High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage Padht

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AND

ENVIRONMENTAL MICROBIOLOGY, Jan. 1992,

p. 187-193

Vol. 58, No. 1

0099-2240/92/010187-07$02.00/0 Copyright © 1992, American Society for Microbiology

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage Padht R. R. RAYA't AND T. R. KLAENHAMMERl 2* Departments of Food Science' and Microbiology,2 Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624 Received 16 June 1991/Accepted 21 October 1991

Plasmid transduction can be mediated by lytic or temperate bacteriophages. The basal frequency of plasmid transduction can be increased by providing phage-plasmid DNA homology, either by inserting a phage DNA fragment into the plasmid DNA molecule or vice versa (5). The effect of this shared homology upon transduction frequency depends on the transduction system under study. In some systems, the cloning of any phage DNA fragment increases the frequency of plasmid transduction (1). In others, the greatest effects are observed when the transducible plasmid contains specific sequences of phage DNA, such as cohesive ends (22, 27, 37), a pac site (17, 34), or a tertiary origin of replication (13). Plasmid transduction is a useful tool for moving plasmids among different strains. Thus, it has been used as a simple and rapid alternative to the transformation of plasmid DNA, particularly for cells that are poorly transformed (31) or not transformed at all. In the latter case, plasmid transduction has provided the first reliable method for the introduction of P1-mediated recombinant DNA into Myxococcus xanthus (30). Plasmid transduction has also been used as an efficient means to define the host range of a bacteriophage, mainly among cells that do not support lytic growth of the phage (24, 26), as well as to identify the DNA sequences required for the packaging of bacteriophage T7 DNA (4) or P22 pac-like signals on the chromosome of Salmonella typhimurium (35). The significantly improved transduction frequency of a plasmid provided by DNA-DNA homology between plasmid and phage has also been exploited to develop in vivo screening methods for the identification of the desired clone from a

genomic DNA library (32, 36, 39). Finally, plasmid DNA transduction has been instrumental in the isolation of cointegrates and the characterization of a site-specific recombination system in Staphylococcus aureus plasmids (29). Generalized transduction mediated by the Lactobacillus salivarius temperate bacteriophage PLS-1 (43) and plasmid transduction mediated by bacteriophage 4~adh are, so far, the only two reports of transduction in the genus Lactobacillus. Bacteriophage (adh, a temperate phage of Lactobacillus gasseri ADH (formerly named Lactobacillus acidophilus ADH [11]), has recently been characterized in our laboratory (33). The genome of 4adh is a linear double-stranded DNA molecule (43 kb) with cohesive ends. Phage Xadh mediates plasmid transduction in ADH at frequencies of 10-8 to 10-10 transductants per PFU (33). In the present study, BglII fragments of phage Xadh DNA were cloned in plasmid vector pGK12 and the effects on plasmid transduction by 4adh were determined. Transduction of the recombinant pGK12 plasmids was significantly improved. The enhanced frequency allowed us to define an expanded host range for 4adh and deliver plasmid DNA to other L. gasseri strains by transduction. MATERIALS AND METHODS Bacteria, phage, and plasmids. The bacteria and plasmids used in this study are listed in Tables 1 and 2, respectively. Lactobacillus and Escherichia coli strains were propagated at 37°C in MRS (Difco Laboratories, Detroit, Mich.) broth and LB (21) broth, respectively. Agar (BBL Microbiology Systems, Cockeysville, Md.) was used at 1.5% to make solid media. When appropriate, chloramphenicol and erythromycin were added at concentrations of 6 and 2 p.g/ml, respectively, for Lactobacillus strains and 20 and 200 jig/ml, respectively, for the E. coli strain. All stock cultures were maintained at -20°C in growth medium with 10% glycerol. Bacteriophage 4adh and transducing particles were induced

* Corresponding author. t Paper FS91-23 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624. t Present address: Laboratoire de Gdndtique Microbienne, Institut de Biotechnologie, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.

