Conjugative Mobilization as an Alternative Vector Delivery System

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1987, p /87/ $02.00/0 Copyright 1987, American Society for Microbiology Vol. 53, No. 10 Conjugative Mobilization as an Alternative Vector Delivery System for Lactic Streptococci DENNIS A. ROMERO,t PHILIPPE SLOS, CATHERINE ROBERT, ISABELLE CASTELLINO, AND ANNICK MERCENIER* Transgene S.A., Strasbourg, France Received 28 January 1987/Accepted 7 July 1987 Due to the current variability in applying polyethylene glycol-mediated protoplast transformation to lactic streptococci, a study was undertaken to assess the feasibility of conjugative mobilization as an alternative method for vector delivery. By using the broad-host-range conjugative plasmid pva797, the partially homologous cloning vector pva838 was successfully introduced into various strains of Streptococcus lactis, Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, Streptococcus thermophilus, and Streptococcus faecalis. Frequencies ranged from 10-2 to 10-6 transconjugants per recipient. Both pva797 and pva838 were acquired intact, without alteration in functionality. Also, the shuttle vector psa3, which shares partial homology with pva797, was mobilized via conjugation. The use of S. lactis LM2301 as the intermediate donor allowed the use of physiologic and metabolic characteristics for recipient differentiation. The construction of a vector containing a "DNA cassette" conferring mobilization and the resolution, segregation, and stability of the cointegrates, pva797, pva838, and psa3, are also reported. The lactic streptococci Streptococcus lactis, Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Streptococcus thermophilus represent a group of microorganisms important in the manufacture of fermented dairy products. The identification of essential and advantageous plasmid-encoded properties and the development of gene transfer systems in these organisms have generated considerable interest in the application of recombinant DNA technology for strain improvement (12, 20, 26). A prerequisite to using this approach is the ability to enter plasmid-cloning vectors into the desired host. In 1982, Kondo and McKay (18) successfully adapted polyethylene glycol-mediated protoplast transformation to S. lactis, providing the means for directed strain modification. It has been used to clone plasmid-encoded lactose metabolism (19) and UV resistance (6) directly into S. lactis. Although the efficiency has since been optimized, protoplast transformation remains a viable method for only a few strains of S. lactis (19, 31, 32, 36, 37). Simon et al. (31) have reported transformation of a single S. cremoris strain, although at an efficiency of four transformants per microgram of DNA. Geis (15) has demonstrated transfection of S. lactis subsp. diacetylactis F7/2 protoplasts; however, to our knowledge transformation of this strain has not been reported. For S. thermophilus, conjugation is the only form of genetic exchange described (14). Furthermore, for S. lactis, various investigators have had difficulty in adapting or applying the existing transformation protocols to specific host strains (19, 36, 37). This apparent specificity often requires extensive examination of numerous parameters which could become a time-consuming proposition. An alternative gene transfer mechanism which occurs naturally in the lactic streptococci is conjugation, whereby genetic exchange is mediated through close cell-to-cell contact. The conjugal transfer of lactose and sucrose metabolism, proteinase, bacteriophage resistance, and bacteriocin * Corresponding author. t Present address: Department of Food Science, North Carolina State University, Raleigh, NC production and resistance within the mesophilic group N streptococci has been demonstrated. Furthermore, the transfer of broad-host-range antibiotic resistance plasmids such as pamp1 and pip501 within the lactic streptococci has also been reported (for reviews, see references 7, 12, and 20). Conjugation would therefore have the advantage of technical feasability and broad host range for disseminating genetic information. Recently, Smith and Clewell (33) described a means of introducing cloned DNA into Streptococcus faecalis by using conjugative mobilization. The method utilizes the Escherichia coli-streptococcal vector pva838 (24) and the conjugative plasmid pva797 (11) that is partially homologous to the cloning vector. Streptococcus sanguis containing pva797 was first transformed with pva838. Through homologous recombination, pva797 and pva838 formed a cointegrate plasmid which was then transferred to S. faecalis by conjugation. Subsequent resolution of the cointegrate and segregation due to replicon incompatibility resulted in transconjugants containing only pva838. Using this mobilization technique, they were able to return S. faecalis DNA which had originally been cloned in E. coli back to the original host. In this study, we have used a transformable S. lactis strain as an intermediate donor to extend pva797-mediated pva838 mobilization to the dairy streptococci. Transfer of chloramphenicol (pva797) and erythromycin (pva838) resistance was observed at up to 10-2 transconjugants per recipient. Also, we have introduced another shuttle vector, psa3 (8), which shares partial homology with pva797, into various recipients via conjugative mobilization. (This work was presented at the Second ASM Conference on Streptococcal Genetics, Miami, Fla., 21 to 24 May 1986.) MATERIALS AND METHODS Bacterial strains, plasmids, and media. The bacterial strains used in this study are listed in Tables 1 and 2. Several strains not listed are simple derivatives and are described in the text. The mesophilic group N streptococci were grown at 30 C in M17 (35) medium containing 0.5% (wt/vol) glucose or lactose. S. thermophilus was propagated at 42 C in M17 with

