Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

PLASMID

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Tn917 Transposition in Lactobacillus plantarum Using the Highly Temperature-Sensitive Plasmid pTV1 Ts as a Vector W. MARK COSBY,] LARS T. AXELSSON,* ANDWALI.ER J. DOBROGOSZ~-' tDepartment of Microbiology, North Carolina State University, Raleigh, North Carolina, 27695-7615; and *Department of Microbiology, Swedish University ofAgricultural Sciences,Box 7025, S-75007 Uppsala. Sweden Received July 10, 1989: revised December 10, I989 pTV ITS, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn9 17. was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40°C and subsequent plating on solid medium, two strains, L. plantarum NC4Tsl and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSCm’ isolates from both strains revealed Tn9 17 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLS%mS isolates from NC7Ts5 indicated four different k.eftiOII SiteS. 0 1989 Academic Press. Inc

Genetic analysis and strain improvement in the genus Lactobacillus and other lactic acid bacteria has been impeded by the lack of a reliable system of genetic transfer and vector DNA. Much effort has been directed to developing a protoplast transformation method similar to that developed by Chang and Cohen (1979) for Bacillus. Several laboratories have reported success but transformation frequencies are low and often strain specific (Lin and Savage, 1986; Morelli et al., 1987; ShimizuKadota and Kudo, 1984; Cosby et al., 1988; Posno et al., 1988). Recent reports of transformation by electroporation have shown transformation frequencies of 1 X IO3 to 1 X 10’ per microgram of DNA for several species of Lactobacillus using different plasmid DNA, including the Escherichia coli-Steptococcus shuttle vectors pSA3 and pGKl2 (Luchansky et al., 1988; Chassy and Flickinger, 1987). These developments now make the study of Lactobacillus genetics by recombinant DNA methodologies possible. This will also

’ Present address: Department of Chemistry, University of Texas, Austin, TX 787 12. 2 To whom correspondence should be addressed.

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Copynghf 0 1989 by Academic Press, Inc. All rights of nproduaion in any form reserved.

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make the delivery of transposons and subsequent genetic mapping by transpositional mutagenesis in Lactobacillus feasible. Several transposons from gram-positive organisms have been used in this way (Luchansky and Pattee, 1984; Hill et al., 1987; Courvalin and Carlier, 1987; Rubens and Heggen, 1988) but perhaps the most extensive use of transpositional mutagenesis for genetic analysis in a gram-positive organism has been that of the transposon Tn9 17 in Bacillus. Youngman et al. (1983) constructed pTV1, a heat-sensitive plasmid, which carries Tn9 17 and in addition encodes for chloramphenicol resistance. This plasmid and its derivatives have been used to study Bacillus subtilis sporulation (Sandman et al., 1987) and germination (Sammons et al., 1987), auxotrophy (Youngman et al., 1983) and the barnase gene in Bacillus amyloliquefaciens (Hartley and Paddon, 1986). pTV 1 has been introduced into Lactobacillus (Luchansky et ul., 1988) and evidence of Tn9 17 transposition has been shown (Aukrust and Nes, 1988). However, none of these reports describe any attempt to enrich for transpositional mutants by using high temperature and subsequent scoring for loss of chloram-

Tn917 TRANSPOSITION IN L. plantarum

phenicol resistance, indicative of loss of the vector replicon. In this regard, pTV 1 is limited for use in Lactobacillus, because incubation temperatures of 45°C or greater are required to inhibit replication of the plasmid (Youngman et al., 1983). Lactobacillus plantarum is mesophilic and most strains cannot grow above 43°C. In this work we report successful transformation of two strains of L. plantarum with the highly temperature-sensitive plasmid pTV 1Ts, a derivative of pTV 1 developed by P. Youngman, using the pEl94Ts replicon originally isolated in the laboratory of R. Novick (Gryczan et al., 1982; Youngman, 1987). Using this plasmid we have been able to generate random Tn9 17 insertions into L. plantarum resident DNA and cause the subsequent loss of the plasmid by growth at 40°C. MATERIAL

