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

1 Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela. Caracas, Venezuela 2 Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Correspondence Diane E. Taylor diane.taylor{at}ualberta.ca 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 the tra genes were undertaken at both 30 °C and 37 °C. Screening of 38 tra genes showed that tra genes 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. Abbreviations: CAT, chloramphenicol acetyltransferase; drR27, derepressed R27; RACE, rapid amplification of cDNA ends; T4SS, type IV secretion system; UTR, untranslated region Tables listing oligonucleotide primers (Table S1) and showing SD values for the effect of temperature and H-NS protein on the expression of tra promoters (Table S2) are available as supplementary data with the online version of this paper.