2 2406 ROMERO ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains and plasmidsa Phenotypic markers Strain Relevant plasmid(s) Description (reference[s]) Plasmidb Chromosomal S. lactis LM2301 Lac- Strr Plasmid-free derivative of C2 (27, 34) LM2345 Lac- Rif Spcr Plasmid-free derivative of C2 (2) SL2/9738 pva797, pva838 Cmr Emr Lac- Stri This study MMS362 Lac- Strr Recombination-deficient derivative of ML3 (2) SH4160 Lac- Fusr Rif' (14) S. sanguis V797 pva797 Cmr (11) E. coli 1106 Transformation host (30) (hsdr hsdm supe supf hsds met) 1106(pVA838) pva838 Cmr Emr This study 1106(pSA3) psa3 Cmr Emr Tcr This study 1106(pTG222) ptg222 Apr Emr This study a Lac-, Lactose negative; Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; Fus, fusidic acid; Rif, rifampin; Spc, spectinomycin; Str, streptomycin; Tc, tetracycline. bplasmid-encoded markers are those expressed in the listed host. 1.0% (wt/vol) sucrose. S. faecalis and S. sanguis were grown in brain heart infusion broth (Difco Laboratories) at 37 C. Acid production was determined by plating on bromocresol purple (BCP) indicator agar (28) containing 1.0% (wt/vol) of the desired carbohydrate. In streptococci, pva797 was maintained by the addition of 5,ug of chloramphenicol per ml. For pva838 and psa3, 10,ug of erythromycin per ml was used. Unless otherwise noted, antibiotics were used at the following concentrations (micrograms per milliliter): chloramphenicol, 5; erythromycin, 10; fusidic acid, 50; rifampin, 50; spectinomycin, 300; streptomycin, 1,000; and tetracycline, 15. Stock cultures were maintained by biweekly transfer in the appropriate medium or stored frozen at -80 C by suspending a pellet from 10 ml of a mid-log- TABLE 2. Transfer of pva797 and pva838 by filter matinga Mating pair Transconjugants per recipient selected on: Donor Recipient Chloramphenicol Erythromycin Chloramphenicol and erythromycin S. lactis SL2/9738 x S. lactis [LM2301(pVA797::pVA838)] LM x x x 10-3 C2 5 x X X10-3 ML3 8 x x x10-3 C1o 7 x x x10-4 M18 2 x x x1o x x x10-4 S. cremoris B1 4 x X x10-4 C3 3 x x 10-4 ND HP 4 x x x x x x10-3 S. lactis subsp. diacetylactis F7/2 4 x X X1O-4 NCIB x x X 10-4 NCIB x X X10-4 S. thermophilus A054 6 x x x10-5 A158 5 x x x10-4 S. faecalis JH2-2 ND ND 2 x 1O-3 S. lactis SL5/9738 x S. lactis MMS362 5 x X 1O-3 ND [LM2345(pVA797::pVA838)] S. thermophilus S31 ND 1 x 10-6 ND S33 4 x X x10-5 S x x X 1O-7 a ND, Not determined. NCIB, National Collection of Industrial Bacteria. S. thermophilus A054 and A158 are industrial yogurt starters. S. thermophilus S31, S33, and S19258 are Stri derivatives of industrial yogurt starters A031, A033, and type strain ATCC 19258, respectively.

3 VOL. 53, 1987 VECTOR MOBILIZATION IN LACTIC STREPTOCOCCI 2407 phase culture in 2.0 ml of synthetic medium, 2.0 ml of 87% glycerol with 20 mm MgSO4. 7H20, and 4.0 ml of 10% (wt/vol) reconstituted skim milk. E. coli 1106 was propagated in LB medium (25) at 37 C. pva838 and psa3 were maintained in E. coli by the addition of 25,ug of chloramphenicol per ml. Although expressed in E. coli 1106, the Emr markers of psa3 and pva838 were not found to be suitable for primary selection. Plasmid isolation and characterization. Streptococcal plasmid isolation was performed by the protocol of Anderson and McKay (1) with minor modifications. Cells were grown in M17 broth instead of lysis broth, and for S. thermophilus lysozyme and sodium dodecyl sulfate treatment times were extended to 15 and 10 min, respectively. For S. sanguis, growth was in brain heart infusion broth supplemented with 20 mm DL-threonine and lysozyme digested for 60 min. For S. faecalis, cells were grown to mid-log phase in brain heart infusion broth and lysozyme digested for 30 min. E. coli V517 (22), J53 containing the 23-megadalton (MDa) plasmid psa, and C600 containing the 65-MDa plasmid p624a (D. Herman, personal communication) were lysed by the same protocol and used as molecular mass plasmid standards. Standard E. coli plasmid isolation was by the alkaline lysis method (25), and preparative amounts of DNA were purified through cesium chloride-ethidium bromide density gradient centrifugation. Restriction endonuclease digestion was performed as outlined by the enzyme manufacturer. Plasmid DNA was visualized by horizontal gel electrophoresis on a 0.6% agarose gel in TAE buffer (40 mm Tris, 20 mm acetic acid, 1 mm disodium EDTA, ph 8.0). Electrophoresis was