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 alpha subunits of RNAP, and we show here that these two alpha 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.

Figure 1. MglA and SspA Are Associated with RNAP in F. tularensis

(A) Schematic representation of the TAP-tag integration vector and its use in making β′-TAP. The calmodulin binding peptide (CBP), protein A moieties (ProtA), and TEV cleavage site that constitute the TAP-tag are shown [17], together with the kanamycin resistance determinant (Kan^R) and the mobilization region (mob). (B) SDS-PAGE analysis of proteins that co-purify with SucD-TAP, β′-TAP, and MglA-TAP from F. tularensis. Protein complexes were tandem affinity purified, electrophoresed on a 4%–12% Bis-Tris NuPAGE gel, and stained with silver. Lane 1, proteins purified from strain LVS SucD-TAP. Lane 2, proteins purified from strain LVS β′-TAP. Lane 3, proteins purified from strain LVS MglA-TAP. MglA, SspA, and subunits of RNAP are indicated together with the β′-CBP and MglA-CBP fusions that result following cleavage of each corresponding TAP fusion with TEV protease. MS/MS analyses revealed that many of the proteins that run between the β and σ⁷⁰ subunits of RNAP (in lane 2) are breakdown products of the β subunit (unpublished data). (C) SDS-PAGE analysis of proteins that co-purify with MglA-TAP in a wild-type (WT, lane 1) or in a ΔsspA mutant background (lane 2). Protein complexes were tandem affinity purified, electrophoresed on a 4%–12% Bis-Tris NuPAGE gel, and stained with silver. Lane 1, proteins purified from strain LVS MglA-TAP. Lane 2, proteins purified from strain LVS ΔsspA MglA-TAP. Molecular weights are indicated on the left.

Figure 2. Bacterial Two-Hybrid Analysis of Protein–Protein Interactions Involving MglA and SspA

(A) Schematic representation of the two-hybrid system. Contact between MglA and SspA fused, respectively, to Zif and to the ω subunit of E. coli RNAP activates transcription from the test promoter, driving expression of lacZ. The diagram depicts test promoter placZif1–61, which bears a Zif binding site centered 61 bp upstream of the transcription start site of the lac core promoter (whose −10 and −35 elements are indicated). In E. coli strain KDZif1ΔZ, this test promoter is linked to lacZ on an F′ episome. (B) Transcription activation by MglA-Zif in the presence of the SspA-ω or MglA-ω fusion proteins, and by SspA-Zif in the presence of the SspA-ω fusion protein. KDZif1ΔZ cells harboring compatible plasmids directing the synthesis of the indicated proteins were grown in the presence of different concentrations of IPTG and assayed for β-galactosidase activity.

Figure 3. MglA Co-Purifies with His-Tagged SspA

Immunoblot analyses of proteins co-purifying with SspA-His6. Cell lysates of E. coli containing MglA-S and SspA (i.e., without the His-tag) or MglA-S and SspA-His6 were incubated with a metal chelate affinity resin. Following washing, proteins specifically bound to the resin were eluted with imidazole. Cell lysates and eluted proteins were separated on a 4%–12% Bis-Tris NuPAGE gel and analyzed by immunoblotting using antibodies that recognize the His-tag on SspA-His6 (anti-His; upper panel), the S-tag on MglA-S (anti-S; middle panel), and the α subunit of E. coli RNAP (anti-α; lower panel). Lane 1, lysate containing MglA-S and SspA. Lane 2, lysate containing MglA-S and SspA-His6. Lane 3, proteins purified from the lysate containing MglA-S and SspA. Lane 4, proteins purified from the lysate containing MglA-S and SspA-His6.

Figure 4. Both MglA and SspA Positively Control Virulence Gene Expression in F. tularensis

Quantitative RT-PCR analysis of iglA, iglC, and pdpA transcript levels in wild-type (WT), ΔmglA, ΔsspA, and FTL0951 mutant backgrounds. Transcripts were normalized to tul4 whose expression is not influenced by MglA or SspA; compared to cells of the wild-type strain, cells of the ΔmglA mutant had 0.91 times (with an SE of 0.04) the number of tul4 transcripts, as determined by quantitative RT-PCR. Similarly, compared to cells of the wild-type strain, the ΔsspA, and FTL0951 mutants had 1.02 times (SE = 0.12) and 1.47 times (SE = 0.09) the number of tul4 transcripts, respectively.

Figure 5. MglA and SspA Control the Expression of a Similar set of Genes

Venn diagram representation of the overlap between genes controlled by MglA, SspA, or growth rate. Each circle represents those genes whose expression was altered by a factor of 3 or more (p < 0.01) in the corresponding mutant strain compared to wild-type, and whose expression altered by a factor of 3 or more (p < 0.01) in the other mutant background.

Figure 6. Models for How the MglA–SspA Complex Positively Controls Virulence Gene Expression in F. tularensis

(A) Contact between a DNA-bound transcription activator and the RNAP-associated MglA–SspA complex activates transcription from a virulence gene promoter. The arrow indicates the transcription start site. (B) Contact between promoter DNA and the RNAP-associated MglA–SspA complex activates transcription from a virulence gene promoter. In these models, the virulence gene promoters are depicted as being recognized by RNAP holoenzyme containing σ⁷⁰. The RNAP that co-purifies with MglA-TAP appears to contain σ⁷⁰ in stoichiometric amounts (Figure 1B), suggesting that the promoters of MglA/SspA-dependent genes may be σ⁷⁰-dependent.