Late gene regulation by the alternative sigma factors of Chlamydia trachomatis [sig54_ChIP-seq]

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 a new host cell. 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 C. 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. A σ28 overexpression strain 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-dependent 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 just four late genes suggests that these genes and the independent control of their temporal expression are important for RB-to-EB conversion. Samples collected at four timepoints (hours post infection). Each timepoint replicated twice. Sheared DNA from each sample divided into three parts: Ab (antibody coated bead added to immunoprecipitate)-450 µl, NoAb (bead without antibody for mock precipitation)- 450 µl, and wce (sheared input DNA)- 50 ul. Each sample eluted and decrosslinked in 150 ul sample volume and purified in 50 ul final volume for library preparation. ChIP antibody: anti-sigma54 (ABClonal)