AND METHODS

Bacteria. L. plantarum strains NC4 (ATCC 80 14) and NC7 were routinely cultured in MRS (Difco Laboratories, Detroit, MI) or LCM (Shrago et al., 1986) containing 1%glucose (LCMG) at 30°C and stored at 4°C. L. plantarum pTV 1Ts transformant strains NC4Ts 1 and NC7Ts5 were grown in the same medium containing 5 pg/ml erythromycin, 10 pg/ml chloramphenicol, and 20 pg/ml lincomycin (EmCmLm3; Sigma Chemical Co., St. Louis, MO). Transpositional mutants were grown in the samemedium asstrains NC4Ts 1 and NC7Ts5 except chloramphenicol was omitted (EmLm). B. subtilis PY3 13, which contains plasmid pTVlTs, was grown in L broth (Maniatas et al., 1982) containing 5 pg/ ml erythromycin, 5 pg/ml chloramphenicol, and 25 rg/ml lincomycin at 30°C and stored at 4°C. For cultivation of bacteria on solid medium, 1.5% agar (Difco Laboratories) was added. DNA isolation. Isolation of pTVlTs was conducted according to the procedure of Gry3Abbreviations used: Em, erythromycin; Cm, chloramphenicol; Lm, lincomycin; BSG, buffered saline with gelatin; SDS, sodium dodecyl sulfate; EP, electroporation; MLS, macrolide-lincosamide-streptogramin B.

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czan et al. (1978) and purified by CsCl-ethidium bromide isopycnic centrifugation (Maniatis et al., 1982). Small-scale isolation of L. plantarum plasmid DNA was carried out according to a modification of the method of Chassyet al. (1978). Strains were grown overnight in LCMG with the appropriate antibiotics and 3 ml of this culture was added to 12 ml of fresh medium. This culture was grown to an absorbance at 600 nm (4600)of 1.0-1.5. The culture was then centrifuged, washed in buffered saline with gelatin (BSG; Gerhardt, 198l), and resuspended in 1.5 ml of 0.3 M sucrosein 10 mM Tris-HCl, pH 8.0, containing 10 mg/ml lysozyme (Sigma) and incubated at 37°C for 30 min. The suspension was centrifuged at 5000g at 4°C and the cell pellet was carefully resuspendedin 675 ~1 of 5X TE (TE buffer is 1 mM EDTA in 10 mM Tris-HCl, pH 8.0). Seventy-five microliters of 10% SDS was added and the suspension was mixed by inversion and incubated at 65“C for 10 min. After incubation, the suspension was vortexed for 1 min, 45 ~1 of 2 M Tris-HCl, pH 7.0, was then added, and the suspension was mixed by inversion for 5 min. One hundred five microliters of 5 M NaCl was added and the suspensionwas again mixed by inversion for 1 min. The lysate was extracted twice with 3% NaCl-saturated phenol and once with chloroform-isoamyl alcohol (24: 1). The DNA was ethanol precipitated, dried, resuspended in 400 ~1TE with 50 pg/ml RNase A (Sigma), and incubated at 37°C for 30 min. Phenol and chloroform-isoamyl alcohol extractions were repeatedand the DNA was precipitated and collected as noted above and resuspendedin 30 ~1 TE buffer. Transformation. Transformation by electroporation of L. plantarum strains using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA) was conducted essentially according to the procedure of Luchansky et al. (1988). The electroporation (EP) buffer used consisted of 680 mM sucrose and 2.5 mM MgC12in 17.5 mM sodium phosphate buffer, pH 7.4. L. plantarum cultures were grown in MRS to an A600of 1.O- 1.5, harvested by cen-

