Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies. Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins. The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes. Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C. trachomatis genomic libraries. Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced. The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known. The 5 ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone-like genes (hctA and hctB) were mapped, and a consensus ؊10 promoter region of TATAAT was derived from their upstream DNA sequences. In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial 66 , while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added 66. These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on 66 , which is homologous to the major sigma factors of other eubacteria.

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Extracts of Chlamydia psittaci and Chlamydia trachomatis were used to transcribe molecularly cloned chlamydial genes in vitro. The extracts were prepared by lysing reticulate bodies, obtaining the 10,000 x g centrifugation pellet, and eluting RNA polymerase from the pellet by treatment with 2M KCl to yield a fraction designated SS2. Some in vitro transcription was initiated from non-chlamydial promoters and a small amount of transcription was from endogenous DNA template in SS2. However, optimal transcription from exogenous templates required chlamydial promoter sequences, and primer extension analysis indicated that chlamydia promoter-specific in vitro transcription was initiated from the same start sites recognized in vivo. A monoclonal antibody that was generated against Escherichia coli sigma 70 and which immunologically cross-reacts with C. trachomatis sigma 66 inhibited in vitro transcription of vector and cloned chlamydial DNA, suggesting that transcriptional initiation in the SS2 fraction is mediated by sigma 66. An in vitro transcription assay based on detection of transcripts of specific lengths was applied to the chlamydial system; this assay and others described here should be useful in defining chlamydial promoters and other transcriptional regulatory elements.

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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...