Identification and Structural Modeling of the RNA Polymerase Omega Subunits in Chlamydiae and Other Obligate Intracellular Bacteria

Abstract

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing sigma factor. The core enzyme comprises two alpha subunits, one beta subunit, one beta ' subunit, and one omega subunit. The omega subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an omega subunit for the obligate intracellular bacterium Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial omega subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing omega subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have omega orthologs.IMPORTANCE Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase omega subunit, and it has been hypothesized that the chlamydial RNA polymerase omega subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle. Here, we report the identification of the chlamydial RNA polymerase omega subunit, based on conserved sequence, conserved synteny, AlphaFold-predicted conserved three-dimensional structure, and AlfaFold-Multimer-predicted conserved interactions. Our identification of the previously elusive chlamydial RNA polymerase omega subunit sets the stage for investigation of its roles in regulation of gene expression during chlamydial growth, development, and stress responses, and sets the stage for preparation and study of the intact chlamydial RNA polymerase and its interactions with inhibitors. Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase omega subunit, and it has been hypothesized that the chlamydial RNA polymerase omega subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle.