Insulation of the sigmaF regulatory system in Bacillus subtilis

Abstract

The transcription factors sigmaF and sigmaB are related RNA polymerase sigma factors that govern dissimilar networks of adaptation to stress conditions in Bacillus subtilis. The two factors are controlled by closely related regulatory pathways, involving protein kinases and phosphatases. We report that insulation of the sigmaF pathway from the sigmaB pathway involves the integrated action of both the cognate kinase and the cognate phosphatase.

FIG. 1.

σ^F and σ^B activity are regulated by paralogous pathways. (A) The structure of the spoIIA operon and the σ^F regulatory circuit. (B) The structure of the sigB operon and the σ^B regulatory circuit.

FIG. 2.

RsbW phosphorylates AA in the absence of AB. (A) The in vivo ratio of AA to AA-P in a strain lacking σ^F (KC461 [lane 1]), a strain lacking σ^F and AB (KC454 [lane 2]), a strain lacking σ^F, AB, and SpoIIE (KC472 [lane 3]), and a strain lacking σ^F, AB, SpoIIE, and the entire sigB operon (KC476 [lane 4]). (B) The in vivo ratio of AA to AA-P in strains that lack σ^F, AB, and SpoIIE and harbor either an intact sigB operon (KC472 [lane 1]), an insertion-deletion of the sigB operon (KC476 [lane 2]), a Campbell disruption of rsbW (KC486 [lane 3]), or an in-frame deletion of rsbT (KC487 [lane 4]). Lysates of cells harvested 90 min after the start of sporulation were subjected to isoelectric focusing followed by immunoblotting with anti-SpoIIAA antibodies.

FIG. 3.

SpoIIAA is phosphorylated in vitro by RsbW. (A) The purified substrate AA, RsbV, or RsbS (20 μM) was incubated with [γ-³²P]ATP and the purified kinase indicated, either AB, RsbW, or RsbT (2 μM). After 30 min at 37°C, the reaction mixtures were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. (B) The AA (•), RsbV (▴), or RsbS (○) substrate was incubated with [γ-³²P]ATP and the AB kinase. Reactions were sampled after the indicated times at 37°C and analyzed by SDS-polyacrylamide gel electrophoresis. Amounts of labeled product were determined by phosphorimaging and expressed as percentages of the cognate AA substrate labeled at 30 min. (C) The AA (•), RsbV (▴), or RsbS (○) substrate was incubated with [γ-³²P]ATP and the RsbW kinase. Reactions were treated as described for panel B, with the amount of each labeled product expressed as a percentage of the cognate RsbV substrate labeled at 45 min.

FIG. 4.

Transcription from the ctc promoter is directed by both σ^B and σ^F. Fluorescence of green fluorescent protein (GFP) expressed under the control of the ctc promoter was monitored by fluorescence microscopy 1 and 2 h after the start of sporulation in cells wild type for sigB and sigF (KC495), with a deletion of sigB (KC496), with a deletion of sigF (KC504), or with a deletion of both sigF and sigB (KC505). Cell membranes were stained with trimethylammonium diphenyl-hexatriene p-toluenesulfonate. One-second exposures were captured with Metamorph version 4.6 (Universal Imaging) with autoscaling on.