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trifugation, washed twice in cold EP buffer, and resuspended in EP buffer to a final concentration of approximately 1 X 10” colony forming units per milliliter (CFU/ml). A 0.8ml portion of this cell suspension was mixed with 1 pg of pTV 1Ts (in TE buffer) and placed in an electroporation cuvette with a 0.4-cm interelectrode gap and placed on ice for approximately 10 min. The cuvette was then placed in the Gene Pulser (set at 2500 V and 25 pF) and a single high voltage electric pulse was applied. The cuvette was then incubated on ice for an additional 30 min. For transformant selection, the electroporated cell-DNA suspensions were serially diluted in EP buffer and 100 pug/mlwas spread on MRS solid medium containing 7.5 fig/ml Cm or 5 pg/ml Em and incubated at 30°C. Controls were treated similarly except no DNA was added prior to electroporation. After 3-4 days, colonies were picked from the selection medium and streaked onto MRS solid medium containing EmCmLm and incubated at 30°C. This step was repeated for strain purification. In order to store the strains for further study, a single colony was picked, suspendedin 100 ~1of MRS broth, and spread onto MRS solid medium containing EmCmLm. These plates were incubated at 30°C until a light haze of growth was evident. The cells were harvested by successivelywashing the plate surface with 1.O ml MRS broth containing 10% glycerol (three times) and the cell suspensions were stored at -20°C. The presenceof pTVlTs was determined by agarose gel electrophoresis of small-scale plasmid DNA isolations. Transposition. Tn9 17 insertions were obtained by a modification of the liquid batch culture method by Youngman (1987). L. plantarum strains NC4Tsl and NCTs5 were grown in MRS broth containing EmCmLm at 30°C to mid-log phase (A600of 0.6-0.7). These cultures were used to inoculate (l%, v/v) MRS broth containing EmLm and MRS broth containing EmCmLm and these new cultures were incubated at 40°C. When the cultures containing EmLm reached mid-log

phase, they were used to inoculate (2%, v/v) a second set of MRS plus EmLm and MRS plus EmCmLm broths which were also incubated at 40°C. When the second EmLm broth cultures reached stationary phase(A600= 2.0) they were serially diluted and plated onto MRS, MRS plus EmLm, and MRS plus EmCmLm solid medium and incubated at 30°C. After 3-4 days incubation, colonies from the MRS plus EmLm plates were picked and suspendedin 250 ~1BSG and ~-PI aliquots were placed on MRS, MRS plus EmLm, and MRS plus EmCmLm solid medium and incubated at 30°C to test for the loss of chloramphenicol resistance. Aliquots of L. plantarum strains NC4Ts1, NC7Ts5, NC4, and NC7 were also placed on the solid medium to serve as positive and negative controls. DNA restriction analysis and Southern hybridization. Enzymes for DNA restriction

analysis were purchased from BoehringerMannheim Biochemicals (Indianapolis, IN) and used according to the manufacturer’s recommendations. DNA wasanalyzed by agarose gel electrophoresis (0.7% agrosein Tris borate EDTA or Tris phosphate EDTA buffers; Maniatis et al., 1982) and transferred to and hybridized on GeneScreen Plus membrane (New England Nuclear, Boston, MA) according to the manufacturer’s specifications. The internal 4.2-kilobase pair (kb) XbaI-Hpal fragment of Tn9 17 (Youngman, 1987) was isolated from pTV 1Ts by agarosegel electroelution, labeled with 32Pwith a random primer translation kit (Amersham Corp., Arlington Heights, IL) as directed by the manufacturer, and used as a probe for hybridization. RESULTS

Transformation. Transformation frequencies were low and varied from trial to trial regardlessof whether chloramphenicol or erythromycin was used for the original selection. For strain NC7, we recovered approximately 1 X 1O2chloramphenicol-resistant colonies per microgram of pTV 1Ts in one trial whereasin a subsequenttrial we recovered only a few iso-

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Tn917 TRANSPOSITION IN L. phntarum