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The temperate bacteriophage +adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10-8 to 10-1O transductants per PFU. BglII-generated DNA fragments from phage +adh were cloned into the Bcll site of the transducible plasmid vector pGK12 (4.4 kb). Phage +adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 102_ to 105-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BgIII DNA fragments of +adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the +adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::+adh molecule. In addition to strain ADH, pTRK170 could be transduced via 4adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).

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RAYA AND KLAENHAMMER TABLE 1. Bacteria used in this study Bacterial strain

L. gasseri ADHb NCK101

L. gasseri F. Gasser 63 AM VPI 11092 VPI 11089 VPI 11759 VPI 12601 L. acidophilus VPI 6032

Adhering strain 4adh+ str-10 spc11 pTRK15 Xadh- pTRK15 NCK101/pGK12 NCK101/pTRK166 NCK101/pTRK167 NCK101/pTRK168 NCK101/pTRK169 NCK101/pTRK170 NCK101/pTRK172 NCK101/pTRK173 NCK101/pTRK174 NCK101/pTRK176 NCK101/pTRK177 NCK101/pTRK178

Neotype (ATCC 33323)

Neotype (ATCC 4356)

MSO1 VPI 11088 VPI 7635 VPI 11694

11, 20 33 33 This This This This This This This

Size of insert (kb)

This This This This

No. of Cmr transductantsa Per PFU Per mlb

7.5

8.7 x 10-9

2.8 x 103 6.3 x 103 3.0 x 104 2.4 x 105 7.0 x 102 1.2 x 106 8.8 x 102

3.8 x 10-6 4.8 x 10-6 2.7 x 10-5 2.0 x 10-4 6.8 x 10-7 1.1 x 10-3 1.6 x 10-6

pGK12 work work work work work work work work work work work

American Type Culture Collection 10 10 10 10

10

9 9 9

Source of plasmid

Plasmid transduced

33

2 10 10 10

L. helveticus 481 384 103 E. coli JM110

Origin or reference

33

pGK12

a4adh', 4adh lysogen; 4adh-, cured of the 4adh prophage; str-10, streptomycin resistance (1 mg/ml); spc-1I, spectinomycin resistance (300 ,ug/ml). b NCK, Culture Collection of the Department of Food Science, North Carolina State University, Raleigh. from lysogens with 0.1 ,ug of mitomycin per ml, and phage titers were determined with the prophage-cured strain NCK102 as described previously (33). DNA isolation and hybridization. Phage particles were purified by CsCl discontinuous and equilibrium density gradient centrifugations, and DNA was extracted as described previously (33). Plasmid DNA from Lactobacillus strains was isolated and purified on CsCl gradients as described by Luchansky et al. (19). Plasmid DNA from E. coli was isolated by the alkaline lysis technique and purified on CsCl gradients (21). DNA was digested with restriction enzymes as recommended by the suppliers (Bethesda Research Laboratories, Gaithersburg, Md., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Analytical or preparative gel electrophoresis in Tris-acetate buffer was performed as described by Maniatis et al. (21). DNA fragments were purified from agarose gels with a Prep-A-Gene kit (Bio-Rad Laboratories, Richmond, Calif.). Nucleic acid was transferred from agarose gels to nylon membranes (Magnagraph, 0.45-gLm pore size; Micron Separations Inc., Honeoye Falls, N.Y.) as described by Southern (38). Probes

Plasmids containing a

4adh-BglII

insert:

pTRK172 pTRK173 pTRK168 pTRK169 pTRK174 pTRK170 pTRK167 pTRK166 pTRK176 pTRK178 pTRK177

0.25 0.32

0.65 0.9 (0.65 + 0.25) 1.5 2.2 (1.4 + 0.8) 1.75 (1.5 + 0.25) 4.1 4.4 7.0 7.9

5.0 2.6 1.8 3.9

x x x x

105 6.1 x 10-4 104 2.9 x 10-5 105 1.5 x 10-4 105 4.5 x 10-4

"Average of four transduction experiments. "From mitomycin-induced phage Xadh lysates.

