FUNCTIONAL DIFFERENCE BETWEEN THE 2 OPPOSITELY ORIENTED PRIMING SIGNALS ESSENTIAL FOR THE INITIATION OF THE BROAD-HOST-RANGE PLASMID RSF1010 DNA-REPLICATION

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the ori gin of vegetative DNA replication (oriV). If either ssiA or ssiB was d eleted or inverted, the RSF1010 miniplasmids containing engineered ori Vs were maintained at low copy numbers, replicated abnormally as dimer s, and accumulated specific single strands in the Escherichia coli str ain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins . Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication d eficiency of these miniplasmids with respect to all three aspects desc ribed above. These were also true f or the RSF1 01 0 miniplasmids in w hich either ssiA or ssiB was replaced by the primosome assembly site ( PAS) or by the G4-type ssi signal (G site). Furthermore, comparative a nalysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespectiv e of their types, ssi signals conducting the initiation of DNA chain e longation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional dif ference reflects the inherent properties of the initiation mechanism o f RSF1010 DNA replication.