performed at a constant 4 V/cm and allowed to migrate for 7 cm Ḃacterial transformation. Transformation of S. lactis LM2301 was performed by using the optimized protocol of Kondo and McKay (19). Erythromycin-resistant transformants were selected by adding 5,ug of antibiotic per ml to the regeneration medium. Competent cells of E. coli 1106 were prepared and transformed as described by Hanahan (16). For rapid plasmid analysis of streptococcal transformants and transconjugants, 2 to 5,ll of miniprep DNA was used to transform E. coli. Appropriate selection conditions enabled the isolation of either pva838 or psa3 shuttle vectors from the resident plasmid complement of the various streptococcal strains. Molecular cloning. The cloning vector ptglpoly (R. Lathe and M. P. Kieny, personal communication) is a minipbr322 derivative containing the M13TG131 polylinker (17). Vector DNA was digested to completion with HindIlI and treated with calf intestinal alkaline phosphatase (Boehringer GmbH). pva838 was digested with HindlIl to liberate the pva749 fragment, which was purified by agarose gel electroelution (25). pva749 consists of the MLS (macrolide, lincosamide, streptogramin B) resistance gene of pamp1 and the pva380-1 origin of replication (23). pva749, as a single HindIII fragment, was mixed in a ratio of 1:1 with phosphatase-treated ptglpoly. T4 DNA ligase was added to the mixture and incubated at 15 C overnight. Competent E. coli 1106 cells were transformed with the ligation mixture and selected on LB agar containing 100,ug of ampicillin per ml. Apr transformants were then screened on LB medium containing 150,ug of erythromycin per ml to select for recombinant plasmids. Conjugation. Conjugation experiments were performed by using the filter mating protocol of Gasson and Davies (14) with modifications. Overnight (ca. 18-h) cultures grown in their respective media were harvested, washed once, and suspended in an equal volume of sterile 0.9% NaCl (saline) solution. Routinely, 0.1 or 1.0 ml of donor cells was mixed with 1.0 ml of recipient cells in a total volume of 2.0 ml of saline solution. The mating mixture was collected onto a 25-mm-diameter, 0.45-,um-pore size nitrocellulose filter (type HAWP; Millipore Corp.) which was then placed onto an M17 agar plate. For S. lactis, S. cremoris, and S. lactis subsp. diacetylactis, filters were incubated at 30 C. Matings with S. faecalis, S. sanguis, or S. thermophilus were performed anaerobically (GasPak; BBL Microbiology Systems) at 37 C. After 20 to 24 h of incubation, filters were placed in a test tube and the growth was suspended in 1.0 ml of saline solution by vortexing. Appropriate dilutions were plated to select for donor, recipient, or transconjugant phenotypes. Transconjugants were selected on agar containing chloramphenicol or erythromycin or both antibiotics in combination. Antibiotic selection used concentrations previously described except for streptomycin, which was increased to 2,000 jig/ml. Differentiation by positive lactose fermentation was done by plating on BCP lactose indicator agar containing the appropriate antibiotic. For S. thermophilus, selection was performed at 45 C on indicator M17 agar in which,-glycerophosphate was reduced to 5 g/liter and 40 mg of BCP per liter was added. The authenticity of the transconjugants was verified by phage typing and by determining the sugar utilization profile. Plasmid stability and segregation. The stability of pva797, pva838, and psa3 in S. lactis LM2301 under nonselective conditions was tested. An overnight culture was inoculated at 2% (wt/vol) into antibiotic-free M17 broth and incubated at 37 C for 24 h. A series of sequential overnight propagations in antibiotic-free broth were performed, using the culture from each day as an inoculum into fresh medium. A single overnight broth propagation under these conditions was calculated to be approximately six generations. Samples were taken periodically and plated for single colonies onto M17 agar. At least 100 individual colonies were then stabbed onto M17 agar containing chloramphenicol or erythromycin to assess the loss of plasmid markers. Resolution and segregation of the pva797::pva838 cointegrate in intermediate donor S. lactis SL2/9738 were similarly tested by sequential propagation in M17 broth containing chloramphenicol, erythromycin, or no antibiotic. The acquisition of plasmid markers in conjugation/mobilization experiments was assessed by taking colonies directly from conjugal selection plates and stabbing into agar containing chloramphenicol or erythromycin. In all experiments, colonies were scored for growth or no growth and representative isolates were analyzed for plasmid content. RESULTS The conjugative plasmid pva797, 20 MDa (30.7 kilobases [kb]), was constructed by Evans and Macrina (11) by replacing the erythromycin resistance of pip501 with the replication origin of Streptococcus ferus cryptic plasmid pva380-1 (Fig. 1). pva797 confers only chloramphenicol resistance and has two functional streptococcal origins of replication which, however, do not lead to structural instability. The E. coli-streptococcal shuttle vector pva838, 6.1 MDa (9.2 kb), is composed of the MLS resistance gene of pamp1, the replication origin of pva380-1, and the entire pacyc184 plasmid (Fig. 1). In the construction of pva838, the tetracycline resistance determinant of pacyc184 was inactivated, leaving the chloramphenicol and erythromycin resistance markers functional ine. coli. In streptococci, only the

