Curing a large endogenous plasmid by single substitution of a partitioning gene

Abstract

To investigate whether plasmid-free cells of pathogenic Escherichia coli can be isolated by disrupting a single gene in an endogenous plasmid without further treatment, the effect of the disruption of partitioning genes on the inheritance of the endogenous plasmid pUTI89 of the uropathogenic E. coli strain UTI89 was studied. We found that mutation of parB, which encodes a type Ib partitioning protein, could cause loss of the endogenous plasmid at a ratio of about 1%. Clones derived from parB mutants, identified by antibiotic sensitivity, were all plasmid free. Plasmid instability caused by the parB mutation was found to correlate with a negative effect on host cell growth. Thus, in this pathogenic E. coli, an endogenous plasmid as large as 114 kbp could be cured effectively by targeting a single type Ib partitioning gene followed by passaging, which may facilitate further investigations on the function of endogenous plasmids in their natural hosts.

Keywords: Escherichia coli; Lambda recombination system; Partitioning gene; Plasmid curing; Plasmid-free cells; Transposon insertion mutants.