Differential Effects of DNA Supercoiling on Chlamydia Early Promoters Correlate with Expression Patterns in Midcycle

Abstract

Changes in DNA supercoiling levels during the chlamydial developmental cycle have been proposed as a global mechanism to upregulate midcycle genes, but the effects on early genes are not known. We examined the promoters for 10 Chlamydia trachomatis early genes and found that they could be separated into two subsets based on their responses to DNA supercoiling in vitro . Furthermore, the type of supercoiling response correlated with the in vivo expression pattern for each early gene. One subset of seven early genes had promoters that were transcribed in a supercoiling-insensitive manner over the physiologic range of supercoiling levels that have been measured in Chlamydia . In vivo transcripts for these genes were detected at similar levels during early-stage and midstage times. In contrast, a second subset, represented in our study by three early genes, had supercoiling-dependent promoters that were transcribed at higher levels from more-supercoiled templates, which is the response observed for midcycle genes. Genes in this subset were expressed at higher levels at midstage times than at early times in vivo . We propose that this second subset represents a novel class of chlamydial developmental genes with features of both early and midcycle genes. We hypothesize that expression of these supercoiling-dependent early genes is upregulated by increased chlamydial supercoiling levels in midcycle via their supercoiling-responsive promoters in a manner similar to that for midcycle genes. Thus, we propose that DNA supercoiling is utilized in Chlamydia as a general mechanism to regulate genes in the midstage of the developmental cycle and not just midcycle genes.

Functional analysis of three topoisomerases that regulate DNA supercoiling levels in Chlamydia

“…All reactions were performed as three independent experiments, and the error bars represent standard deviations. Comparison graphs showing the supercoiling response for a supercoiling‐insensitive ( euo ) and a supercoiling‐sensitive ( dna K P 1) promoter are adapted from Cheng and Tan, . B.…”

“…We treated Chlamydia ‐infected cells with novobiocin for 30 min at 16 hpi, which is before the peak in chlamydial supercoiling at about 18–24 h (Niehus et al ., ), and measured transcript levels of selected genes by quantitative RT‐PCR. In validation experiments, novobiocin treatment decreased transcription of dnaK , groEL , ompA , pgk and fliF , which each have a supercoiling‐sensitive promoter (Niehus et al ., ; Case et al ., ; Cheng and Tan, ), but did not affect rRNA, euo and incD , early genes with supercoiling‐insensitive promoters (Cheng and Tan, ). Transcription of all three chlamydial topoisomerases was decreased by novobiocin treatment, with transcript levels of gyrB , topA and parE reduced to 4.5%, 30.7% and 11% of untreated levels respectively.…”

“…This model is supported by the finding that promoters of representative midcycle genes are transcribed at higher levels from more supercoiled templates (Niehus et al ., ; Case et al ., ). In addition, a subset of early genes that are upregulated in midcycle also have supercoiling‐dependent promoters (Case et al ., ; Cheng and Tan, ). DNA supercoiling co‐ordinates the expression of 7–15% of the genome in other bacteria (Champoux, ; Gmuender et al ., ; Niehus et al ., ; Peter et al ., ; Ferrandiz et al ., ; Schroder et al ., ), but it has the potential to be the global regulator of gene expression in Chlamydia by controlling the expression of the majority of genes in midcycle.…”

Regulation of Chlamydia Gene Expression by Tandem Promoters with Different Temporal Patterns

“…For example, chlamydial genes with supercoiling-responsive promoters have been proposed to be upregulated in midcycle by increased chlamydial DNA supercoiling levels at this stage of the developmental cycle (7,8,11). In contrast, late genes have been proposed to be repressed during early times and midcycle by the transcription factor EUO, until this repression is relieved at late times by an as-yet-undefined mechanism (12,13).…”

“…DNA supercoiling, which peaks in midcycle, is proposed to regulate genes with supercoilingresponsive promoters, which include midcycle genes and a subset of early genes (7,8). Late genes consist of two subsets that are transcribed by either the major chlamydial RNA polymerase, which contains the sigma factor 66 , or an alternative RNA polymerase containing 28 (7, 9-11).…”