4 h%4 #I ROMERO ET AL. PU I Hind III FIG. 1. Restriction endonuclease maps of pva797 (11), pva838 (24), and psa3 (8) adapted from the original reports showing the regions of homology (_, =z) existing between each plasmid. erythromycin resistant determinant is expressed. In S. sanguis, pva797 and pva838 replicate by using the pva380-1 origin and are mutually incompatible (11, 24). The region of homology between pva797 and pva838 spans the pva380-1 DNA segment and is approximately 3 kb in length (Fig. 1). The shuttle vector psa3, 6.8 MDa (10.2 kb), was constructed by Dao and Ferretti (8) by inserting AvaIlinearized pgb305 into the single AvaI site of pacyc184 (Fig. 1). pgb305 is a deletion derivative of pip501 which confers erythromycin resistance only and is no longer self- A B C D E F G transmissible (4). From comparative restriction map analysis of psa3 and pva797, we determined the single longest stretch of homology between the two plasmids to span the HindIlI fragment at approximately 4.3 to 6.0 kb on the psa3 map and 23.8 to 25.5 kb on the pva797 map (Fig. 1). This region of approximately 2 kb encompasses the replication/ copy functions of the parental pip501 plasmid present in both psa3 and pva797. Construction of an intermediate S. lactis donor. S. lactis C2 derivatives LM2301 and LM2345 were used for preliminary testing of pva797/pva838 mobilization in lactic streptococci. Both strains are transformable and complementarily marked for easy counterselection and provide a plasmidfree, prophage-free, isogenic background for conjugation experiments. To construct an intermediate S. lactis donor, LM2301 was first mated with S. sanguis V797. Cmr Strr transconjugants were isolated at 8.2 x 10-5 per recipient and a representative isolate designated SL2/797A was stocked for future use. pva838 purified from E. coli 1106(pVA838) was used to transform LM2301. Emr transformants were recovered at approximately 102 per,ug of DNA and found to harbor a single 6.1-MDa plasmid. A representive isolate designated SL2/838 was then mated with SL2/797A. Cmr Emr transconjugants were recovered at approximately 10-3 per recipient and contained pva797, pva838, and a large plasmid bandingjust above the chromosome. The size of this plasmid was estimated to be approximately 26 MDa and is consistent with the formation of a pva797::pva838 cointegrate as described by Smith and Clewell (33). The comparative plasmid profiles of S. lactis SL2/797A, SL2/838, and intermediate donor SL2/9738 [LM2301(pVA797::pVA838)] are shown in Fig. 2. Mobilization of pva838 was tested in a homologous system by mating SL2/9738 with LM2345. Transconjugants were selected on agar containing rifampin and spectinomycin with chloramphenicol or erythromycin or both antibiotics in combination. Transfer of Cmr or Emr or both occurred at approximately 1o-3 transconjugants per recipient. Transduction was ruled out since both strains originate from a prophage-cured parent and mating efficiency in the presence of 100,ug of DNase I per ml was not significantly affected, excluding transformation. Furthermore, no antibioticresistant colonies corresponding to a recombinant phenotype were observed when donor or recipient cells were plated at 108 to 109 CFU/ml on the appropriate agar. These HI J K L M N 0 APPL. ENVIRON. MICROBIOL. chr pva797 pva838 FIG. 2. Plasmid profiles of S. lactis SL2/797A (lane A), SL2/838 (lanes B and 0), and intermediate donor SL2/9738 (lane C) compared with various S. lactis transconjugants: lane D, S. lactis C10; E, C10(pVA838); F, C1O(pVA797::pVA838); G, C10(pVA797); H, S. lactis M18; I, M18(pVA797::pV838); J, S. lactis 7962; K, 7962(pVA838); L, 7962(ApVA838); M, 7962(pVA797::pVA838); N, 7962(pVA797). The extra bands appearing in pva838-containing strains represent different forms of the plasmid. Southern blot analysis has confirmed, in the case of S. lactis SL2/838 (lanes B and 0), that all of the bands correspond to pva838. m

5 VOL. 53, 1987 VECTOR MOBILIZATION IN LACTIC STREPTOCOCCI 2409 TABLE 3. Transfer of pva797, pva838, ptg219, and ptg222 from various S. lactis donors to S. lactis LM2345 by filter mating Transconjugants per recipient Donor selected on: Chloramphenicol Erythromycin SL2/ x x 10-3 [LM2301(pVA797: :pva838)] ML3/ x x 10-3 [ML3(pVA797: :pva838)] SL3/ x X 10-3 [MMS362(pVA797: :pva838)] SL13/ x 1O-3 2 x 10-3 [MG1363(pVA797: :pva838)] SL2/ x x io-9 [LM2301(pTG219, pva797)] SL2/ x 1O-3 2 x 1o-3 [LM2301(pTG222::pVA797)] SL14/ x 1O-4 4 x 1O-4 [IL1403(pTG222: :pva797)] controls confirmed conjugation as the mechanism of transfer. Putative transconjugants were verified by their susceptibility to streptomycin. Analysis of selected Cmr Emr transconjugants showed their plasmid profiles to be identical to donor SL2/9738 for which pva797, pva838, and a cointegrate plasmid