesis (69), and bacteriocin production (66). DNA sequencing of the four plasmids harbored by Lactococcus lactis strain SK11, a widely used dairy strain, identified a broad repertoire of novel genes that significantly enhance or expand the metabolism, fitness, and stress resistance of the bacterium (62). The ability of plasmids to undergo dissemination by conjugation or other processes underlines their potential importance for contributing significant but variable traits to LAB.Comprising over a hundred species, the genus Lactobacillus represents the largest group within the family Lactobacillaceae (15, 60). The lactobacilli, like LAB in general, are associated with foodstuffs, plants, and animals (27), and many species are used in industrial appl...

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“…Also, it seems reasonable that closely related species must present similar characteristics to survive in the same conditions. This is in agreement with Ben Omar et al (2008) who first reported the detection of plantaricin gene cluster in L. fermentum and L. plantarum isolated from the same niche and Hurtado et al (2011) (Barrett et al, 2007;Li et al, 2007). This feature may be important for the successful establishment of L. salivarius within the GIT (O Shea et al, 2011).…”

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

“…2A and C which do not correspond to linear plasmids in the PFGE are due to the hybridization of probes to another form of the plasmid that had not been linearized completely by nuclease S1. These are not the megaplasmids demonstrated by Li et al (38), as different hybridization patterns were seen when the same membrane was probed with a fragment based on the megaplasmid (data not shown). Therefore, 52% of the tested 27 L. salivarius strains harbor plasmids ranging from 10 to 70 kb, which are all repA 20 -or repA 44 -related plasmids.…”

“…Thus, transferring and expressing large DNA clusters in probiotic lactobacilli can be accomplished through bacterial con- (Fig. 2) and is bacteriocin nonproducing (38). Failure of the mobilization of pLS208 from L. plantarum NCIMB8826 to L. salivarius LS201 may be due to the bactericidal effect on the donor of bacteriocin produced by the L. salivarius recipient or the incompatibility of two plasmids containing a similarly functioning traA gene ( Fig.…”

“…The preparation of agarose gel plugs of high-molecular-weight DNA for pulsed-field gel electrophoresis (PFGE), cell treatment, and electrophoresis was performed following the protocol described by Li et al (38). A CHEF-DR II pulsed-field system (Bio-Rad Laboratories) was used to resolve DNA fragments at 6 V/cm in 0.5ϫ Tris-borate-EDTA running buffer at 14°C for 20 h. A time setting of 3 to 50 s was employed for the linear ramped pulse.…”

Genetic tools for investigating the biology of commensal lactobacilli

“…Approaches are also desirable for DNA manipulation in Lactobacillus strains with atypical genome architectures. For example, megaplasmids are widely present in L. salivarius strains and a few other species (162). Thus, efficient gene disruption systems need to be developed to inactivate genes of interest which are located on megaplasmids.…”