Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein

Abstract

A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein. We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259. The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested. In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE. All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein. These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence. We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity. This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

“…Apparently, the wild-type RepE bound to ori2 DNA with a very low efficiency as reported (11)(12)(13), whereas RepE54 bound with a much higher efficiency (Fig. 2), in agreement with the high initiator activity of RepE54 found in vivo (26). The Stoichiometry of the RepE Binding to ori2 DNA.…”

“…The majority of mini-F mutants that can replicate in E. coli strains defective in &2 or DnaJ carried a single amino acid change within the RepE segment spanning residues 92-134 and exhibited increased initiator activity and reduced repressor activity (26,31). All these RepE mutants except RepE54 have been purified as dimers that exhibited enhanced ori2 binding (3-to 12-fold ofwild-type binding) and concomitantly reduced operator binding (0.3-to 0.5-fold of wild-type binding), in agreement with their properties in vivo (ref.…”

“…E. coli BL21 (ADE3) producing T7 RNA polymerase (27) was used as the host for RepE production; pLysS and the repE expression plasmid, pBK815, were as described (13). A similar plasmid carrying the repE54 mutant allele, pBK815 (repE54), was constructed by replacing the Xma I-EcoRV fragment of pBK815 with the equivalent fragment from pKV739 carrying repE54 (26), and the structure was confirmed by nucleotide sequencing.…”

“…To further examine the role of heat shock proteins in mini-F plasmid replication, we have recently isolated and characterized mini-F mutants that can replicate in the dnaJdefective host (26). Among them was a mutation (repE54) that caused production of RepE54 with a markedly enhanced initiator activity but little or no repressor activity as a result of the Arg-117 -+ Pro replacement (26).…”

“…Among them was a mutation (repE54) that caused production of RepE54 with a markedly enhanced initiator activity but little or no repressor activity as a result of the Arg-117 -+ Pro replacement (26).…”