were observed. To determine the structure of pva838 acquired via conjugative mobilization, miniprep DNA from randomly selected transconjugants were used to transform E. coli 1106 to chloramphenicol resistance. When analyzed by restriction endonuclease digestion, these transformants were found to contain a single 9.2-kb plasmid which was indistinguishable from authentic pva838. A representative isolate, designated SL5/9738 [LM2345 (pva797::pva838)] was later used as a donor in matings with S. thermophilus, indicating that the plasmids were acquired without functional alteration. Mobilization of pva838. To demonstrate the versatility of conjugative mobilization, S. lactis SL2/9738 was used in matings to various recipients, with selection based on differential metabolic or physiologic characteristics as opposed to antibiotic resistance. In homologous matings with parental S. lactis C2 and closely related strain ML3 (9), positive lactose fermentation (Lac') was used to differentiate donor and recipient cells. On BCP lactose indicator agar containing chloramphenicol or erythromycin or both, yellow Lac' Cmr/Emr transconjugants were easily identified against a background of white donor cells. Transfer occurred at about 10' transconjugants per recipient, which was equivalent to the frequencies observed with LM2345 (Table 2). Because C2 and ML3 contain a lactose plasmid transferable via transduction (29) or conjugation (27), respectively, control platings on BCP lactose agar containing 2,000,ug of streptomycin per ml were performed to verify the direction of genetic exchange. No Lac' Strr colonies were observed, excluding the transfer of lactose metabolism to the donor SL2/9738. Plasmid analysis of randomly selected transconjugants confirmed the acquisition of the relevant plasmids (data not shown). pva838 structure was verified via transformation and restriction analysis through E. coli. Furthermore, an ML3(pVA797: :pva838) transconjugant (ML3/ 9738) was capable of conjugally transferring Cmr and Emr to LM2345 (Table 3). To broaden the host range of pva797/pva838 mobilization, matings were performed to S. lactis C10, M18, and 7962, S. cremoris Bi, C3, HP, and 205, and S. lactis subsp. diacetylactis F7/2, 10484, and For S. lactis C10, M18, and 7962, efficiency of plaquing of virulent c2 phage indicated significant modification-restriction barriers between these strains and the S. lactis C2/ML3/712 family (A. Mercenier and I. Castellino, unpublished results). Furthermore, C10 and M18 were previously reported as nontransformable by Kondo and McKay (19). Transfer of Cmr or Emr or both from SL2/9738 was accomplished to all recipients tested and ranged from 10-2 to 10-4 transconjugants per recipient (Table 2). Differentiation was based on lactosefermenting ability, while appropriate controls verified that lactose metabolism had not been transferred to SL2/9738. Plasmid analysis of selected isolates confirmed transconjugant identity and the presence of pva797 and pva838 (Fig. 2). The host range was further extended by mating S. lactis SL2/9738 or SL5/9738 to S. thermophilus A054, A158, S31, S33, and S19258 and S. faecalis JH2-2. For S. thermophilus, selection was either based on resistance to streptomycin (S31, S33, and S19258) or by growth at 45 C (A054 and A158). Since S. lactis SL2/9738 is unable to ferment sucrose, BCP sucrose indicator agar was also used as a nonselective secondary marker to follow S. thermophilus recipients. Cmr and Emr transconjugants were recovered from all mating pairs at 10-3 to 10-8 per recipient (Table 2). Selection at 45 C was effective in removing any S. lactis background up to a bacterial population equivalent to 107 CFU/ml. The plasmid profiles of S. thermophilus S31 and S33 transconjugants are shown in Fig. 3. Transfer to S. faecalis JH2-2 occurred at 1.4 X 10-3 transconjugants per recipient, using Fusr Rif' selection (Table 2). Plasmid analysis confirmed the presence of pva797 and pva838. The need for homologous recombination was demonstrated in attempted mobilization of Emr shuttle vector ptg219 (A. Mercenier and C. Robert, unpublished results) by pva797. ptg219 is essentially pva838 with the pva380-1 region of homology replaced by insertion of S. thermophilus plasmid pa2 into the ori-probe vector pva891 (21). S. lactis SL2/1997 [LM2301(pTG219, pva797)] was mated with LM2345 and scored for Cmr or Emr transconjugants (Table 3). Transfer of Cmr occurred at about 10-3 per recipient, which is equivalent to pva797 transfer between different S. lactis strains. In contrast, only a single Emr transconjugant was isolated, representing a frequency of 10' per recipient compared to 10-3 observed for pva838 mobilization. This isolate was also Cmr and contained a large plasmid of about 24 MDa which would be expected for a ptg219::pva797 cointegrate. A subsequent construction, ptg222 (see below), containing the pva380-1 chr pva797 - A BC D E F G H PVA838-_ FIG. 3. Plasmid profiles of S. lactis SL2/838 (lane A), donor S. lactis SL2/9738 (lane B), and S. thermophilus transconjugants: Lane C, S31; D, S31(pVA838); E, S31(pVA797::pVA838); F, S33; G, S33(pVA838); H, S33(pVA797::pVA838).

