Gene, 154 (1995) 93-98 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50 93 GENE 08614 Transcriptional regulation in the Chlamydia trachomatis pCT plasmid (Promoter; RNA processing; sigma factor) Stefano Ricci, Giulio Ratti and Vincenzo Scarlato Imrnunobiological Research Institute Siena ( I.R.LS.), 53100 Siena, Italy Received by H.M. Krisch: 1 April 1994; Revised/Accepted: 26 September/4 October 1994; Received at publishers: 14 November 1994 SUMMARY We have analyzed tran,;criptional regulation of the chlamydial plasmid pCT. Transcription of a full-length 2.9-kb ORF1-ORF2 mRNA is likely to be regulated by the ~66 transcription factor which recognizes the TATAAT and TNGNCA sequences at the - 10 and - 35 DNA regions, respectively. RNA synthesis starts 39 nucleotides (nt) upstream from the ATG start codon of ORF1 and terminates within the downstream ORF3 DNA region. A 2.8-kb transcript transverses the ORF3-6 DNA region, while two transcripts of 2.2 and 1.9 kb cover the ORF4-6 DNA region. These mRNAs overlap two abundant transcripts which regulate the expression of the ORF3 and ORF4 genes. The accumulation of transcripts associated with these ORFs is likely to be regulated at the level of RNA synthesis by an unknown factor which could select the RTTTAAA and TTYTTR sequences located at the - 10 and - 35 DNA regions, respectively. This new promoter consensus sequence could be unique to the gene expression machinery of Chlamydiae. INTRODUCTION The study of gene expression in Chlamydia trachomatis (Ct) is of particular interest because Chlamydiae are obligate intraceUular parasites of eukaryotic cells with a unique developmental cycle involving differentiation between an extracellular, metabolically inert form, and an intracellular replicative form (Ward, 1988; Moulder, 1991 ). With the exception of a single Ct isolate (Peterson et al., 1990), all human Ct isolates described contain a conserved plasmid, pCT. This plasmid, which has been previously described as pCHL1, pCTT1 and pLGV440 Correspondence to: Dr. V. Scarlato, I.R.I.S., Via Fiorentina 1, 53100 Siena, Italy. Tel. (39-577) 243-239; Fax (39-577) 243-564. Abbreviations: aa, amino acid(s); bp, base pair(s); Ct, Chlamydia trachomatis; dNTP, deoxyribonucleoside triphosphate; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame(s); ORF, gene(s) (DNA, RNA encoding a given ORF); ori, origin of DNA replication; P, promoter; PAGE, polyacryl- amide-gel electrophoresis; PCR, polymerase chain reaction; p.i., post infection; T, terminator of transcription; tsp, transcription start point(s); [], denotes plasmid-carrier state. (Palmer and Falkow, 1986; Sriprakash and MacAvoy, 1987; Hatt et al., 1988; Comanducci et al., 1988; 1990), is approx. 7.5 kb in length and comprises eight major ORFs. Downstream from the origin of DNA replication (or/) of pCT (Tam et al., 1992) two convergent ORFs, ORF7 and ORF8, encode putative proteins which share aa homology with each other and have C-terminal domains typical of the phage integrase family of proteins (Ricci et al., 1993). These ORFs are likely to be finely regulated at the transcriptional level by their own pro- moters and short anti-sense RNAs complementary to the 3' end of the ORF8 mRNA (Ricci et al., 1993). ORF3 was shown to encode a 28-kDa antigen and to be co-transcribed with the downstream ORF4 in the late phase of Ct infection from the P3 promoter (Comanducci et al., 1993). Furthermore, ORF4 appears to be tran- scribed from its own promoter P4 (Comanducci et al., 1993; and this work). In this paper we analyze RNA transcripts from the ORFI-ORF6 DNA region of pCT. The results suggest that transcriptional regulation of pCT involves at least two classes of promoters. SSDI 0378-1119(94)00825-6