were labeled with digoxigenin-11-dUTP by use of a Genius kit (Boehringer). Hybridization reactions were performed in accordance with the supplier's specifications. Transduction assay. Transduction of pGK12 and pGK12 derivatives to recipient cells was carried out as described by Raya et al. (33) with the following modifications. Cells were propagated in 10 ml of MRS broth to an optical density at 590 nm of 0.5, after which 1.0 M CaC12 was added to a final concentration of 20 mM. A 0.2-ml portion of recipient cells was mixed with 0.2 ml of the phage suspension (ca. 0.6 x 109 to 2.2 x 109 PFU/ml), and the mixture was incubated at 37°C for 1 h to allow for the phenotypic expression of antibiotic resistance. Antibiotic-resistant transductants were selected on MRS agar supplemented with chloramphenicol after 48 h of aerobic growth at 37°C. Cloning and transformation experiments. Phage DNA fragments were ligated to plasmid pGK12 with T4 DNA ligase (Bethesda Research Laboratories) by standard recombinant DNA techniques (21). E. coli and Lactobacillus cells were transformed by electroporation with a Gene Pulser (BioRad) in accordance with the specifications of the manufacturer and the method of Luchansky et al. (19), respectively.

RESULTS Construction of pGK12::4+adh recombinant plasmids. Broad-host-range plasmid pGK12 is 4.4 kb long and carries genes conferring Cmr and Emr (12). This plasmid is transduced in an ADH isogenic background by 4adh particles at a frequency of 10-9 Cmr transductants per PFU (33). To evaluate the effect of DNA-DNA homology between plasmid and bacteriophage on ~adh-mediated plasmid transduction, we constructed pGK12::+adh recombinant plasmids. BglII DNA fragments from phage Padh were ligated in the unique BclI site of plasmid pGK12 to inactivate its Emr gene, and the ligation mixture was introduced into ADH cells by electroporation. Recombinant plasmids from Cmr Ems transformants were isolated and characterized by size, restriction mapping, and hybridization analyses. The phage Xadh BglII DNA fragments which were cloned in pGK12 are shown in Fig. 1. In recombinant plasmids pTRK167, pTRK169, and

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NCK102 NCK111 NCK370 NCK371 NCK372 NCK373 NCK374 NCK376 NCK377 NCK378 NCK442 NCK443 NCK444

characteristics"

TABLE 2. Effect of plasmid-phage DNA homology on the transduction of recombinant pGK12 plasmids

