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 (KanR) 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of the proteins that run between the β and σ70 subunits of RNAP (in lane 2) are breakdown products of the β subunit (unpublished data).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.
Table 1.
Microarray Analysis of Genes Whose Expression Changes by a Factor of 3 or More in either a ΔmglA or a ΔsspA Mutant Background Compared to Wild-Type
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.