Late gene regulation by the alternative sigma factors of Chlamydia trachomatis [sig28_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, s28 and s54, that alter the promoter specificity of C. trachomatis RNA-polymerase. s28 ChIP-seq identified hctB and tsp as the only promoters bound by s28, and binding only occurred late, around the time of RB-to-EB conversion. A s28 overexpression strain confirmed that these genes are transcribed in a s28-dependent manner. s54 ChIP-seq showed that s54 only bound ctl0021 and ctl0052, and only at late times. This s54 regulon appears to be conserved as in silico analysis identified s54 promoter sequences upstream of ctl0021 and ctl0052 homologs in all Chlamydia spp. The genes encoding s28 and s54 were only transcribed at late times, but ChIP analysis with the late regulator Euo showed that Euo only controls s28 expression, and late transcription of s54 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. Overall design: 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-sigma28 (ABClonal)