Transcriptional analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and σ⁷⁰-selected promoters

Summary

We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5′ end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3′ end of ORF8 mRNA, with identical 5′ ends but different 3′ ends. In vitro and in vivo experiments in Escherichia coli showed that the σ⁷⁰-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at −10 and −35 nt upstream of the RNA 5′ ends resemble σ⁷⁰ consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters. The same AS-RNA 3′ ends found in C. trachomatis were detected in E. coli, at two putative ϱ-dependent termination sites, and further downstream, at a C-independent termination structure. The results provide the first demonstration of transcriptional mechanisms that are potentially shared by E. coli and Chlamydiae.