6 2410 ROMERO ET AL. TABLE 4. Segregation of chloramphenicol and erythromycin markers in pva797::pva838 transconjugantsa No. of transconjugants/ Recipient Selection total with: Cmr Emr S. lactis LM2345 Cm Rif Spc 13/50 Em Rif Spc 49/50 S. lactis C2 Cm Lac 7/25 Em Lac 31/32 S. lactis ML3 Cm Lac 7/30 Em Lac 29/30 S. lactis MMS362 Cm Str 35/100 Em Str 91/100 S. lactis C10 Cm Lac Em Lac 49/50 S. lactis 7962 Cm Lac 10/30 Em Lac 128/130 S. thermophilus S31 Cm Str Em Str 10/15 S. thermophilus S33 Cm Str 4/50 Em Str 79/83 S. thermophilus S19258 Cm Str 4/37 Em Str 36/41 a Individual colonies were taken directly from conjugal selection plates, stabbed into agar containing chloramphenicol or erythromycin, and scored for growth after 24 h of incubation at 30 C. The donor strain was SL2/9738 or SL5/9738. replication origin and MLS resistance gene of pva838 inserted into a pbr322-based plasmid was mobilized by pva797 at an efficiency similar to that for pva838 (Table 3). Smith and Clewell (33) had been unable to mobilize pva838 to a recombination-deficient S. faecalis recipient. However, we were able to conjugally mobilize the vector into S. lactis MMS362, a Strr recombination-deficient derivative of ML3 (2). The pva797::pva838 cointegrate was efficiently transferred from SL5/9738 to MMS362 (Table 2), where it was resolved and segregated (Table 4; see below). Furthermore, a transconjugant designated SL3/9738 [MMS362(pVA797: :pva838)] transferred both Cmr and Emr markers to LM2345 at efficiencies comparable to SL2/9738 and ML3/9738 donors (Table 3). The recombination-deficient phenotype of MMS362 and SL3/9738 was confirmed by their marked UV sensitivity compared with parental ML3. At this time we have no explanation for these results. In addition to C2 and ML3 derivatives, two commonly used S. lactis strains, MG1363 (13) and IL1403 (5), were also tested as intermediate donors for conjugative mobilization of pva838 or ptg222. Strains SL13/9738 [MG1363(pVA797:: pva838)] and SL14/2297 [IL1403(pTG222::pVA797)] were constructed through transformation and conjugation as described for SL2/9738. In matings with LM2345, SL13/9738 and SL14/2297 conjugally transferred Cmr and Emr at 10-4 to 0-3 transconjugants per recipient (Table 3). pva797::pva838 resolution and segregation. As previously described (33), the pva797: :pva838 cointegrate could be resolved into two separate plasmids which segregated due to mutual replicon incompatibility. In S. lactis SL2/9738, resolution and segregation were observed with preferential loss of Emr. Under nonselective growth conditions, one overnight propagation resulted in 40 of 100 colonies tested losing Emr. This increased to 85 of 100 colonies after 5 days. No Cms Emr colonies were isolated from antibiotic-free propagation. Expectedly, loss of Emr was also seen when SL2/9738 was grown in the presence of chloramphenicol. Conversely, loss of Cmr was not observed during growth APPL. ENVIRON. MICROBIOL. under erythromycin selection. Isolation of either Cms Emr or Cm' Ems colonies from SL2/9738 was not observed. Plasmid analysis of randomly selected Ems isolates confirmed the loss of pva838 and the presence of pva797. Isolation of Cms Emr transconjugants (i.e., containing only pva838) was possible when colonies were taken directly from erythromycin conjugation selection plates and stabbed onto agar containing chloramphenicol or erythromycin to assess marker acquisition. For the conjugal recipients shown in Table 4, the frequency of isolating such colonies ranged from 1.5 to 12.5% of erythromycin-selected transconjugants. The inability to isolate Cms Emr transconjugants from other recipients was likely due to an insufficient number of colonies tested. When colonies from chloramphenicol conjugation selection plates were similarly tested, Cmr Ems transconjugants which had acquired only pva797 (Fig. 2) were readily isolated. Plasmid analysis of Cm' Emr isolates shown in Fig. 2 and 3 for selected S. lactis and S. thermophilus strains confirmed the presence of intact pva838. Plasmid stability. Under nonselective growth conditions, both pva797 and pva838 were stably maintained in S. lactis LM2301 during growth at 37 C. After 10 sequential propagations (approximately 60 generations), no loss of either plasmid marker was observed. In contrast, psa3 in LM2301 was rapidly lost at 37 C, although it was stably maintained in this host at 32 C. After a single overnight propagation, 45% of the colonies tested had become Ems. After 6 sequential propagations (approximately 36 generations), 100% of the colonies tested had become Ems. S. thermophilus S31 transconjugants containing either pva797 or pva838 were similarly tested for plasmid stability during growth at 42 C. After approximately 60 generations over 10 sequential overnight propagations, no loss of antibiotic resistance could be observed, indicating stable plasmid inheritance. Construction of a mobilization cassette. It had previously been shown that recombinant plasmids based on pva380-1 could be efficiently mobilized by pva797 (33). The grampositive region of pva838 was liberated as a single HindlIl fragment (pva749) and inserted into the single HindIll site of cloning vector ptglpoly (2 kb). The resulting plasmid designated ptg222 (7.0 kb) contains the pva380-1 replicon and the MLS resistance gene in the middle of the M13TG131 polylinker and confers Apr Emr in E. coli. Restriction endonuclease analysis showed the EcoRI, SmaI, BanII, SacI, SphI, KpnI, XbaI, and BamHI sites remained unique in ptg222, allowing for asymmetric cloning of the "mobilizable DNA segment." Furthermore, ptg222 is fully functional in S. lactis (having the same properties as pva838), confers Emr, is stably inherited without selective pressure, and is mobilizable by pva797, resulting in precise resolution and segregation. S. lactis strains containing ptg222 were susceptible to as little as 1,ug of ampicillin per ml, indicating the Apr gene to be nonfunctional. Mobilization of psa3. S. lactis SL2/797A was mated to S. lactis SL2/A3 [LM2301(pSA3) transformant], resulting in the intermediate donor SL2/A397 [LM2301(pSA3, pva797)]. Plasmid profiles of these three strains are shown in Fig. 4. In contrast to SL2/9738, a cointegrate plasmid was not observed in SL2/A397. To assess the ability of pva797 to mobilize psa3, SL2/A397 was mated to S. lactis LM2345 and SH4160 (Table 5). Transfer of Cmr occurred at the expected frequency of 10-3 per recipient. However, selection for Emr resulted in a marked decrease in conjugation efficiency. Plasmid analysis of randomly isolated Cmr Emr