lates per microgram of pTV ITS. Only 2-5 chloramphenicol or erythromycin-resistant colonies per microgram of pTV 1Ts were recovered per trial for strain NC4. Six resistant colonies from pTV 1Ts transformation for each strain were picked and found to be resistant to erythromycin, lincomycin, and chloramphenicol. Two isolates, designatedL. plantarum NC4Tsl and L. plantarum NC7Ts5, were selected for further study and after plasmid DNA isolation and agarose gel electrophoresis were found to contain plasmid pTVlTs (Figs. 1 and 4). Transposition. The frequency of Tn9 17 transposition in L. plantarum strains NC4Tsl and NC7Ts5 was first tested according to the method of Youngman (1987). Cultures were grown in MRS broth containing EmCmLm at 30°C to mid-log phase, serially diluted, and plated on solid MRS plus EmLm medium and incubated at 30 and 40°C. The titer of CFU at these two temperatures was then compared. Unlike B. subtilis where the ratio of CFU at 4O”C/CFU at 30°C (the transposition frequency) ranges from 5 X 10e5 to 5 X 1O-4 (Youngman, 1987), our Lactobacilfus strains exhibited no difference in CFU at 40°C versus 30°C. In the liquid batch culture method, both strains would grow to stationary phasein MRS broth containing EmCmLm at 40°C when inoculated with a mid-log phase culture incubated at 30°C in the samemedium. However, after two passagesat 40°C in MRS broth containing EmLm, both strains lost chloramphenicol resistance but retained MLS resistance by a factor of 100 or more as determined by the ratio of CFU/ml on MRS plus EmLm/ CFU on MRS plus EmCmLm (data not shown). For each strain, 80 colonies were picked from the MRS plates containing EmLm and examined for their antibiotic resistances.One hundred percent of the colonies picked for strain NC7Ts5 and 97% of the colonies picked for NC4Tsl were MLS resistant and chloramphenicol sensitive. Five MLSCm” isolates,designatedL. plantarum NC4Tp 1 and L. plantarum NC7Tp 1-Tp4, were picked and analyzed for their DNA content.

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FIG. 1. Southern blot hybridization analysis of EcoRI digest of plasmid DNA from L. pluntarum NC7Ts5 and NC7Tp4 using “P-labeled XbaI-HpuI internal fragment of Tn917 as the probe. (A) Agarose gel electrophoresis: lanes 1 and 8, X Hind111digest; lane 2, pTV1Ts; lane 3, recipient NC7; lane 4, transformant NC7Ts5; lane 5, NC7Tp4; lane 6, NC7 chromosomal DNA; lane 7, NC7Tp4 chromosomal DNA. (B) Corresponding autoradiogram after DNA transfer to membrane and hybridization to the Tn9 17 fragment probe.

Evidence for Tn9 17 transposition in strain NC7 is presented in Figs. 1, 2, and 3. Figure 1 shows the presence of the transposon in NC7s5 and NC7Tp4. An EcoRI digest was used since both the resident plasmid and pTV 1Ts contain single sites for this enzyme. The only signal detectedin lane 7 representing chromosomal DNA from NC7Tp4 was very weak and corresponded in size to the signal in lane 5. As expected, the signal in lane 4 represents the vector pTV ITS. This was controlled by an additional restriction digest with NcoI which has no site in the resident plasmid designated~256 but hasthree sitesin pTVlTs, one which is present in Tn9 17 (Youngman, 1987) and is shown in Fig. 2B. Lanes 6 and 7 show the EcoRI-NcoI restriction patterns of pTVlTs and ~256, respectively. Lane 8 shows the expected EcoRI-NcoI pattern of pTV 1Ts and ~256 coexisting in strain NC7Ts5. Lane 9 showsthat the plasmid from strain NC7Tp4 differs from both pTV 1Ts and P256. The major band in lane 9 is actually two bands since NcoI cuts the EcoRI linearized plasmid within Tn917 so that two equal sized fragments of

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I.0 .0.6 FIG. 2. (A) BclI restriction analysis of ~256 from L. pluntarurn NC7 and p256::TnY 17 from strains NC7Tpl through NC7Tp4. Lane I. NC7Tpl; lane 2. NC7Tp2; lane 3, NC7Tp3; lane 4, NC7Tp4; lane 5, NC7; lane 6, pTVITs: lane 7, EcoRI-Hind111 digest of X DNA; band sizes identified in kilobases. (B) EC&NcoI restriction analysis of ~256 and pTVlTs isolated from NC7Ts5. p256::TnY 17 from NC7Tp4. ~256 from NC7. and pTV1Ts. Lane 1, EcoRI-Hind111 digest of X DNA; lane 2, pTVlTs, EcoRI: lane 3, ~256, EcoRI; lane 4, plasmid DNA from NC7Ts5, EcoRI: lane 5, p256::Tn9 17, EcoRI: lane 6. pTV I Ts EcoRINcoI; lane 7, ~256, EcoRI-NcoI; lane 8, plasmid DNA from NC7Ts5, EcoRI-NcoI; lane 9, p256::Tn917, EcoRI-NcoI.

