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

Abstract

On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a ؊35 hexamer region of ؊42 aaaaaga TATACAaa ؊28 and an unusual, GC-rich ؊10 hexamer region of ؊13 tTATCGCt ؊6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5 extent of P2 was located at bp ؊42. Replacement of wild-type sequence with two G's at positions ؊41 and 40, ؊35 and 34, and ؊29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the ؊35 and ؊10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position ؊13, ؊10, ؊7, or ؊6 had little or no effect on transcription, whereas substitution of G at ؊11 or ؊12 significantly decreased promoter strength. Triple point mutations which changed the ؊10 hexamer from TATCGC to TATTAT, TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis 66 -RNA polymerase used in this study, purified Escherichia coli 70 -RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with ؊10 hexamers that resembled the 70 consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis 66 -RNA polymerase has special promoter recognition properties not found in E. coli 70 -holoenzyme.Chlamydiae are obligate parasitic bacteria. They progress through an intracellular developmental cycle that is characterized by the interconversion of infectious, metabolically inert elementary bodies and noninfectious, dividing reticulate bodies. The mechanisms by which gene expression is regulated during the developmental cycle are not known. The genes encoding the chlamydial RNA polymerase core subunits have been either partially or completely sequenced, and their predicted peptides bear significant similarity to the ␣, ␤, and ␤Ј subunits of RNA polymerase of other bacteria (7,10,14). Only one chlamydial sigma factor, 66 , has been identified (5, 7, 14). 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. The resemblance of the putative Ϫ35 hexamer of these ge...

Identification and Mapping of Sigma-54 Promoters in Chlamydia trachomatis

“…Our recent investigations (18) identified temporal expression of the three C. trachomatis RNA polymerase sigma factors, 66 (major 70 homolog), 28 , and 54 . Several reports have characterized 66 promoters (8,17,29), but to date, no promoters have been identified for the developmental-stage-specific sigma factors, 28 and 54 . The present study utilized the complete Chlamydia trachomatis genome sequence (27) and a modified fluorescence-based primer extension (PE) assay to identify and map the first 54 promoters for Chlamydia.…”

Conservation of Putative Promoter Sequences Located Upstream of Chlamydial Major Sigma Factor Gene, sigA among Chlamydia Spp.

“…However, a previous study showed that the ompA putative ial strains, although the upstream and downstream Arich sequences are conserved around the putative -35 element (3,29). The sigA promoter sequences are the only ones so far shown to be conserved among Chlamydiae .…”

“…Many chlamydial promoter regions include not only 670 -like -35 and -10 elements and the appropriate spacer region but also adenine (A) or adenine-thymine (AT)-rich sequences upstream and downstream of the -35 sequence (3,24,26). An upstream A-rich sequence was found 87 to 83 by upstream from the first ATG codon of GCP-1 sigA, however, a downstream AT-rich sequence was not found (Fig.…”

“…The upstream A-rich sequence was identical in all strains. A-and AT-rich sequences around the -35 element in the ompA and rRNA operons have been shown to be essential for effective transcription by in vitro transcription assay (3,24,26). Interestingly, an A-rich sequence upstream of the -35 element has also been reported to be conserved in Gram-positive organisms, and to be necessary for the high rate of transcription in the B. subtilis promoters (9, 14, 19).…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…Functional evidence has been demonstrated for several σ 66 promoters and one σ 28 promoter using mutational analysis and in vitro transcription assays (e.g. Ricci et al, 1993;Fahr et al, 1995;Douglas and Hatch, 1996;Tan et al, 1996;Tan et al, 1998;Ochiai et al, 1999;Schaumburg and Tan, 2003;Yu et al, 2003;Yu et al, 2006). Chlamydial promoters share homology with each of the eubacterial consensus sequences with certain exceptions, including the presence of a cisacting, A/T spacer region in the majority of σ 66 promoters analyzed (Schaumburg and Tan, 2000).…”