Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.
The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.
Romero
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Department of Microbiology, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695.
Klaenhammer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids; D011815:R Factors; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004251:DNA Transposable Elements