approximately 6.4 kb result. The faint upper band is partially cut, linearized plasmid. Additional digests on this plasmid using the enzymes XbaI, PvuII, NW111 (not shown), and BcII (Fig. 2A) show that the plasmid is ~256 with an insert corresponding to Tn9 17 in both size and restriction sites. The apparent similarity in size of pTV1 Ts and the plasmid in NC7Tp4 was expected assuming a Tn9 17 insertion in ~256. A Be/I restriction analysis of the plasmids from four MLS%m” strains (labeled NC7Tpl-Tp4) isolated after the 40°C incubation is shown in Fig. 2A. A map of p256 and the insertion points of Tn917 deduced from these analyses is shown in Fig. 3. Figure 4 shows a Southern blot analysis of plasmids from strain NC4, the pTV 1Ts transformant, NC4Ts7, and MLSCM” isolate, NC4Tpl. Similar to strain NC7, the transposon is present in a new plasmid, approximately 14 kb in size, and pTVITs is absent in NC4Tp 1. The resident 9.4 kb plasmid is missing in NC4Tp 1, indicating transposition to this plasmid. However, this plasmid is also missing in the transformant NC4Tsl and a signal is

detected at 14 kb. This would be the result assuming that transposition already had occurred. A signal is also detected for a plasmid of about 8.0 kb in this strain. This plasmid is apparently lost in NC4Tpl.

Pvu

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FIG. 3. Partial restriction map ofp256 showing the points of insertion of Tn9 17 in NC7Tpl and NC7Tp4. Arrows indicate the orientation of Tn9 17 and are pointing toward the urn-proximal end of the transposon. Sizes in kilobases.

Tn917 TRANSPOSITION IN L. pluntnrum 6

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FIG.4. Southern hybridization analysisof plasmids from L. pluntarum strains NC4, NC4Ts1, and NC4Tpl using the label as described in Fig. 1. (A) Agarose gel electrophoresis; lanes 1 and 6, E. coli V5 17 reference plasmids; lane 2, pTV ITS; lane 3, NC4; lane 4, NC4Tsl; lane 5, NC4Tp I. (B) Corresponding autoradiogram after DNA transfer to membrane and hybridization to the Tn917 probe. Sizes in kilobases of pTV ITS (12.4) and the four resident plasmids of NC4 are indicated.

DISCUSSION

The introduction of Tn9 17 into L. plantarum DNA and subsequent loss of the vector pTV 1Ts will greatly facilitate genetic analysis in this group of microorganisms in much the same way it has been used in B. subtilis. It is not clear why we could not determine a transposition frequency using the method of Youngman (1987) but the fact that two passagesat 40°C in MRS plus EmLm were required before chloramphenicol resistancewas lost may point to a higher copy number of pTV ITS in L. plantarum in B. subtilis. It could also be that replication of pTV 1Ts is not entirely inhibited, but rather that cells containing the plasmid grow slower and will finally be diluted out of the system. The rep and cop genes (or their products), required for stable replication and copy control of pTV 1Ts (Gryczan et al., 1982) may operate differently in these two genera. The DNA analysesof strains NC7, NC7Ts5, and NC7Tpl-Tp4 show evidence of Tn9 17 transposition to the resident plasmid, ~256. The similarity in size between the vector

