Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3

Abstract

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.

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris

“…Such a structure suggests that the lactose operon was originally part of a transposable element whose ends were ISSJ elements that transposed to a smaller plasmid. A similar structure of two ISSJ elements surrounding a lactose operon has been reported on the lactose plasmid of L. lactis ML3 (29). Our results support the idea that ISSJ played a significant role in the acquisition by lactococci of genes necessary for maximum growth and acid production in milk.…”

“…Southern transfers were performed as described previously (29), by using Nytran membranes (Schleicher & Schuell, Inc., Keene, N.H.) prepared as suggested by the manufacturer. Probe labeling, hybridization, and detection were conducted by using the Genius kit (Boehringer Mannheim Biochemicals) as recommended by the manufacturer.…”

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030

“…The IS946 insertions from pTRK24, pTRK25, and pTRK27 (Fig. 5) were mapped within the pC194 (14) and pE194 (13) 18-bp terminal sequence (5'-GGTTCTGTTGCAAAGTTT-3') identical to the terminal inverted repeat of lactococcal insertion sequence ISSI (31). The additional HpaII (GGCC) restriction site in pTRK25 resulted from an insertion adjacent to a CC dinucleotide within the ermC gene.…”

Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. Iactis M189 which is transcribed from an extended -10 promoter.

“…From From o cu ac is 71 the sequence data obtained, it is likely that the plasmid pND301 carries the insertion elements 1S981 and ISS1T. In L. lactis, various insertion sequences are widely distributed among plasmids and they may play an important role in the formation of plasmid integrates, generating deletions and rearrangements (Haandrikman et al, 1990;Novel et al, 1988;Polzin and Shimizu-Kadota, 1987;Romero and Klaenhammer, 1990). When pND301 was mobilized from M189 by a conjugative plasmid (pND300) (Duan et al, 1996), large novel plasmids were also produced which appeared to be cointegrates of the two plasmids (data not shown).…”