We have found previously that, in contrast to the free O initiator protein of  phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the O present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the O fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of  plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from  DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to cro⁺, in cro plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the crots plasmid-harboring cells, a Cro repressor-independent control of  DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to  DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the cro plasmid replication control.