Transcriptional organization of the temperature-sensitive transfer system from the IncHI1 plasmid R27

Abstract

One of the characteristic features of IncHI1 plasmids is a thermosensitive process of conjugation, which is optimal between 22 °C and 30 °C but inhibited at 37 °C. R27, the prototypical IncHI1 plasmid, contains transfer genes clustered in two regions of the plasmid, Tra1 and Tra2. In the present study, transcriptional analyses of thetragenes were undertaken at both 30 °C and 37 °C. Screening of 38tragenes showed thattragenes are transcriptionally linked in six operons, three in each Tra region. RT-PCR analysis showed that gene expression was reduced at 37 °C relative to that observed at 30 °C. The transcription start sites of the six transcripts were identified, promoters and upstream regions were cloned, and transcription was tested at both temperatures. In cells grown at 37 °C, in the presence of R27, the promoters were inhibited, except for promoters of the H operon and AN operon. Conditions that influenced DNA topology, such as osmolarity, anaerobiosis, quorum sensing and acidity, showed no significant influence on transfer frequency. These results should facilitate future understanding of the basis of temperature-sensitive transfer in this large conjugative plasmid.