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189

cut pTRK170 (lane 14). Therefore, EcoRI should not cut a

concatemeric form of pTRK170. As lane 13 shows, tp-¢adh DNA digested with EcoRI showed a hybridizing band migrating at ca. 18 kb, a position between those of the uncut and the BglII-cut hybridizing bands of tp-4+adh DNA. These data suggest that plasmid pTRK170 was integrated in the (adh genome. To confirm this result, we digested tp-4adh DNA and pTRK170 with Sacl and StuI. These restriction enzymes cut the pGK12 sequences but not the BglII inserts of pTRK170. Therefore, plasmid-phage junction fragments should be detected in the tp-4adh DNA sample when probed with pGK12. Figure 3 shows that a junction fragment of approximately 16 or 27 kb was detected when tp-4adh DNA was digested with StuI (lane 1) or Sacl (lane 5), respectively. A second junction fragment was not visualized in either experiment. Lanes 5 and 6 of Fig. 3 also show a hybridizing band of 6.6 kb, corresponding to a monomer of pTRK170, in both the StuI and Sacl digests of tp-4adh DNA. In all experiments, the 18-kb hybridizing band of tp-+adh DNA was present at extremely low concentrations relative to Xadh. The 18-kb band was not visualized in the corresponding agarose gels stained with ethidium bromide. On the basis of these observations, we conclude that a multimer of plasmid pTRK170 (not greater than a trimer, because of the size of pTRK170 and because of the 18-kb EcoRI-cut hybridizing band from tp-4adh DNA) integrates into the Xadh genome and that this cointegrate molecule is packaged in phage particles. Analysis of pTRK170 in transductants. Plasmid DNA analysis of 13 transductants of NCK101 showed that pTRK170 was present as a monomer without apparent deletions or rearrangements due to the transduction event (data not shown). Similar results have been reported for the transduction of plasmid pGK12 (33). Transductants of the Xadh prophagecured derivative strain, NCK102, were further evaluated for their response to phage 4adh infection. Three derivatives of each NCK102 transductant carrying plasmid pGK12, pTRK170, pTRK167, or pTRK174 were found sensitive to (adh infection in MRS broth or a standard plaque assay, indicating that new lysogens capable of providing superinfection immunity had not been formed during transduction. These data suggest that the pTRK170 multimer present in pTRK170: :4adh recombinant molecules is resolved, after transduction, into a pTRK170 monomer in recipient cells. Transduction of plasmids into L. gasseri strains by 4adh. The high frequency with which pTRK170-containing transduction particles are formed (ca. 1.3 per 1,000 PFU) prompted us to investigate whether phage (adh can transduce plasmid DNA from ADH cells to other Lactobacillus strains. The host range for transduction by bacteriophage 4~adh was tested against the strains listed in Table 3. None of these strains supported lytic growth of (adh in a standard plaque assay. Phage 4adh did transduce pGK12 into ADH but not into any of the other strains examined. Plasmid pTRK170 was transduced at frequencies of 10' to 108 transductants per PFU into strains ADH, VPI 6033, VPI 11089, VPI 11759, VPI 12601, MSO1, and VPI 11088. In selected transductants, pTRK170 was detected as an intact 6.6-kb plasmid. Other L. acidophilus strains, including the neotype strain, ATCC 4356 (VPI 6032), and Lactobacillus helveticus strains did not show plasmid transduction by 4adh. These data indicate that the plasmid-transducing ability of Xadh is limited to strains within DNA homology groups Bl and B2 of Johnson et al. (10) or group Ila of Lauer et al. (14), classified now within the species L. gasseri (15). Transduction of pTRK170 to the neotype strain L. gasseri 63 AM (ATCC 33323) confirmed this observation.

