Exploitation of Plasmid pmrc01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2001, p Vol. 67, No /01/$ DOI: /AEM Copyright 2001, American Society for Microbiology. All Rights Reserved. Exploitation of Plasmid pmrc01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains RITA M. HICKEY, 1,2 DENIS P. TWOMEY, 1,2 R. PAUL ROSS, 1 * AND COLIN HILL 2,3 Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, 1 and Microbiology Department 2 and National Food Biotechnology Centre, 3 University College Cork, Cork, Ireland Received 19 September 2000/Accepted 21 March 2001 Genetic analysis of the 60.2-kb lactococcal plasmid pmrc01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (orit). This orit-like sequence was amplified and cloned on a 312-bp segment into pci372, allowing the resultant plasmid, prh001, to be mobilized at a frequency of transconjugants/donor cell from an MG1363 (reca mutant) host containing pmrc01. All of the resultant chloramphenicol-resistant transconjugants contained both prh001 and genetic determinants responsible for bacteriocin production and immunity of pmrc01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pmrc01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the orit-containing fragment was exploited to mobilize prh001 alone to a number of lactococcal hosts. These results demonstrate that orit of pmrc01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform. Most lactococcal starter strains harbor a rich plasmid complement which encodes many of their industrially significant traits, including lactose utilization, bacteriocin production, bacteriophage resistance, and exopolysaccharide production (21, 25, 27, 47). Many of the larger plasmids, including pnp40 (36), plp712 (20), ptr2030 (28), prs01 (37), and pmrc01 (12), have been shown to be self-transmissible, a feature which has been exploited by researchers in the development of new strains over the last 20 years. Such an approach, however, has limitations in that these natural plasmids must contain a suitable selectable marker and must be compatible with resident plasmids. Allied to this, many desirable plasmids cannot be transferred via conjugation. Electroporation, an alternative means of plasmid transfer to cells, may also be unsuitable, given that many industrial strains appear difficult to transform, and this method may not be regarded as a food-grade approach in some countries. Self-cloning (a process in which the recombinant DNA comes from the host species) using conjugative vectors may provide an alternative approach to genetic improvement of strains (31). Consequently, it would be very desirable to exploit the lactococcal conjugative machinery to efficiently transfer recombinant self-cloned plasmids between strains. This study concerns the characterization and exploitation of the origin of transfer (orit) of the lactococcal plasmid pmrc01, from Lactococcus lactis subsp. lactis DPC3147 (44). In addition to conjugative and bacteriophage resistance functions, this plasmid encodes the production and immunity functions of a broad-spectrum bacteriocin, lacticin To date, * Corresponding author. Mailing address: Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. Phone: Fax: pross@moorepark.teagasc.ie. this plasmid s industrially significant traits have been successfully transferred to more than 30 lactococcal starter strains by taking advantage of the self-transmissible nature of pmrc01 (7, 40, 44). In a previous study, Dougherty et al. (12) determined the complete sequence of this 60.2-kb plasmid and the conjugative transfer region was identified based on the similarity with other transfer regions of gram-positive organisms. This region comprises an 18-gene operon-like structure and two divergently transcribed genes (Fig. 1A). When comparisons are made between this region and equivalent regions from other plasmids of gram-positive organisms, it is evident that the gene encoding the probable orit nickase of pmrc01 is similar to equivalent genes from several staphylococcal plasmids, including pip501 (49), psk41 (3), and pg01 (6), and the lactococcal plasmid pk214 (42). In addition, a putative orit locus was identified between these divergently transcribed loci. orit is the only cis-acting site required for DNA transfer which has the ability to convert a nontransmissible plasmid into a mobilizable plasmid (29). A number of lactococcal plasmid-derived orit loci have been identified, and these may be acted upon and mobilized by the ML3/712 sex factor element (33, 38). Cloning of the origin of transfer of pmrc01. The complete sequence of pmrc01, a 60.2-kb conjugative plasmid from L. lactis DPC3147, was determined previously, and a putative orit locus was identified (12). This putative orit locus includes a 13-nucleotide sequence which is similar to known and putative orit genes from the IncQ plasmids of gram-negative organisms, ptic58, psc101, R1162, RSF1010, and ptf1, and the staphylococcal plasmids pip501, pgo1, and psk41 (Fig. 1C). A similar sequence is also present in the self-transmissible conjugative lactococcal plasmids prs01 (38) and pci528 (33) and in several lactococcal plasmids, including pah33 (41), 2853

2 2854 HICKEY ET AL. APPL. ENVIRON. MICROBIOL.