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pTV ITS and the hybrid p256:Tn9 17 made the Southern blot analysis somewhat inconclusive. However, the restriction digests clearly revealed the insert as a 5.3-kb moiety with restriction sites matching the pattern of Tn9 17 (Shaw and Clewell, 1985). There is still a possibility that very small parts of pTV ITS just outside the transposon may have been cointegrated together with Tn9 17. This seemsvery unlikely, due to (i) the restriction data we have and (ii) the broad range of speciesTn9 17 has been shown to function in. Even if this occurred, the purpose of using Tn9 17, namely gene inactivation, can still be achieved. We overexposed an autoradiogram of the hybridization presented in Fig. 1 in order to determine if Tn9 17 also inserted into chromosomal DNA. The only signal detected corresponded in size to the signal in lane 5 and is most likely contaminating p256:Tn9 17. The BclI restriction analysis of p256:Tn9 17 reflects a certain degree of randomness as to the site of Tn9 17 insertion. Although detailed restriction maps were constructed only for the Tp I and Tp4 plasmids, it is evident that Tn9 17 has inserted to yet different points in Tp2 and Tp3. Youngman points out that one disadvantage with the liquid bath method is that the isolatesrecoveredmay not be derived from different transposition events (Youngman, 1987). The two transfers used in our method could enhance this problem. This seems not to be the case,since the isolates analyzed here clearly come from different transposition events. It may be necessaryto analyze more isolates to address this question more stringently. Due to the complexity with multiple plasmids in strain NC4, a detailed analysis was not performed. The Southern blot in Fig. 4 does show a pattern in this strain similar to that for strain NC7. The loss of Cm resistance, loss of vector (at least as an intact plasmid), absenceof a resident 9.4-kb plasmid, and appearance of a new plasmid, containing the transposon and approximately 5 kb larger than the missing resident plasmid, strongly suggest transposition of Tn9 17 to the 9.4-kb plasmid.

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The presence of multiple signals in the transformant NC4Tsl may reflect some capacity for plasmid rearrangements in addition to the transposition, which apparently already had occurred at this stage. This is very similar to the observation by Aukrust and Nes (1988) that transposition could be detected in 2 of 25 randomly selectedisolates recovered from the initial transformation with pTV 1. Our results also show that it is possible to select against the vector replicon by using pTVlTs and elevated temperature. Our preliminary efforts with the Tn9 17 system showed that this could not be done by using the original vector pTV 1. In summary, we have presented data establishing the usefulness of plasmid pTVlTs as a vector for Tn9 17 transpositional mutagenesis in L. plantarum and possibly other lactic acid bacteria. These data and the report by Aukrust and Nes (1988) show that Tn9 17 will insert into different resident plasmids in a particular strain and even into different sites on a single plasmid. It seemsthat Tn917 has a preference for transposition into plasmids, which is consistent with what others have found (P. J. Youngman, personal communication). We are, however, hopeful that random insertions will also occur in chromosomal DNA. We have been successfulin transforming L. plantarum NC8, a plasmidless strain, with pTVlTs and are currently working on the isolation and identification of Tn9 17 insertions into chromosomal DNA and their resulting mutations. ACKNOWLEDGMENTS We thank P. Youngman for providing plasmid pTV1 Ts. This research was supported by the U.S. Department of Agriculture, Competitive Research Grants Office, Biotechnology, Molecular Biology and Growth and Development Program Agreement 85-CRCR-I- 1728.Paper No. 1166I of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. REFERENCES AUKRUST,T., AND NES, I. F. (1988). Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation. FEMS Microbial. Lett. 52, 127-l 32.

CHANG, S., AND COHEN,S. N. (1979). High frequency transformation of Bacillus subtilus protoplasts by plasmid DNA. Mol. Gen. Gene?.168, 11l-l 15. CHASSY,B. M., GIBSON, E. M., AND GIUFFRIDA, A. (1978). Evidence for plasmid-associatedlactose metabolism in Lactobacillus caseisubspcasei.Curr. Microbial. 1,141-144. CHASSY,B. M., AND GIUFFRIDA,A. (1980). Method for the lysis of gram positive, asporogenousbacteria with lysozyme. J. Bacterial. 139, 153-l 58. CHASSY,B. M., AND FLICIUNGER,J. (1987). Transformation of Lactobaciilus casei by electropomtion. FEMS Microbial. Lett. 44, 173- 117. COSBY,W. M., CASAS,1. A., AND DOBROGOSZ,W. J. (1988). Formation, regeneration, and transfection of Lactobacillus plantarum protoplasts. Appl, Environ. Microbial. 54, 2599-2602. COURVALIN,P., AND CARLIER, C. (1987). Tn1545: A conjungative shuttle transposon. Mol. Gen. Genet. 206, 259-264.

