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

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.

RelA regulates virulence and intracellular survival of Francisella novicida

“…Stress-induced (p)ppGpp production in Helicobacter pylori is required for survival during acid exposure and aerobic shock (Mouery et al, 2006;Wells & Gaynor, 2006), conditions typically encountered during the course of infection. In Francisella, intracellular replication and virulence are dependent on MglA and SspA, which regulates the FPI (Guina et al, 2007;Nano & Schmerk, 2007;Charity et al, 2007). In this study, we have identified a global regulatory system that plays a role in F. novicida virulence, where a relA mutant had reduced survival in macrophage-like cells, and was attenuated more than 100-fold in a BALB/c mouse model.…”

“…Disruption of many of the genes in the FPI, such as iglA, iglC, pdpA and pdpD, results in attenuation in vitro or in vivo (Baron & Nano, 1998;Golovliov et al, 2003;Gray et al, 2002;Lauriano et al, 2004;Lindgren et al, 2004;Nano et al, 2004). Genes of the FPI have been shown to be regulated by MglA, encoded by mglA (Lauriano et al, 2004), and SspA, encoded by sspA (Charity et al, 2007). MglA and MglB are essential for intracellular replication and virulence in mice (Baron & Nano, 1998;Lauriano et al, 2004), and share homology with the Escherichia coli SspA and SspB proteins, which are regulators of stationary phase transcription and are involved in stress survival (Williams et al, 1994).…”

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH

“…As reported by Charity et al (2007), the F. tularensis genome contains two genes, designated rpoA1 (FTL_0261) and rpoA2 (FTL_0616), which encode two different a subunits of RNAP that differ from one another in regions that are predicted to be critical for dimer formation, promoter recognition, and activator interaction. FTL_1744 and FTL_1743 (rpoB and rpoC) encode the b and b9 subunits, respectively, and FTL_1533 (rpoZ) the v subunit.…”

Identification of two substrates of FTS_1067 protein – An essential virulence factor of Francisella tularensis

“…Overnight cultures were diluted in fresh LB medium supplemented with antibiotics and 0.5 mM IPTG and grown to OD 600 0.5-0.6 and assayed for β-galactosidase activity [9]. As a negative control, E. coli KDZif1ΔZ were cotransformed by empty pACTR-Ap-Zif and pBRGP-ω plasmids; and as a positive control by plasmids pACTR-Ap-MglA-Zif and pBGRP-SspA-ω [15]. The empty plasmid pBRGP-ω together with FTS_1067-Zif was used as another negative control.…”