7 VOL. 53, 1987 VECTOR MOBILIZATION IN LACTIC STREPTOCOCCI 2411 Pf5kr A B C D E F G- H pva GINTEGRATE psa3 FIG. 4. Plasmid profiles of S. lactis SL2M97A (lane C), SL2/A3 (lane D), and intermediate donors S. lactis SL2/A397 (lane E) and SL2/A397C (lane F containing the 25-MDa cointegrate) which are compared to those of S. thermophilus transconjugants S31 (lane G) and S31/A397C (lane H). Molecular mass reference plasmids are shown in lanes A (E. coli V517) and B (E. coli J53 [23-MdA psa]/c600 [65-MDa p624a]). SH4160 transconjugants found that the majority had acquired a single plasmid of approximately 26 MDa. This would be expected for the formation of a psa3::pva797 cointegrate. A representative transconjugant designated SL4/ A397C [SH4160(pSA3::pVA797)] conjugally transferred Cmr Emr to LM2301 with concurrent transfer of the 26-MDa plasmid (Fig. 4). In third-generation matings, SL2/A397C [LM2301(pSA3: :pva797) transconjugant containing a 26-MDa plasmid] transferred Cmr Emr to LM2345 at approximately 2 orders of magnitude greater than SL2/A397 when psa3 and pva797 exist separately. The reason for this difference is unknown at this time, although we hypothesize that the recombination event between psa3 and pva797 may disrupt the expression of a gene(s) necessary for efficient conjugal transfer. Nevertheless, once a stable recombinant is formed, transfer occurs at frequencies equivalent to pva797. From the original SL2/A397 x SH4160 mating, one of the colonies isolated from erythromycin selection plates was found to be Cm', indicating possible resolution and segregation of markers. Interestingly, this Cm' Emr transconjugant contained a single plasmid approximately the same size as pva797 (data not shown). Furthermore, Emr could be conjugally transferred to LM2301 at 10-3 per recipient with concurrent transfer of this plasmid. At this time the recombinational event resulting in an Emr Tra+ Mating pair TABLE 5. (self-transmissable) plasmid of about 20 MDa (30 kb) is unknown. To further examine resolution and segregation, transconjugants from SL2/A397 or SL2/A397C to LM2345 matings were tested for acquisition of plasmid markers. From SL2/A397 x LM2345 matings, 100 of 100 colonies from chloramphenicol selection plates were Ems, indicating the transfer of pva797 only. Of the 100 colonies tested from erythromycin selection plates, 2 were found to be Cms. These isolates, however, contained a plasmid similar in size to pva797 and not psa3 as expected. For SL2/A397C x LM2345 matings, 3 of 100 transconjugants from chloramphenicol selection plates were Ems, indicating resolution of the 26-MDa cointegrate. No Cms Emr transconjugants were isolated from erythromycin selection plates. Resolution experiments were also performed on SL2/ A397C and SL4/A397C by propagating each strain with only chloramphenicol or erythromycin selection at 37 C. After one overnight propagation followed by plating for single colonies on the appropriate agar, no Cms Emr colonies were isolated. Only 1 of 100 colonies tested from chloramphenicol propagation of each strain was found to have lost Emr, indicating cointegrate resolution. Plasmid analysis of these two strains revealed a plasmid comigrating with pva797, suggesting precise resolution and then loss of psa3. The structure and functionality of this plasmid have not been tested at this time. Intraspecific matings demonstrated that the Emr marker of psa3 could be conjugally mobilized from S. lactis to other streptococci. SL2/A397C transferred the 26-MDa cointegrate with Cmr Emr to S. faecalis JH2-2 at 10-3 transconjugants per recipient. Furthermore, SL4/A397C also transferred Cmr Emr to S. thermophilus S31 and S33 at 10-5 per recipient. The plasmid profile of a representative Cmr Emr S31 transconjugant (S31/A397C), shows the acquisition of the 26-MDa cointegrate (Fig. 4). Although not visible in this photograph, we observed a very faint band in S31/A397C comigrating with psa3, indicating the presence of free psa3 in addition to the cointegrate plasmid. To examine this possibility, miniprep DNA from S31/A397C, SL2/A397C, and SL4/A397C (no free psa3 was visible in the latter two strains) was used to transform E. coli. Compared to DNA derived from pva838-containing strains, relatively few Cmr transformants were obtained. These transformants were also Tcr Emr and contained a 10.2-kb plasmid indistinguishable from psa3. This would suggest the presence of intact free Transfer of pva797 and psa3 by filter mating Transconjugants per recipient selected on: Donor Recipient Chloramphenicol Erythromycin Chloramphenicol and erythromycin S. lactis SL2/A397 x S. lactis [LM2301(pSA3, pva797)] LM x X 10-5 NDa SH x x x 10-6 S. lactis SL4/A397C x S. lactis [SH4160(pSA3::pVA797)] LM2301 ND ND 3 x 10-3 LM2345 ND ND 7 x 10-3 S. thermophilus S31 1 x x x 10-6 S33 2 x x x 105- S. lactis SL2/A397C x S. lactis LM2345 ND ND 2 x 10-3 [LM2301(pSA3::pVA797)] S. faecalis JH2-2 ND ND 1 X 10-3 a ND, Not determined.

