Effect of integration host factor of RNA II synthesis in replication of plasmid containing orip 15A.

The synthesis rates of the replication control RNAs of plasmid orip15A. RNA I, an inhibitor of replication, and RNA II, the primer, have been determined using lacZ fusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) in Escherichia coli integration host factor-positive (IHF+) and -negative (IHF-) strains containing pACYC184 plasmid (orip15A). In the absence of IHF (E. coli IHF-), expression of the lacZ gene from the PRNAII promoter increased by a factor of 4 compared with the E. coli wild type (IHF+). Also, the increase in expression was more pronounced when the IHF protein was mutated in the ihfB gene than in the ihfA gene. For the PRNAII promoter of oripMB1 (pBR322), no significant differences were found in expression of the lacZ gene in he E. coli strains examined. The level of beta-galactosidase expression from the PRNA promoter of orip 15A shows that the absence of functional IHF in the transformed strains has no effect on expression of the lacZ gene. The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 for E. coli IHF+ and 4.4 for E. coli IHF-. Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I in E. coli IHF+ and IHF-, are equal, but the relative level of RNA II in E. coli IHF is about four times higher than in E. coli IHF+. These results indicate that the IHF protein inhibits transcription from the PRNAII promoter of orip15A plasmid.