Plasmid DNA studies in Lactobacillus plantarum strains isolated from olive fermentations: production of and immunity to plantaricin OL15 is associated to a 9.6 Kb plasmid (pOL15)

En un estudio previo, se aislaron 12 cepas de Lactobacillus plantarum a partir de aceitunas fermentadas. Entre ellas, solo L. plantarum OL15 produjo bacteriocinas (plantaricin OL15). En este estudio, se examinó el contenido de AND plásmido en las 12 cepas citadas. Entre ellas, 9 cepas han mostrado de una a tres bandas de plásmido con tamaños en el rango de 5.4 a 12.2 kb. L. plantarum OL15 exhibió un plásmido (9.6 kb) que se denominó pOL15. Después del curado con novobiocina y bromuro de etidio, la pérdida del plásmido pOL15 asociada a la pérdida de su facultad para producir plantaricin OL15, sugiere que la producción de plantaricina OL15 está ligada al plásmido. La plantaricin OL15 no se inactivó por amilasa ni por lipasa sugiriendo que su actividad no es dependiente de la presencia de carbohidratos o lípidos. La plantaricina OL15 mostró actividad frente a bacterias lácticas de diferentes especies y también frente a residuos de aceitunas y bacterias fitopatogénicas, incluyendo Pseudomonas y Erwinia


INTRODUCTION
Lactobacillus plantarum is considered to be the most frequent and the most important specie in the process of lactic fermentation in olives (Jimenez-Diaz et al., 1993 andKacem andKaram, 2006).Some strains of this specie produce bacteriocins (Maldonado et al., 2002).Bacteriocins are proteins or protein complexes displaying a bactericidal mode of action towards sensitive closely related species (Klaenhammer, 1988).
Several reports of plasmid-associated bacteriocin production in lactic acid bacteria have been reported (Ray et al., 1989;and Mørtvedt and Nes, 1990).It is well known that the association between plasmids and bacteriocin production is generally, determined by the use of chemical curing agents, including novobiocin, ethidium bromide, acriflavin, and others, which eliminate plasmid (s) in the tested strains (Bringel et al., 1989 andFloriano et al., 1998).
In previous studies, L. plantarum strains were isolated from fermented green olives (Kacem et al., 2004).Only L. plantarum OL15 produced bacteriocin (plantaricin OL15) which showed a bactericidal mode of action towards Gram negative and positive bacteria (Kacem et al., 2005).The aims of this work were to determine (i) the plasmid profiles of these L. plantarum strains, and (ii) whether the genetic determinant for plantaricin OL15 is plasmid associated.
The bacterial strains were maintained as a frozen stock at Ϫ20 °C in distilled water plus 20% (v/v) glycerol and propagated twice in Man Rogosa Sharpe (MRS) broth (Oxoid Ltd., UK) (de Man et al., 1960) at 30°C before use.

Plasmid DNA isolation
Plasmid DNA was isolated based on the method described by Anderson and Mc Kay (1983).DNA preparations were analysed by electrophoresing (3.5 V cm Ϫ1 for 5 h) on 0.7% agarose gels.Plasmids size were estimated by using commercial covalently closed circular (CCC) DNA markers containing plasmid species of known molecular weigh (18.2, 14.2, 12.1, 10.1, 8.1, 4.0, and 3.0 Kb) (Bathesda Research Laboratory, Product No 5639, USA).

Plasmid curing
Novobiocin (Sigma, Mo, USA) and ethidium bromide (Sigma, Mo, USA) were used to cure the indigenous plasmid (s) of the wild-type L. plantarum OL15 according to Ruiz-Barba et al. (1991) and Floriano et al. (1998) methods with minor modifications.
Prior to curing, we determined the sublethal concentrations of the two curing agents for the L. plantarum OL15 strain.The sublethal concentration was defined as the highest concentration allowing for the detectable, albeit reduced, growth of a L. plantarum OL15 strain (OD 600 =0.1-0.4).The sublethal concentrations of novobiocin and ethidium bromide for L. plantarum OL15 strain were found to be 1.8 μg/ml and 2.5 μg/ml, respectively.Overnight cultures (approximately 10 6 cfu/ml) of the wild-type L. plantarum OL15 were inoculated into fresh MRS broth containing a sublethal concentration of the respective curing agent.At appropriate intervals (7, 14, and 21 days), the cultures were serially diluted, then, plated onto MRS agar to yield individual colonies.After 18 h at 30 °C, MRS soft agar (0.6% agar) containing the indicator strain Lactococcus lactis BO8 (10 5 cfu/ml) was poured onto the plates, which were incubated at 30 °C for 24 h.
Colonies without clear zones of inhibition, indicating loss of bacteriocin activity, were isolated on MRS agar and repeatedly transferred into MRS broth.Crude extracts (CE) from these liquid cultures were essayed for bacteriocin activity as described below.As controls, MRS broth cultures of L. plantarum OL15 that had not been treated with novobiocin and ethidium bromide as well as those treated cultures which didn't lack the antagonistic phenotype on MRS agar were processed at the same time.The immunity of the nonproducing variants to plantaricin OL15 was examined by spotting active L. plantarum OL15 MRS culture supernatants on lawns of these derivatives (Floriano et al., 1998).Both producing and nonproducing variants of L. plantarum OL15 were analysed for plasmid DNA content.