3 VOL. 67, 2001 EXPLOITATION OF orit FROM pmrc TABLE 1. Summary of conjugal matings Plasmid(s) of donor L. lactis VEL1122 Recipient L. lactis strain Frequency of transfer/donor Presence of proteinase K % Transconjugants with prh001 and without pmrc01 prh001 MG a NA b pci372 MG a NA pci372 pmrc01 MG a NA prh001 pmrc01 MG a 0 MG DRC DPC DPC DPC DPC DPC DPC DPC DPC a Frequency data are the averages of three separate experiments. b NA, not applicable. pnz4000 (48), psrq900 (16), pbl1 (34), and pwv01 (three copies present; 30). Plasmids from other lactic acid bacteria, such as the Lactobacillus plantarum phage resistance plasmid plks (15), the Lactobacillus helveticus cryptic plasmid plh2 (43), and the Leuconostoc mesenteroides plasmid ptxl1 (accession no. AJ272077), also contain a similar element. The majority of the nic regions in the well-characterized plasmids of gram-negative and gram-positive organisms are preceded by an inverted repeat structure centered around the trinucleotide sequence GAA, a structure believed to interact with specific DNA-binding proteins (6, 29). pmrc01 contains an appropriately spaced GAA sequence and is flanked by an imperfect inverted repeat structure ( G 3.2 kcal/mol; 45). The putative orit loci of psrq900 (16) and the citrate permease plasmid, pcit264 from L. lactis lactis lactis biovar diacetylactis CRL264 (32), also contain an appropriately spaced GAA site upstream of their putative nic sites, which are flanked by imperfect inverted repeat sequences. Analysis of the sequence surrounding the putative pmrc01 nic site revealed a series of inverted repeat structures (IR1 through IR4; Fig. 1B) which may have a role in the functionality of this putative orit region. To investigate if this pmrc01 sequence could be exploited as the basis for conjugative vectors in lactococci, a suitable donor strain was constructed which contained the orit-like sequence cloned on a nonmobilizable vector accompanied by pmrc01, which should allow conjugal transfer functions to be supplied in trans. A 312-bp fragment containing the proposed orit of pmrc01 was amplified by PCR and cloned into the Escherichia coli-lactococcal shuttle vector pci372 (Cm r ; 24). The primers used for amplification were 5 -AACTGCAGCTT GATTTTTATTGACCG-3 and 5 -GCGGATCCGCACTTCT TCCCTTACC-3, which incorporated the restriction sites PstI and BamHI (italicized), respectively. The integrity of the insert was confirmed by sequencing, and this construct was designated prh001. Mobilization of the orit plasmid, prh001. Using the method described by Holo and Nes (26), prh001 was electroporated into a reca mutant derivative of MG1363 called L. lactis VEL1122 (14). L. lactis MG1363 is known to contain a sex factor element (22) that therefore should be present in L. lactis VEL1122, which was used in this study. To investigate if this sex factor can act upon the orit gene of pmrc01, L. lactis VEL1122 containing prh001 was used as a donor in a solid surface mating (36) with L. lactis MG1614, using VEL1122 cells containing pci372 as a negative control. Transfer of chloramphenicol resistance was unsuccessful for both matings, suggesting that the sex factor of MG1363 cannot mobilize prh001 or pci372 (Table 1). L. lactis VEL1122 containing prh001 was then used as a recipient in a mating, with L. lactis MG1363 containing pmrc01 (44). Transconjugants resistant to chloramphenicol (5 g/ml), tetracycline (3 g/ml; resistance encoded by Tn916 on the chromosome of VEL1122), and lacticin 3147 ( 160 arbitrary units/ml) were selected. As expected, all of the resulting transconjugants examined produced lacticin 3147 when tested by means of the well diffusion assay described by Ryan et al. (44). Importantly, the resulting strain contained two plasmids of 60 and 6 kb, corresponding to pmrc01 and prh001, respectively. In parallel, a suitable negative control, L. lactis VEL1122 containing pmrc01 and pci372 without the cloned insert, was also constructed. In order to assay for mobilization, both the positive control FIG. 1. (A) Diagrammatic representation of the conjugative region of pmrc01. Open reading frame designations are as