8 2412 ROMERO ET AL. psa3 in the transconjugants or the precise resolution of the cointegrate plasmid by E. coli. Southern blot analysis of putative psa3::pva797 cointegrate-containing transconjugants, using pacyc184 as a probe, showed hybridization to the 26-MDa plasmid band as well as a faint signal where psa3 would normally be found in agarose gels (data not shown). No hybridization occurred between the pacyc184 probe and pva797 or the 20-MDa Emr Tra+ plasmid. This confirmed the existence of free psa3, although at very low levels, in Cmr Emr transconjugants and that psa3 is an integral part of the cointegrate plasmid. DISCUSSION In this study, we demonstrated the utility of conjugative mobilization for entering plasmid-cloning vectors in the lactic streptococci. Following the original report of Smith and Clewell (33), we have extended the host range of pva797-mediated mobilization of pva838 to include dairy streptococcal starter cultures. We have also shown that the shuttle vector psa3, which shares a 2-kb region of homology with pva797, could also be mobilized in some form. The principal advantages to using psa3 are as follows: functional Cmr and Tcr markers in E. coli, allowing for insertional inactivation; and the ability to replicate and express Emr in Bacillus subtilis. Furthermore, we constructed a novel shuttle vector, ptg222, which contains the DNA segment conferring mobilization by pva797 on a convenient multiplesite polylinker. Although we do not advocate the use of Apr-based shuttle vectors in streptococci, we introduced ptg222 into S. lactis to demonstrate that the mobilization properties had not been affected. In any case, the pbr322 Apr gene of ptg222 is nonfunctional in S. lactis. Although the selective basis of the plasmids used in this work rely on antibiotic resistance, making them unsuitable for direct industrial application, pva797-mediated mobilization should be useful for rapid assessment of cloned gene function in the desired streptococcal host. The protoplast transformation methods described for the streptococci are usually strain dependent. We thus applied the conjugative mobilization system to provide an alternative means for gene cloning into various dairy starter cultures. S. lactis was chosen as the intermediate donor since this species is closer to the dairy streptococci than S. sanguis or S. faecalis. In addition, intraspecific conjugal transfer from S. lactis donors to various streptococci has been previously demonstrated (14). Moreover, S. lactis is the only species of dairy streptococci that can be efficiently transformed at this time. We have introduced pva797- and pva380-1-based vectors into the S. lactis strains most commonly used for molecular cloning: C2 derivatives LM0230 and LM2301 (10, 27, 34), NCDO712 derivative MG1363 (13), and IL594 derivative IL1403 (5). These strains possess several useful characteristics as intermediate donors for delivering cloned genes to industrial starters. They are transformable with current protocols at up to 104 transformants per microgram of DNA (19, 31, 36); for IL1403, Simon et al. (32) have reported transformation as high as 5 x 106 transformants per microgram of DNA. These strains are also plasmid-free, simplifying analysis of recombinant plasmids, and prophage-free, ensuring against the possible introduction of bacteriophage into the recipient strain. Finally, LM0230 and LM2301, MG1363, and IL1403 are lactose negative, which was essential to the differential scheme we used for conjugal matings. At the frequencies observed for pva797/pva838 transfer, Lac' colonies are easily distin- APPL. ENVIRON. MICROBIOL. guishable from white donor cells. Since most industrial strains are likely to be Lac', it is unnecessary to genetically mark prospective recipient strains for counterselection. In matings with S. thermophilus, we took advantage of its ability to grow above 42 C to effectively select against mesophilic donor cells. Therefore, using the proper combination of metabolic and physiologic characteristics indigenous to the recipient with the appropriate intermediate donor, the need to mutagenize industrial strains is avoided. Recently, the,-galactosidase gene from S. thermophilus has been subcloned into ptg222. This chimeric plasmid was transformed into S. lactis and subsequently transferred to various recipients via pva797-mediated conjugative mobilization. Transfer occurred at the expected frequencies and without alteration of plasmid structure (P. Slos, C. Robert, and A. Mercenier, unpublished results). By combining established E. coli and B. subtilis cloning techniques with existing methods for S. lactis, conjugative mobilization will allow for the study of cloned gene function in the desired lactic streptococcal host. ACKNOWLEDGMENTS We thank D. B. Clewell, J. J. Ferretti, M. J. Gasson, F. L. Macrina, and L. L. McKay for their generous contribution of strains and plasmids used in this study. We also thank J. P. Lecocq and Y. Lemoine for their scientific support and encouragement of this project. We are grateful to P. Kourilsky and P. Chambon for continued interest in this work and to A. Fazel for critical reading of the manuscript. We thank F. Daul for the preparation of figures, and we especially thank L. Schneider for her expertise in preparing this manuscript. This work has been supported by BSN-Gervais-Danone. LITERATURE CITED 1. Anderson, D. G., and L. L. McKay Simple and rapid method for isolating large plasmid DNA from lactic streptococci. 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