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pTRK170, inserts were composed of two Xadh BglII DNA fragments. The 4.1-kb fragment inserted in plasmid pTRK166 remained after a deletion of the 9-kb BglII fragment (Fig. 1). We were unable to isolate pGK12 recombinant plasmids containing the 4.5- and 6.0-kb BglII fragments (shown in Fig. 1) in either E. coli or ADH. Transduction of pGK12 derivative plasmids by 4)adh. Phage 4adh lysates induced from ADH lysogens carrying pGK12 or a recombinant plasmid were used to transduce L. gasseri ADH to Cmr. These experiments (Table 2) revealed that the transduction frequencies of the recombinant plasmids were 102_ to 105-fold higher than that of pGK12. The highest transduction frequency was observed with plasmid pTRK170 (1.1 x 10'- transductants per PFU). In general, transduction frequencies correlated with the extent of homology between plasmid and phage DNAs (Table 1), with the exception of plasmids pTRK167 and pTRK174, which contained the 1.5-kb BglII 4adh fragment. These plasmids were transduced at relatively low frequencies (ca. 10-6 and 1i-' transductants per PFU), even though they shared extensive regions of homology with the 4adh genome, 1.75 and 1.5 kb, respectively. The reason for this effect is unknown. The 1.5-kb BglII fragment probably did not encode a "repressor" of phage DNA replication: similar titers (PFU per milliliter) of phage Xadh could be obtained from either Oadh lysogens or 4adh prophage-cured derivatives carrying these plasmids, relative to plasmid-free cells (data not shown). In the SPO2 (22), R4 (27), and lambda (37) transduction systems, recombinant plasmids containing cohesive end sequences (cos) of these phages are transduced more efficiently. In this study, plasmid pTRK177 (Fig. 1), which contains the cos sequences of 4adh within the 7.9-kb BglII insert, exhibited a 5 x 104-fold increase in transduction frequency relative to pGK12. The high efficiency of transduction observed with this plasmid relative to pGK12 could be attributed either to the size of the insert or to the presence of cos sequences or to both. Characterization of plasmid pTRK170 and pTRK170-containing phage 4adh particles. Plasmid pTRK170, which showed the greatest response to Padh-mediated plasmid transduction, was selected to further analyze the physical structure of a hybrid plasmid in transducing particles. DNADNA hybridization and restriction analyses indicated that the 0.8- and 1.4-kb 4Xadh BglII fragments present in pTRK170 were noncontiguous on the phage genome (Fig. 1). A restriction map of pTRK170 is shown in Fig. 2. To determine the physical state of pTRK170 in transducing particles, we digested plasmid DNA, phage DNA obtained from purified Xadh particles, and DNA extracted from purified transducing particles (tp-4adh DNA) with different restriction enzymes and analyzed them by DNADNA hybridization with digoxigenin-11-dUTP-labeled pGK12 (Fig. 3). The pGK12 probe hybridized with pTRK170 (lane 10) and tp-4+adh DNA (lanes 9 and 11) but not with Xadh (lane 8). The hybridizing band from uncut tp-Iadh DNA (lanes 9 and 11) migrated at the position of uncut 4Xadh (43 kb) (lane 8) but not at the position of uncut pTRK170 (6.6 kb) (lane 10). In contrast, when pTRK170 and tp-4adh DNA were digested with BglII (lanes 6 and 7, respectively), a common pGK12-hybridizing band of 6.6 kb was observed. These results indicate that pTRK170 sequences in tp-kadh DNA were present in a high-molecular-weight form, either as a concatemer or as part of a phage-plasmid recombinant molecule. To distinguish between these two possibilities, we treated pTRK170 and tp-4adh DNA with EcoRI, a restriction enzyme which cuts the Xadh genome in 12 sites but does not

TRANSDUCTION IN L. GASSERI

190

n°~ O°n ~ m

APPL. ENVIRON. MICROBIOL.

RAYA AND KLAENHAMMER

CZO-L

0

th = 8~~~~~~~~~~~~~~~~~~~~

| _ 1 m

k~~~~~~~~~~~~~~~~~~

l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~C

ol n

*

C

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2 =O~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~C

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co

VOL. 58, 1992

StUI

_

TRANSDUCTION IN L. GASSERI TABLE 3. Transduction of plasmids pGK12 and pTRK170 into lactobacilli

ClaI

L. gasseri ADH VPI 6033 (ATCC 19992) VPI 11092 VPI 11089 VPI 11759 VPI 12601 F. Gasser 63 AM

FIG. 2. Restriction map of pTRK170. The positions of the 1.4-kb (closed box) and 0.8-kb (open box) BglIl fragments of 4adh DNA cloned into pGK12 are indicated. The flanking BgII sites (denoted by arrowheads) were lost during ligation to the BclI site of pGK12.

Table 3 also shows that transduction frequencies similar to those for ADH were obtained with strain VPI 6033 (ATCC 19992). ATCC 19992 harbors a temperate bacteriophage which is inducible by mitomycin (3, 45). Therefore, to study the relatedness between ADH and ATCC 19992, we compared phage DNA isolated from ATCC 19992 with 4adh DNA. Restriction analysis revealed identical restriction patterns for both phages. Also, hybridization experiments re-

1

2 3 4

Kb

L. helveticus 481 103 384

pTRK170

NDb Bi (Ila)

5.0 x 10-9