Construction of an IS946-based composite transposon in Lactococcus lactis subsp. lactis.

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RESUMO

An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+. When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA). pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01. An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L. lactis LM2345. Transfer of either marker alone occurred at frequencies of ca. 2 x 10(-4) per input donor. Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker. No Cmr Ems transconjugants were recovered (n = 550). Plasmid analysis showed that the Cms Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element. A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids. DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA. This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms.

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