described by Dougherty et al. (12) and are denoted by arrows. The location of the putative origin of transfer is denoted by an X. (B) Nucleotide sequence of the region spanning bp 1675 to 1986 of pmrc01 containing the origin of transfer (orit). Inverted repeats (IR) are indicated by solid arrows. Sequences homologous to primers (RH001 and RH002) used in PCR to amplify the region are highlighted by dashed arrows. A 13-bp segment similar to that of other orit loci is denoted by asterisks. (C) Comparison of the putative orit of pmrc01 with orit genes from other plasmids. A conserved 13-bp sequence common to all these elements is boxed and shaded, and a consensus sequence is provided. Where known, the nic site is indicated by a vertical arrow. Inverted repeats are indicated by solid arrows. A GAA sequence central to many of the inverted repeat structures is highlighted in bold. The accession number of pmrc01 is AE

4 2856 HICKEY ET AL. APPL. ENVIRON. MICROBIOL. FIG. 2. (A) Plasmid profiles of lactococcal strains to demonstrate the acquisition of prh001 by transconjugants: P, the parent strain;, transconjugants containing prh001;, transconjugants containing prh001 and pmrc01. The positions of the chromosome, pmrc01, and prh001 are indicated. Plasmids isolated from L. lactis DRC3 (37) were used as standard molecular sizes. (B) Gel obtained from PCR using pmrc01-specific primers indicating the presence of a 490-bp product specific for this plasmid. (C) Well diffusion assay showing zones of inhibition whenever pmrc01 is present as lacticin 3147 is being produced. Downloaded from (pmrc01 plus prh001) and the negative control (pmrc01 plus pci372) were mated with a streptomycin-resistant derivative of MG1363 (L. lactis MG1614). Putative transconjugants were selected for on medium containing chloramphenicol and streptomycin (500 g/ml). In addition, donors were distinguished from recipients based on their tetracycline-resistant phenotype. The efficient mobilization of chloramphenicol resistance in the positive control ( transconjugants/ donor cell) relative to the negative control (0 transconjugants/ donor cell) demonstrated that the 312-bp insert contains an orit locus and could be acted upon in trans by the pmrc01 nickase (Table 1). However, 100% of the resulting transconjugants analyzed also produced lacticin 3147, suggesting that pmrc01 had been comobilized in all cases. Obviously, a desirable trait of a mobilizable vector would be its ability to be transferred on its own to a recipient strain without comobilization of other plasmids. To investigate if prh001 could be mobilized without comobilizing pmrc01, the bacteriocin selective pressure was relieved by the addition of proteinase K (5 mg/ml) to both the growth medium and the mating mixture. In this case, the frequency was lower ( transconjugants/donor cell) than that of the matings without proteinase K. A possible explanation for this might be that such a high concentration of proteinase K may affect the conjugative machinery and therefore inhibit plasmid transfer. However, only 53% of the resulting transconjugants produced the bacteriocin. The absence of pmrc01 in the nonproducing strains was verified by PCR using primers specific for the lacticin 3147 structural peptides. A 490-bp product was evident for constructs which produced lacticin 3147, while no product was obtained for those which did not produce the bacteriocin, demonstrating that the orit plasmid can be mobilized by itself. Mobilization of prh001 into industrial starter strains. To determine if prh001 could be mobilized into other lactococcal strains, including strains used for commercial cheddar cheese production, nine lactococcal starter strains (L. lactis DRC3, DPC5020, DPC220, DPC143, DPC743, DPC778, DPC4272, DPC4935, and DPC745) were chosen for similar conjugations incorporating proteinase K. Lactose indicator agar was used to discriminate lactose-positive recipients (yellow) from lactosenegative donors (white). Chloramphenicol-resistant, tetracycline-sensitive, lactose-positive transconjugants were selected. Mating frequencies varied from to transconjugants/donor cell (Table 1). The presence of prh001 was determined by plasmid profile analysis (2) (Fig. 2) and PCR using orit-specific primers (data not shown). While the presence of the lacticin 3147 genetic determinants could be confirmed by PCR, a product of the expected size was present only for strains which produced lacticin 3147 (Fig. 2). A significant percentage of the transconjugants obtained from each of the matings involving the commercial strains contained prh001 alone. Indeed, 100% of the transconjugants tested from the DPC143, DPC743, DPC4272, DPC4935, and DPC745 on October 20, 2018 by guest

