Twin RNA Polymerase–Associated Proteins Control Virulence Gene Expression in Francisella tularensis

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Abstract
The MglA protein is the only known regulator of virulence gene expression in Francisella tularensis, yet it is unclear how it functions. F. tularensis also contains an MglA-like protein called SspA. Here, we show that MglA and SspA cooperate with one another to control virulence gene expression in F. tularensis. Using a directed proteomic approach, we show that both MglA and SspA associate with RNA polymerase (RNAP) in F. tularensis, and that SspA is required for MglA to associate with RNAP. Furthermore, bacterial two-hybrid and biochemical assays indicate that MglA and SspA interact with one another directly. Finally, through genome-wide expression analyses, we demonstrate that MglA and SspA regulate the same set of genes. Our results suggest that a complex involving both MglA and SspA associates with RNAP to positively control virulence gene expression in F. tularensis. The F. tularensis genome is unusual in that it contains two genes encoding different α subunits of RNAP, and we show here that these two α subunits are incorporated into RNAP. Thus, as well as identifying SspA as a second critical regulator of virulence gene expression in F. tularensis, our findings provide a framework for understanding the mechanistic basis for virulence gene control in a bacterium whose transcription apparatus is unique. The Gram-negative bacterium Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. In F. tularensis, the MglA protein is the only known regulator of virulence genes that are important for intracellular survival, yet it is not known how MglA functions. F. tularensis also contains an MglA-like protein called SspA whose function is not known. In this study, we show that both MglA and SspA associate with RNA polymerase (RNAP) and positively regulate virulence gene expression in F. tularensis. Our study provides evidence that MglA and SspA interact with one another directly and that the association of MglA with RNAP is dependent on the presence of SspA. We also show that, unlike RNAP from any other bacterium, RNAP from F. tularensis contains two distinct α subunits. Given the fundamental roles the α subunit plays in transcription regulation, this may have far-reaching implications for how gene expression is controlled in F. tularensis. Our study therefore uncovers a new critical regulator of virulence gene expression in F. tularensis (SspA), provides mechanistic insight into how MglA and SspA cooperate to control virulence gene expression, and reveals that the F. tularensis transcription machinery has an unusual composition.