Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci

Abstract

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...

Relationship between Lactococcus lactis subsp. lactis 952, an industrial lactococcal starter culture and strains 712, ML3 and C2

“…Hybridization experiments using the well-characterized IS Aements, ISSl (Polzin and Shimizu-Kadota 1987), IS904 Dodd et al 1990) and IS981 (Polzin and McKay 1991) -evealed almost identical patterns of homology between Mind111 digests of total DNA from 952, 712, and ML3 Fig. 4).…”

“…T o determine if the co-integration event observed in this study was IS-mediated, the well-characterized elements ISS 1 (Polzin and Shimizu-Kadota 1987), IS904 (Dodd ct al. 1990) and IS981 (Polzin and McKay 1991) were used to probe two pGU301-containing co-integrate plasmids, pCI014 and pCI017. Surprisingly, no homology was detected, although several copies of each of these IS elements are known to be located on both plasmid and chromosomal DNA from 952 (results not shown).…”

Characterization of an insertion sequence-like element identified in plasmid pCIT264 from Lactococcus lactis subsp. lactis biovar diacetylactis

“…These kinds of genetic elements are widely distributed in both the chromosome and the plasmid complement of lactic acid bacteria. Four families of IS elements in lactococci have been defined [3] and their copy numbers, host ranges and positions have been shown to vary widely among strains [I]. The existence of multiple copies of IS elements within the genomes of lactic acid bacteria is particularly interesting and accounts for some specific DNA rearrangements.…”

Chromosome mapping in lactic acid bacteria

“…They may also have a positive evolutionary role in conferring an increase in relative fitness in the environment in which the bacteria are maintained [95]. Two of the three insertion sequences known for L. laetis [96][97][98] have been mapped: ISS1RS [99], which is closely related to ISS1 [96], and IS1076 [100], which is related to IS904 [98]. ISSIRS is absent from the chromosome of IL1403 but one copy was found in strain MG1363.…”