Late gene regulation by the alternative sigma factors of Chlamydia trachomatis

The pathogenic bacterium Chlamydia reproduces via two specialized forms inside a eukaryotic host cell. The dividing form called the reticulate body (RB) must convert at late times into the infectious elementary body (EB) for spread to new host cells. Late genes are a temporal class of chlamydial genes believed to be responsible for RB-to-EB conversion, but late gene regulation is incompletely understood. In this study, we used chromatin immunoprecipitation (ChIP) to investigate two alternative sigma factors, σ²⁸ and σ⁵⁴, that alter the promoter specificity of Chlamydia trachomatis RNA polymerase. σ²⁸ ChIP-seq identified hctB and tsp as the only promoters bound by σ²⁸, and binding only occurred late, around the time of RB-to-EB conversion. Overexpression of σ²⁸ confirmed that these genes are transcribed in a σ²⁸-dependent manner. σ⁵⁴ ChIP-seq showed that σ⁵⁴ only bound ctl0021 and ctl0052 and only at late times. This σ⁵⁴ regulon appears to be conserved as in silico analysis identified σ⁵⁴ promoter sequences upstream of ctl0021 and ctl0052 homologs in all Chlamydia spp. The genes encoding σ²⁸ and σ⁵⁴ were only transcribed at late times, but ChIP analysis with the late regulator Euo showed that Euo only controls σ²⁸ expression, and late transcription of σ⁵⁴ is regulated in an Euo-independent manner. Thus, multiple mechanisms regulate late genes, including Euo and different forms of RNA polymerase. The dedicated use of two alternative RNA polymerases to control a small subset of late genes suggests that these genes and the independent control of their temporal expression are important for RB-to-EB conversion.

In this study, we performed chromatin immunoprecipitation-seq to identify genes transcribed by alternative forms of RNA polymerases in Chlamydia trachomatis. Under normal growth conditions, the sigma factors, σ²⁸ and σ⁵⁴, bound only two genes each, and binding was only detected at late times. In addition, the late regulator Euo controls the expression of σ²⁸ but not σ⁵⁴. Thus, Chlamydia utilizes multiple mechanisms to regulate late gene expression and uses alternative forms of RNA polymerases for specialized control of specific late genes that likely have important roles in reticulate body to elementary body conversion. This genome-wide binding approach can be applied to identify target genes of alternative sigma factors in other pathogenic bacteria.