Functional reconstitution of an unusual Firmicutes σ factor into a Gram-negative heterologous host.

Sigma (σ) factors are single-subunit proteins that reversibly bind RNA polymerase and play an important role in the transcription initiation process. An unusual 2-subunit σ factor, consisting of proteins SigO and RsoA, activates transcription from a group of related promoters in Bacillus subtilis. These 2 proteins specifically interact with each other and with RNA polymerase subunits. This system is widespread among species in several Bacillus-related genera, but otherwise appears restricted to the Firmicutes. Here, we reconstituted SigO-RsoA, and a cognate promoter, into the distantly related heterologous host Escherichia coli to examine whether this system can function in bacteria outside of the Firmicutes. We show that these proteins can productively associate with E. coli RNA polymerase and activate transcription, demonstrating that there are no structural barriers to function. In parallel, we tested a wide array of protein-protein interaction mutations and promoter mutations that impact SigO-RsoA function in both B. subtilis and E. coli and conclude that the SigO-RsoA system behaves, in most instances, similarly in both genetic backgrounds. These data raise the possibility of genetically isolating the system in this heterologous host and away from unknown B. subtilis factors that may also be playing a role in SigO-RsoA regulatory pathways, thus facilitating further study of the system. As a result of this work, we also provide a comprehensive mutational analysis of a SigO-RsoA promoter and report the preliminary identification of amino acids in SigO that play a role in mediating the SigO-RsoA protein-protein interaction.