Degree Type


Date of Award


Degree Name

Doctor of Philosophy




An improved system for transforming protoplasts of Staphylococcus aureus with plasmid DNA was developed to facilitate the construction of an integrable plasmid. Protoplast transformants were recovered at higher frequencies when the protoplasts were warmed to 56 C immediately prior to the addition of cesium chloride-purified plasmid DNAs. More transformants were recovered when the protoplast-DNA mixture was plated on R agar than on DM3 medium, and when the selective agent was incorporated directly in the medium rather than supplied in an agar overlay. More transformants were recovered when the protoplast-DNA mixture was incubated at 30 C for 3.5 h before plating than when this mixture was plated immediately. The protocol developed in this study yielded higher transformation frequencies than similar procedures for protoplast transformation of S. aureus;The use of a restriction-deficient recipient strain and an improved protocol for protoplast transformation facilitated the isolation of a recombinant plasmid, designated pPQ126, directly in S. aureus. pPQ126 is a novel, temperature-sensitive integrable plasmid 13.6 kilobase pairs in length. Plasmid pPQ126 contains genes encoding for resistance to erythromycin and chloramphenicol (from plasmid pTV1(,ts)) and resistance to gentamicin (from transposon Tn4001). Plasmid pPQ126 is established as an autonomous replicon when introduced into an appropriate recipient strain at the permissive temperature (30 C). Integration of pPQ126 is then directed into homologous target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 C (non-permissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of a chromosomal target. It was also observed that pPQ126 excises from the chromosome under permissive conditions, and excision often generates derivatives of this plasmid that contain additional fragment(s) of DNA.



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Copyright Owner

John Bernard Luchansky



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243 pages