5 VOL. 67, 2001 EXPLOITATION OF orit FROM pmrc matings contained only prh001. This demonstrated that prh001 can be successfully transferred into the industrial strains without pmrc01 when proteinase K is used to reduce the selective pressure of the bacteriocin. This work shows through mobilization studies that the pmrc01 orit is located within a 312-bp segment between an 18-gene cluster and two divergently transcribed genes (Fig. 1A). Significant similarities are found between the orit of pmrc01 and other orit loci, including multiple inverted repeats and a region homologous to the IncQ family nic sites, indicating similarities between conjugal systems of gram-positive and gram-negative bacteria. Furthermore, significant identity was also found between the deduced TraA protein of pmrc01 and equivalent proteins from several conjugative transfer systems, such as those from the antibiotic resistanceencoding lactococcal plasmid pk214 (97%) and the staphylococcal plasmids pip501 (22%), pg01 (22%), and psk41 (22%) (12, 18). These similarities suggest that other conjugative nickases may be able to act upon the pmrc01 orit with the result that other conjugative plasmids may be able to mobilize prh001. Indeed, we have recently found that another conjugative plasmid, pcbg104 (10), encoding an undefined conjugative transfer system, is capable of mobilizing prh001 (R. M. Hickey, D. P. Twomey, R. P. Ross, and C. Hill, unpublished work). However, in contrast to the orit loci of pci528 and prs01, which can be mobilized by the ML3/712 class sex factor (33, 38), the pmrc01 orit is apparently not mobilized by this element. This inability to mobilize the pmrc01 orit may be a reflection of inherent differences in the secondary structure of this element. While pci528, prs01, and pmrc01 all have similar nic sites (Fig. 1C), pmrc01 is preceded by a GAA sequence flanked by an inverted repeat structure and in this respect is similar to many orit genes of IncQ plasmids of gram-negative organisms and staphylococcal plasmids. In recent years, electroporation as a means of plasmid transfer has become a popular method for transforming lactococci. However, there are a number of limitations to this strategy, including the difficulty in transforming many lactococcal plasmids (particularly if the plasmids are large) into particular strains. While conjugation may overcome some of these problems, this approach also has limitations, which includes plasmid incompatibility or lack of suitable selectable markers. To date, the majority of markers used in cloning vectors are based on resistance to antibiotics and therefore cannot be accepted in starter cultures. Alternative selectable markers include genes for lactose metabolism (8), bacteriocin resistance and immunity (1, 7, 19, 35), bacteriophage insensitivity (23), and suppressor mutations for selection (46). In this study, the location and functionality of the pmrc01 orit has been characterized. Linkage of this element to an appropriate selectable marker (i.e., lacticin 3147 immunity) should offer an efficient system for food-grade improvement of starter and probiotic strains. This research was partly funded by grant aid under the Food Sub- Programme of the Operational Programme for Industrial Development, which is administered by the Irish Department of Agriculture, Food and Forestry and supported by national and EU funds. R.M.H. was supported by the Teagasc Walsh Fellowship Programme. REFERENCES 1. Allison, G. E., and T. R. 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