Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1

Abstract

The promiscuous streptococcal plasmid pLS1 encodes for the 5.1 kDa RepA protein, involved in the regulation of the plasmid copy number. Synthesis of RepA was observed both in Bacillus subtilis minicells and in an Escherichia coli expression system. From this system, the protein has been purified and it appears to be a dimer of identical subunits. The amino acid sequence of RepA has been determined. RepA shows the αhelix-turn-αhelix motif typical of many DNA-binding proteins and it shares homology with a number of repressors, specially with the TrfB repressor encoded by the broad-host-range plasmid RK2. DNase I footprinting revealed that the RepA target is located in the region of the promoter for the repA and repB genes. Trans-complementation analysis showed that in vivo, RepA behaves as a repressor by regulating the plasmid copy number. We propose that the regulatory role of RepA is by limitation of the synthesis of the initiator protein RepB.