Bacteriocin assays
The producer and nonproducing variants of L. plantarum OL15 were propagated in 500 ml MRS broth for 24 h at 30 °C.Then, the culture extract samples, partial purification, ultrafiltration and titre determination of bacteriocin were done by the same methods described by Kacem et al. (2005) for plantaricin OL15.After these steps, the resulting material was referred to as "crude extract" (CE).The CE activity was determined by the agar well diffusion assay (Schillinger and Lücke, 1989).Prepoured MRS agar plates were overlaid with 7 ml MRS soft agar containing 0.2 ml of indicator culture (Lactococcus lactis BO8).In order to standardize the assay, the inoculum was approximately 10 6 indicator cfu/ml.Wells of 5 mm in diameter were cutted into the agar plate by using a cork borer and aliquots of 50 μl from each CE solution samples were placed into the wells.Plates were incubated at 30 °C for 18 h, and examined for the presence of 1.5 mm or larger clear zones of inhibition around the wells.Each assay was performed in duplicate.

Search for bacteriocin spectrum
For the determination of the inhibition spectrum of CE, several lactic acid bacteria and non-lactic acid bacteria strains were used (Table 1) with the agar diffusion test, using the appropriate agar media and incubation conditions for their growth: lactococci, streptococci, enterococci and lactobacilli were tested in MRS agar at 30 °C for 18 h, Propionibacterium strains in YGL agar medium at 37 °C for 48 h, Pseudomonas aeruginosa in Brain Heart Infusion agar at 32 °C for 48 h, and E. coli and Erwinia in Nutrient agar at 37 °C for 3 days.

