Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis

Abstract

We determined that the putative upstream promoter of the long transcript (P1) of ompA, the gene encoding the major outer membrane protein of Chlamydia trachomatis L2, is recognized in vitro by the major sigma factor of chlamydiae, 66 . We found no evidence that the putative downstream promoter of the short ompA transcript (P1) is functional in vitro. RNase protection of guanylyltransferase-capped transcripts made in vivo confirmed that P2 is a primary transcript while P1 is a processed product of a longer transcript, possibly P2.The major outer membrane protein (MOMP) of Chlamydia trachomatis constitutes about 50% of the total protein content of the outer membrane in both developmental forms of this obligately intracellular parasite (2, 6). Although the MOMP is present during all stages of the chlamydial life cycle, transcription of the gene encoding the MOMP, ompA, appears to be developmentally regulated. A short transcript (designated P1), with a 5Ј end 25 bp upstream from the AUG translation initiation codon, predominates early in the cycle, and an additional long transcript (designated P2), with a 5Ј end 247 bp upstream from the AUG codon, can be detected during the middle and late stages (8). By 15 to 18 h postinfection (p.i.), the steadystate ratio of short to long transcripts, as measured by RNA Northern blot analysis, is about 1 to 1 (8). Stephens et al. (8) suggested that the two transcripts are initiated from different promoters, P1 for the P1 transcript and P2 for the P2 transcript.Analysis of ompA transcripts generated in vitro. A map of the ompA upstream region, with the locations of the 5Ј ends of transcripts P2 and P1, is shown at the top of Fig. 1. The putative Ϫ10 and Ϫ35 promoter sequences of the P2 and P1 transcripts (8) are shown at the bottom of the figure. We used the oligonucleotides LMPE1 and LMPE2 ( Fig. 1) to map by primer extension (PE) the 5Ј ends of ompA transcripts generated in vivo (by using RNA isolated from infected HeLa cells harvested at 20 h p.i.) and in vitro (by using RNA polymerase prepared from reticulate bodies harvested and purified at 20 h p.i.). The in vitro transcription and PE assays were carried out as described previously (3, 7). The reaction mixtures included pFEN50 (1), a DNA template that encodes the entire MOMP gene and over 1 kb of upstream sequence, and recombinant Chlamydia psittaci 66 , a homolog of the Escherichia coli major sigma factor, 70 (3). The major 5Ј ends that we identified for transcripts generated in vivo were identical to those identified by Stephens et al. (8) (Fig. 2). A strong PE signal was observed when in vitro-generated RNA served as the template and LMPE2 was used as the primer (Fig. 2B); this signal was identical to the 5Ј end of the long in vivo transcript, suggesting that the P2 promoter region functions in our in vitro system. The same 5Ј end was also identified when LMPE1 was used as the primer (Fig. 2A). In addition, several bands which roughly correspond to the 5Ј end of the short P1 transcript were noted when RNA generated i...

Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis

“…2, lanes 3 and 5). This result confirms previous observations (4,5,19) that the antisense and ompA promoters are recognized by 66 , despite significant differences between their core promoter regions (Fig. 1A).…”

“…The putative Ϫ35 and Ϫ10 regions of the P2 promoter are TATACA and TATCGC, respectively. The P2 promoter appears to be recognized by 66 , since transcription in vitro is enhanced by the addition of recombinant 66 to a reaction mixture containing a crude preparation of chlamydial RNA polymerase (4). In contrast to the Ϫ10 regions of the other chlamydial genes for which a 66 requirement has been demonstrated, the Ϫ10 region of the ompA P2 promoter is unusually GC rich.…”

“…All mutant templates were prepared for in vitro transcription by CsCl gradient centrifugation. The predicted length of the transcript generated in vitro from the wild-type P2 promoter template (pALR210C) is 145 nucleotides; the observed transcripts have a mobility corresponding to 140 nucleotides relative to a DNA sequencing ladder (4).…”

“…The amino acid identity between Chlamydia trachomatis 66 and Escherichia coli 70 is 91% in region 2.4 and 82% in region 4.2, the regions thought to be important in the recognition of the Ϫ10 and Ϫ35 hexamers of promoters (16). The requirement of 66 for the expression of four late-stage and two growth-phase genes of C. trachomatis has been established by demonstrating that either the addition of recombinant 66 enhances transcription or the addition of a monoclonal antibody which reacts with 66 on immunoblots inhibits transcription of these genes in vitro (4,5,9,19). The putative Ϫ10 regions of the late genes (crpAB, ltuA, ltuB, and hctA) and one of the growth-phase genes (the gene encoding the antisense transcripts of the chlamydial plasmid) are AT rich and resemble the E. coli 70 consensus recognition sequence of TATAAT in a minimum of four of six positions.…”

DNA Supercoiling-Dependent Gene Regulation in Chlamydia

“…The Ϫ35 and Ϫ10 elements of an optimal chlamydial 66 promoter (39,45) and the tested promoters are indicated by bold uppercase letters. The ompA promoter is ompA P2 (11). A promoter was considered supercoiling responsive if its relative promoter activity dropped below 50% over the range of superhelical densities tested.…”