# Late gene regulation by the alternative sigma factors of Chlamydia trachomatis

## TL;DR

This study identifies how two alternative sigma factors in Chlamydia trachomatis regulate specific genes during the conversion of bacterial forms inside host cells.

## Contribution

The study reveals that σ28 and σ54 sigma factors regulate only a small subset of late genes in Chlamydia, with distinct regulatory mechanisms.

## Key findings

- σ28 binds to hctB and tsp genes only at late stages of Chlamydia's life cycle.
- σ54 binds to ctl0021 and ctl0052, with conserved promoter sequences across Chlamydia species.
- Euo regulates σ28 expression but not σ54, indicating multiple mechanisms for late gene regulation.

## Abstract

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.