Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor sigmaF in Bacillus subtilis

Abstract

SigmaF, the first compartment-specific sigma factor of sporulation, is regulated by an anti-sigma factor, SpoIIAB (AB) and its antagonist SpoIIAA (AA). AB can bind to sigmaF in the presence of ATP or to AA in the presence of ADP; in addition, AB can phosphorylate AA. The ability of AB to switch between its two binding partners regulates sigmaF. Early in sporulation, AA activates sigmaF by releasing it from its complex with AB. We have previously proposed a reaction scheme for the phosphorylation of AA by AB which accounts for AA's regulatory role. A crucial feature of this scheme is a conformational change in AB that accompanies its switch in binding partner. In the present study, we have studied three AB mutants, all of which have amino-acid replacements in the nucleotide-binding region; AB-E104K (Glu104-->Lys) and AB-T49K (Thr49-->Lys) fail to activate sigmaF, and AB-R105A (Arg105-->Ala) activates it prematurely. We used techniques of enzymology, surface plasmon resonance and fluorescence spectroscopy to analyse the defects in each mutant. AB-E104K was deficient in binding to AA, AB-T49K was deficient in binding to ADP and AB-R105A bound ADP exceptionally strongly. Although the release of sigmaF from all three mutant proteins was impaired, and all three failed to undergo the wild-type conformational change when switching binding partners, the phenotypes of the mutant cells were best accounted for by the properties of the respective AB species in forming complexes with AA and ADP. The behaviour of the mutants enables us to propose convincing mechanisms for the regulation of sigmaF in wild-type bacteria.

Scheme 1. Interactions of AB with σ^F and AA

(A) Phosphorylation pathway of AA by AB. (B) Induced release of σ^F.

Figure 1. Positions of mutations in the structure of AB

The monomers of the AB dimer are coloured light blue and violet, and the ADP molecules are coloured blue. Thr⁴⁹ is coloured red, Glu¹⁰⁴ is coloured purple and Arg¹⁰⁵ is coloured green. This Figure was prepared from the co-ordinates of AB deposited in the Protein Data Bank under the accession code 1L0O.

Figure 2. Time course of the phosphorylation of AA by wild-type AB (♦), AB-E104K (▴), AB-T49K (•) and AB-R105A (▪)

The inset shows the reaction catalysed by AB-T49K and has the same dimensions on the axes as the main Figure

Figure 3. Inhibition by σ^F of the phosphorylation of AA

(A) Reactions catalysed by AB-E104K and AB-T49K. (B) Reactions catalysed by AB-R105A. AA (40 μM) was phosphorylated by 0.5 μM AB-E104K (triangles), AB-T49K (circles) and AB-R105A (squares). Closed symbols show reactions in the absence of σ^F and open symbols show reactions in the presence of 5 μM σ^F. In (B), 5 μM AA was phosphorylated by 0.5 μM AB-R105A in the absence (×) and presence (+) of 5 μM σ^F.

Figure 4. Changes in fluorescence intensity from a fluorescent probe in σ^F

The fluorescence intensities were recorded in real time during the formation of σ^F-W46L·AB complexes and the dissociation of this complex on the addition of AA. σ^F-W46L (1.3 μM) and AB (1 μM) were incubated together at zero time, and a baseline value was determined (see the Experimental section). After 1 min, 100 μM ATP was added, followed after a further 5 min by SpoIIAA. (A) A reaction with wild-type AB (blue line) is compared with reactions with AB-T49K (red line) and AB-E104K (purple line). AA (5 μM) was added to the reaction with wild-type AB, and 10 μM AA was added to the reactions with AB-T49K and AB-E104K. (B) Comparison of results from wild-type AB reacting with 5 μM AA (blue line) with those from AB-R105A reacting with 2.5 μM AA (dark green line), 5 μM AA (light green line) and 10 μM AA (brown line). The black points show a simulation of the dissociation derived from the off-rate of the AB-R105A·σ^F complex (see text for details).

Figure 5. Changes in fluorescence intensity in AB proteins on binding σ^F and on phosphorylating AA

In all experiments, the proteins were incubated together at zero time, and a baseline value was determined (see the Experimental section). After 1 min, 100 μM ATP was added. (A) Fluorescence changes of 1 μM AB proteins on binding to 1.3 μM σ^F-W46L,W190L. (B) Fluorescence changes during phosphorylation reactions of 7.5 μM AA by 1 μM AB proteins. Blue line, AB-F97W; purple line, AB-F97W,E104K; red line, AB-F97W,T49K; green line, AB-F97W,R105A.

Scheme 2. Interactions of σ^F and AA with either wild-type or mutant AB proteins

Schemes for the interactions of wild-type AB (A), AB-E104K and AB-T49K (B), and AB-R105A (C). The phosphorylation pathway is shown in normal text and the interactions with σ^F are shown in outline.