The citrate synthase-encoding gene of Rickettsia prowazekii is controlled by two promoters

Abstract

The transcripts of the citrate synthase-encoding gene (gltA) in Rickettsia prowazekii (Rp), an obligate intracellular parasitic bacterium, were analyzed by RNase protection (RP), primer extension (PE) and in vitro transcription assays. Analysis of the 5' end of the gltA mRNA by RP and PE assays revealed that there were two gltA mRNAs with the 5' ends located at 16 bp and 307 bp upstream from the gltA coding region. Since these two mRNAs might represent two species of mRNA transcribed from two different promoters or a single transcript that was processed to give two mRNAs, an in vitro transcription analysis with purified Rp RNA polymerase (RNAP) was performed to distinguish these two possibilities. Purified Rp RNAP catalyzed the formation of two transcripts initiated from the same nucleotides indicated by RP and PE. Sequence analysis identified Escherichia coli (Ec) promoter-like sequences immediately upstream from both transcription start points (tsp). The first promoter (promoter P1) had the core sequence TTCTAA-N17-TATACT, was 6 bp upstream from the tsp (base A) and was centered at 37 bp upstream from the coding region. The second promoter (promoter P2) had the core sequence ATGAAA-N17-TAAAGT, was 7 bp upstream from the tsp (base T) and was centered at 329 bp upstream from the coding region. This is the first demonstration of multiple promoters in this obligate intracellular parasite which has implications concerning transcriptional regulation.