RESULTS AND DISCUSSION
In this study, 12 strains of L. plantarum from fermented olives were examined for plasmid DNA content.Of these, 9 strains have shown one to three plasmid bands ranging in size from approximately 5.4 to 12.2 Kb (Figure 1, lane 1 to 12).It was found that some strains showed the same plasmid DNA profiles.Among these, 3 strains (L.plantarum OL2, OL53 and OL54) showed two bands of similar size (5.4 and 11.4 Kb), 3 strains (L.plantarum OL16, OL33 and OL40) had three plasmids (5.4,10.9 Kb and 12.2 Kb), L. plantarum OL12 and OL36 showed a single band of 5.4 Kb, while L. plantarum OL15 exhibited a quite distinct single band corresponding 1 : Inhibition essays were done according to the well agar diffusion test (Schillinger and Lücke, 1989) PLASMID DNA STUDIES IN LACTOBACILLUS PLANTARUM STRAINS ISOLATED FROM OLIVE FERMENTATIONS… to 9.6 Kb which was named pOL15 (Figure 1, lane 1).In contrast, no plasmid DNA was observed in L. plantarum OL9, OL7 and OL23 strains.
Our results were in accordance with those of Ruiz- Barba et al. (1991) who showed that 35 L. plantarum strains, isolated from different green olive fermentors, contained a large number of plasmids in the CCC form (from 5 to 16) ranging from 2.0 to 68 kb in size.Xanthopoulos et al. (2000) also have found different plasmids in several L. plantarum strains isolated from Feta cheese.Delgado and Mayo (2003) have reported that L. plantarum LL441 strain harbours seven plasmids and produces a bacteriocin.Similar results were obtained by Kanatani and Oshimura (1994) with L. plantarum strains, isolated from fermented sausage.However, our results may not reflect the actual number of plasmids in our L. plantarum strains, as we know; plasmid DNA in lactic acid bacteria is not always easily detected.This may be due to growth temperature, copy number and isolation procedure (Casey and Jimeno, 1989).
L. plantarum OL15 was treated by combination of sublethal concentrations of novobiocin and ethidium bromide in attempts to free this strain of its plasmid.Of 632 treated colonies screened for bacteriocin production, 16 (2.5%) of them produced no zone of inhibition in the overlays containing the sensitive strain (Lactococcus lactis BO8).When these suspected non-bacteriocin (plantaricin OL15) producers were isolated on MRS agar and repeatedly subcultured in MRS broth (at least three transfers in MRS broth), none of them regained the ability to produce bacteriocin.Lactobacillus plantarum strains (OL15, OL2, OL7, OL9, OL12, OL16, OL23, OL33, OL36, OL40, OL53 and OL54), Lanes A to G: non producing derivatives from the wild type L. plantarum OL15 isolated after novobiocin and ethidium bromide treatment.
L. plantarum OL15 producer culture was not inhibited by its own CE, while all bacteriocin deficient derivatives cultures were sensitive to CE from the wild type L. plantarum OL15 (results not shown).This indicated the presence of an immunity mechanism.
Plasmid profile analysis of seven non producing derivatives, isolated after novobiocin and ethidium bromide treatment, showed that the 9.6 Kb plasmid pOL15 harbored by parental strain OL15 had been lost in all cases (Figure 1, lane A to G).This result indicates that the loss of the bacteriocin-producing trait could be correlated with a loss of the plasmid (pOL15), and therefore, that the bacteriocin produced by L. plantarum OL15 is plasmid encoded.This result is in agreement with those reported by other authors (Scherwitz et al., 1983 andMørtvedt andNes, 1990) who reported that bacteriocins production is plasmid-encoded.On the contrary, in other cases, bacteriocins production in L. plantarum strains has been linked to chromosomal DNA (Kato et al., 1994 andFranz et al., 1998).
In order to further characterize plantaricin OL15 as described in Kacem et al. (2005), we tested the CE sensitivity (loss of activity) to another enzymes (lysozyme, ficin, proteinase K, ␣-amylase and lipase).The antimicrobial activity was inactived only with proteinase K, thus indicating his proteinaceous nature.The fact that the inhibitory compound was not inactivated by treatment with lipase or ␣amylase, suggests that activity was not dependent on the presence of either a carbohydrate or lipid moiety.
Bacteriocin produced by L. plantarum OL15 exhibited similar biochemical properties to other bacteriocins produced by lactic acid bacteria, including producer strains isolated from fermenting olives such as L. plantarum LB17.2b (Delgado et al., 2001) or L. plantarum NC8 (Maldonado et al., 2003).On the other hand, plantaricin OL15 production differs from plantaricin B (West and Warner, 1988) and plantaricin F (Fricourt et al., 1994) since this bacteriocin is produced either on solid medium or in liquid culture, while the latter two are produced on solid medium only.In addition, plantaricin OL15 differs from plantaricin B (West and Warner, 1988), as the later appears to require a carbohydrates moiety for activity.Plantaricin OL15 differs from plantaricin F because it is active at pH values of 3 to 8, whereas plantaricin F is active only at pH 4.5 or lower (Fricourt et al., 1994).Plantaricin S is inactivated by lipolytic and glycolytic enzymes but not by proteinase K (Jimènez Diaz et al., 1993), and thus differs in these characteristics from plantaricin OL15.Similar result was reported by (Gonzàlez et al., 1994) for plantaricin C.
Plantaricin OL15 (CE of L. plantarum OL15) showed inhibition against a wide range of lactic acid bacteria strains of different species and other nonlactic acid bacteria (Table 1).In addition, inhibitory activity was directed against the natural flora present in olive fermentations, including L. plantarum and L. lactis strains and also against olive spoilage and phytopathogenic bacteria organisms, including, Pseudomonas, Erwinia, and Propionilbacterium.This type of result was also described by Visser et al. (1986), Floriano et al. (1998) and Kacem et al. (2004).
In conclusion, further work will be necessary to test the efficiency of plantaricin OL15 against more undesirable bacteria in olive fermentation and in the biological control of phytopathogenic bacteria in vivo.Finally, further experiments concerning physical mapping of the plasmid pOL15 are in progress.

Table 1 Antimicrobial spectrum of plantaricin OL15 crude extracts (CE) against a wide range of indicator strains.
NCPPB: National Collection of Plant Pathogenic Bacteria.Plant Pathology Laboratory, Hatching Green, Harpenden, England, U.K PDDCC: Culture Collection of Plant Diseases Division.New Zealand Department of Scientific and Industrial Research, Auckland.New Zealand.LBMB: Laboratoire de Biologie des Microorganismes et Biotechnologie, Faculté des Sciences, Université d'Oran, Algeria.* : Our strains collection.