1887

Abstract

The integrating plasmid pCOA18, comprising pUC18 linked to a mutated coagulase () gene from , and constructed by substituting sequences with a tetracycline (Tc)-resistance marker (∆::Tc), was transformed into RN4220, where it underwent recombination with the chromosomal locus. Allele-replacement mutants were recovered at a low frequency directly after transformation. The majority of transformants carried pCOA18 integrated in the chromosome by a single Campbell-type recombination event. The majority of integrants contained tandem repeats of pCOA18 and expressed high levels of resistance to Tc (> 30 μg ml) compared to the single-copy integrants and allele-replacement mutants (15 μg ml). Transduction of a single-copy integrant to a Coa recipient allowed the cointegrant to be resolved and allele-replacement recombinants to be selected. In addition, growth of a single-copy integrant on high concentrations of Tc (> 30 μg ml) selected for amplified derivatives at a frequency of 10. It was estimated that up to 19 copies of pCOA18 could occur in a tandem array in the chromosome.

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1993-04-01
2021-10-18
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