Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A

Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131^T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.Lactobacillus species are normal inhabitants of the human gastrointestinal tract, and Lactobacillus gasseri is one of the most commonly detected of these species (37, 47). Health-promoting effects of this species, such as immunomodulation (35), suppression of Helicobacter pylori-induced interleukin-8 production (44), and improvement of intestinal conditions (34), have been reported, and some L. gasseri strains are used in commercial probiotic products.Bacteriocins are antimicrobial peptides, proteins, or protein complexes produced by bacteria and active mainly against related bacterial species (38). Several bacteriocins also inhibit the growth of food-borne pathogens, such as Listeria, Bacillus cereus, and Clostridium perfringens. Production of bacteriocin is thought to be a desired feature for probiotic strains, since bacteriocin is believed to provide an advantage for survival in the ecological niche and to prevent the growth of pathogens. Several L. gasseri strains are known to produce bacteriocins (18). The classification of bacteriocins remains controversial. We use the definition proposed by Maqueda et al. (30), where bacteriocins are classified into class I (lantibiotics), class II (nonlantibiotics), class III (large heat-labile bacteriocins), and class IV (circular bacteriocins linked at the N- and C-terminal ends). Among these, the class IV circular bacteriocins have attracted increasing attention, since they are the simplest prokaryotic representatives of the ubiquitous circular peptides with various physiological activities (6). Enterocin AS-48 from Enterococcus faecalis strain S-48 is the first and most vigorously characterized member of the class IV bacteriocins (30). L. gasseri strain LA39 (JCM 11657) produces a 58-amino-acid (aa) circular bacteriocin, gassericin A (18). Gassericin A is a representative of the non-AS-48-like circular bacteriocin group including butyrivibriocin AR10 from Butyrivibrio fibrisolvens AR10 (15) and carnocyclin A from Carnobacterium maltaromaticum UAL307 (32), as well as reutericin 6 from Lactobacillus reuteri LA6 (17) and acidocin B from Lactobacillus acidophilus M46 (26). The last two bacteriocins have nearly identical amino acid sequences to that of gassericin A. Though the number of reported circular bacteriocins has been increasing, their primary sequences and the genes responsible for production of and immunity to them are diversified (for a review, see reference 31). Recently, we isolated and sequenced seven genes (gaaBCADITE) from LA39 deduced to be responsible for production of and immunity to gassericin A (20). The gaa genes add new information to the complex world of the class IV bacteriocin genes.The structural gene of gassericin A, gaaA, was reported to be located on the chromosome of LA39 (19). However, the high amino acid sequence identity of gassericin A to reutericin 6 (100%) and to acidocin B (98%) suggests recent horizontal gene transfers of the relevant bacteriocin genes, possibly via mobile elements. In fact, the acidocin B genes were reported to be located on a plasmid, namely, pCV461 (26). Many Lactobacillus strains are known to harbor one or more plasmids of various sizes, and several Lactobacillus plasmids have been reported to contain genes for production of bacteriocins (48). To our knowledge, however, only three have been sequenced entirely: these are pLA103 from Lactobacillus acidophilus TK8912 (16), pRC18 from Lactobacillus curvatus (previously known as Lactobacillus casei) CRL705 (7), and pMP118 from Lactobacillus salivarius subsp. salivarius UCC118 (5). Thus, genetic information about bacteriocin-producing Lactobacillus plasmids is still limited. Furthermore, little has been known about plasmids of L. gasseri, even though the existence of plasmids in a few strains has been reported, including a 26.5-kb anonymous plasmid in strain ADH (27) and pK7 in strain K7 (28).Here we describe a 33.3-kb plasmid, designated pLgLA39, from L. gasseri LA39. The gaa genes are located on this plasmid. pLgLA39 carries a set of genes for conjugative transfer and was shown to be transmitted to another L. gasseri strain. L. reuteri LA6 also harbors a plasmid almost identical to pLgLA39. We demonstrated that production of gassericin A increased the apparent segregational stability of a plasmid carrying the gaa genes. A pemIK homolog in pLgLA39 was also functional as a plasmid-stabilizing mechanism. This is the first report describing the entire nucleotide sequence and detailed genetic analysis of an L. gasseri plasmid, which contains functional genes for circular bacteriocin production, conjugation, and plasmid maintenance.