GERHARDT,P. (Ed.) (1981). “Manual of Methods for General Bacteriology,” 1sted, pp. 504-507. Amer. Sot. Microbiology, Washington, DC. GRYCZAN,T. J., CONTENTE,S., AND DUBNAU,D. (1978). Characterization of Staphylococcusaureus plasmids introduced by transformation into Bacillus subtilus. J. Bacterial. 134, 3 18-329. GRYCZAN,T. J., HAHN, J., CONTENTE,S., AND DUBNAU, D. (1982). Replication and incompatability properties of plasmid pE 194 in Bacillus subtilis. J. Bacteriol. 152, 122-735.

HARTLEY,R. W., AND PADDON,C. J. ( 1986).Use of plasmid pTV1 in transpositional mutagenesis and gene cloning in Bacillus amyloliquefaciens. Plasmid 16,4551. HILL, C., DALY, C., AND FITZGERALD,G. F. (1987). Development of high-frequency delivery system for transposon Tn9 19 in lactic streptococci: Random insertion in Streptococcus lactis subsp diacetylactis 18-16.Appl. Environ. Microbial. 53, 74-78. LIN, J. H.-C., AND SAVAGE,D. C. (1986). Genetic transformation of rifampicin resistancein Lactobaciltus acidophilus. J. Gen. Microbial. 132, 2 107-2 1I 1. LUCHANSKY,J. B., MAUIANA, P. M., AND KLAENHAMMER, T. R. (1988). Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus. Bacillus, Staphylococcus, Enterococcus, and Propionibacterium. Mol. Microbial. 2, 637-646. LUCHANSKY,J. B., AND PATTEE,P. A. (1984). Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus. J. Bacteriol. 159, 894-899. MANIATIS,T., FRITSCH,E. F., AND SAMBROOK,J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Tn917 TRANSPOSITION IN L. plantarzan MORELLI,L., COCCONCELLI, P. S., BOTTAZZI,B., DAMIANI, G., FERRETU,L., AND SGARAMELLA,V. (1987). Lactobacillus protoplast transformation. Plasmid 17, 73-x

POSNO,M., LEER, R. J., VAN RUN, J. M. M., LOKMAN, B. C., AND POUWELS,P. H. (1988). Transformation of Lactobacillus plantarum by plasmid DNA. In “Genetics and Biotechnology of Bacilli” (A. T. Ganesan and J. A. Hoch, Eds.),Vol. 2, pp. 397-40 1. Academic Press,New York. RUBENS,C. E., AND HECCEN,L. M. (1988). Tn9 16DE: A Tn9 16transposonderivative expressingerythromycin resistance.Plasmid 20, 131- 142. SAMMONS,R. L., SLYNN,G. M., ANDSMITH,D. A. (1987). Genetic and molecular studies on gerM, a new developmental locus of Bacillus subtilis. J. Gen. Microbial. 133,3299-3312.

SANDMAN,K., LOSICK,R., AND YOUNGMAN,P. (1987). Genetic analysis of Bacillus subtilus spo mutations generated by Tn9 17-mediatedinsertional mutagenesis.Genetics 117, 603-617.

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SHAW,J. H., AND CLEWELL,D. B. (1985). Complete nucleotide sequence of macroiide-lincosamide-steptogramin B-resistance transposon Tn9 17 in Streptococcus faecalis. J. Bacterial. 164, 782-796. SHIMIZU-KADOTA,M., AND KUDO, S. (1984). Liposomemediated transfection of Lactobacillus casei spheroplasts. Agric. Biol. Chem. 48, 1105-l 107. SHRAGO,A., CHASSY,B. M., AND DOBROGOSZ,W. J. (1986). Conjugal plasmid transfer (pAMPI) in Lactobacillus plantarum. Appl. Environ. Microbial. 52, 574 576.

YOUNGMAN,P. J. (1987). Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other gram positive bacteria. In “Plasmids: A Practical Approach” (K. G. Hardy, Ed.), pp. 79-103. IRL Press, Washington, DC. YOUNGMAN,P. J., PERKINS,J. B., ANDLOSICK,R. (1983). Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcusfaecalis transposon Tn917. Proc. Natl. Acad. Sci. USA 80, 2305-2309. Communicated by Gary Dunny