Biochemical and genetic characterization of conjugative plasmid SSB's from Escherichia coli

Abstract

The single-stranded DNA binding protein of E. coli (Eco SSB) is required for DNA replication, recombination and repair. The F sex factor was shown to complement the Eco SSB mutation ssb-1, and to encode a protein, FSSB, which appears related to Eco SSB. A variety of conjugative plasmids from various incompatibility (Inc) groups were also found to complement ssb-1. Southern analysis with fssb DNA suggested these plasmids also encoded their own SSB's. Our work has centered on the isolation, characterization and expression of the SSB's from the IncY plasmid, pIP231a, the Inc9 plasmid, pIP71a, and the IncI{dollar}\alpha{dollar} plasmids, R64 and its derepressed form, R64drd11.;We have cloned the genes encoding each of these proteins, determined the DNA sequence, purified each of the proteins, and confirmed the amino acid sequence by protein chemistry. The conjugative plasmid SSB's share regions of homology with Eco SSB, but are more homologous to each other. The NH{dollar}\sb2{dollar}-terminal regions of the proteins are extremely conserved with Eco SSB, but diverge in the COOH-terminal region, with the exception of the last five amino acid residues. The NH{dollar}\sb2{dollar}-terminal region in Eco SSB contains the DNA binding domain, and all amino acid residues implicated in DNA binding in Eco SSB are conserved in the conjugative plasmid SSB's. Temperature melting studies reveal these proteins lower the Tm of poly (dA:dT) more efficiently than Eco SSB. This was pertinent considering the behavior of these proteins in in vitro assays with DNA PolIII holoenzyme. Whereas Eco SSB stimulates DNA PolIII holoenzyme and reaches a plateau, the conjugative plasmid SSB's were stimulatory at low protein concentrations, but inhibitory at higher protein levels. This is consistent with our finding that the expression of conjugative plasmid SSB's appear significantly less than Eco SSB, as exhibited in Western and Northern analyses.