In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISSIS) which was involved in and duplicated during formation of pPW2. ISSIS was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) Fig. 2A) contain IS elements which mediate cointegration of pSK08 and pRS01, as depicted in Fig. 1. Either putative IS element indicated in this figure should be identifiable as a pSK08 sequence that is duplicated on the cointegrate plasmid with a copy extending from both junction sites in direct-repeat orientation.As shown in Fig. 2A ML3, a lactose-fermenting (Lac') strain (15), contains a 55-kb lactose plasmid (pSK08), a 48.4-kb conjugal plasmid (pRS01), and three cryptic plasmids of 8.5, 3.0, and 1.5 kb (1,15). MMS362 (Lac-) is a derivative of ML3 cured of pSK08 (2). LM2302 (Lac-) is a plasmid-free, prophage-free, streptomycin-and erythromycin-resistant derivative of S. lactis C2 (7). PW2 is a transconjugant strain derived by conjugation of ML3 (donor) and LM2301, another Lac-plasmid-free derivative of S. lactis C2 (7,35), carrying a 104-kb cointegrate plasmid, pPW2. Cultures were grown in M17-lactose, M17-glucose (34), or LB broth (17) containing the appropriate antibiotic for S. lactis Lack, S. lactis Lac-, and Escherichia coli strains, respectively. The antibiotic concentrations used were 15 ,g/ml for erythromycin and 50 pug/ml for ampicillin. S. lactis cultures were grown at 32°C. E. coli cultures were grown at 37°C with shaking.

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The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. Plasmid replication and stability have been extensively studied for several plasmids found in gram-negative species. These studies have led to identification of several plasmidencoded elements which mediate plasmid replication, control plasmid copy number, and stabilize plasmid inheritance (18,24,26,30 the reasons for the differing abilities of L. lactis and L. cremoris strains to maintain pSK11L, we attempted to identify the pSK11L replication origin and regions involved in plasmid maintenance since these regions would be most likely to interact with host plasmid replication and maintenance functions.We report here that a 14.8-kbp PvuII fragment of pSK11L confers the replication ability and the temperature-sensitive pSK11L maintenance phenotype of L. lactis LM0230. We also report identification of several regions on this fragment which appear to affect pSK11L maintenance. Several of these regions affect pSK11L maintenance differently in L. lactis LM0230 and L. cremoris EB5. MATERIALS AND METHODSBacterial strains and plasmids. The L. lactis strains used in this study included LM0230, a plasmid-free, prophage-free derivative of C2 (9), and JF3216, an LM0230 transformant containing pSK11L, a lactose plasmid from L. cremoris SK11 (10). The L. cremoris strain used was EB5, a plasmidfree derivative of KR1 (3). Strains LM0230 and EB5 were grown at 32°C in M17 broth (33) supplemented with 0.5% glucose (M17-G). Transformants derived from these strains were grown at 25°C in M17-G containing erythromycin (Em) (10 jig/ml). Strain JF3216 was grown at 25°C in M17 broth supplemented with 0.5% lactose. The Escherichia coli plasmid used to clone the replication region of pSK11L was pVA891, a derivative of pACYC184 (4) containing the pAM,1 Emr-encoding gene (21). pVA891 and its derivatives were maintained in E. coli XL1-Blue

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, ISSJ, IS150, IS600, IS629, IS861, IS904, and ISLI. * Corresponding author. t Published as paper no. 18,168 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 18-62. bacteriophage infection mechanism cloned into pGB301 (14, 18), and MN14(pMN14), the pGBK17-derivative containing the DNA insertion which inactivated the abi gene(s) (18). The L. lactis strains screened for the presence of IS981 were obtained from the laboratory stock culture collection. Strains GBK17 and MN14 were grown in M17 broth (36) supplemented with 0.5% glucose and erythromycin (5 ,ug/ ml). The L. lactis strains screened for the presence of IS981 were grown in M17 broth supplemented with 0.5% lactose, except for strains LM0230, MMS368, and MG1363 which were grown in M17 broth supplemented with 0.5% glucose. All cultures were grown at 32°C. pGBK17 and pMN14 restriction fragments were subcloned into the vectors pUC118 and pUC119 (19) or pBluescript SK(+) and pBluescript SK(-) (Stratagene, La Jolla, Calif.). Transformation was into Escherichia coli XL-1 Blue (Stratagene). XL-1 Blue and XL-1 Blue transformants were grown in LB broth (15) and LB broth supplemented with ampicillin (50 ,ug/ml), respectively, at 37°C with shaking. Plasmid isolation, restriction enzyme digestion, agarose gel electrophoresis, and cloning. L. lactis plasmid DNA was isolated by the method of Anderson and McKay (1) and purified by CsCl-ethidium bromide (EtBr) density gradient centrifugation. For subcloning and restriction mapping, E. coli plasmid DNA was prepared either by the ethanol-phenol lysis procedure (17), followed by preparation of cleared lysates (15) and CsCl-EtBr density gradient centrifugation, or by boiling lysis (12). E. coli plasmid DNA for sequencing was prepared by boiling lysis or by a rapid, small-scale lysis procedure (24) followed by CsCl-EtBr density gradient centrifugation. Restriction enzymes were purchased from Life Technologies, Inc. (Grand Island, N.Y.), and digestions were performed as recommended by the...