Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E

Abstract

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.

FIG. 1.

Schematic representation of stages of spore formation showing the normal location of activity of sporulation-specific sigma factors.

FIG. 2.

Schematic representation of the spoIIG-spoIIIG region of the chromosome. The promoter for spoIIG requires σ^A and activated Spo0A for expression. The promoter specific to spoIIIG requires σ^F or σ^G for expression. The different transcripts of spoIIIG are indicated. The inverted repeat between spoIIGB and spoIIIG is indicated by opposed arrows.

FIG. 3.

Activity of σ^G in a strain with the spoIIA locus deleted. The activity of σ^G is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains: filled squares, SL10369, spo⁺; open circles, SL12436, spoIIAΔ; open squares, SL11727, spoIIAΔ spoIIIG::neo.

FIG. 4.

The presence of SpoIIAB blocks σ^G activity in a strain that lacks σ^F. The activity of σ^G is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains: filled squares, SL12434, spoIIAC (encoding σ^F) deleted; open triangles, SL12432, spoIIAB and spoIIAC deleted; open squares, SL12436, spoIIAA, spoIIAB, and spoIIAC deleted (spoIIAΔ::neo). For each strain, the extent of the deletion in the spoIIA operon is indicated on the right side, with Δ indicating a deleted gene.

FIG. 5.

Examples of GFP-expressing cells illustrating the patterns of localization of green fluorescence obtained with different strains containing a σ^G-directed sspA-gfp fusion. Bacteria were stained with FM4-64 to visualize membranes (red). A, SL10969 (spo⁺ sspA-gfp); B, SL10034 (spoIIAΔ4 sspA-gfp); C, SL10153 (spoIIAΔ::spc sspA-gfp); D, SL11813 (spoIIAΔ::neo spoIIGBΔ::spc thrC::P_spac(hy)-spoIIGB sspA-gfp); E, SL11815 (spoIIAΔ::spc spoIIIG::neo amyE::spoIIIG sspA-gfp). An arrow is used to indicate a prespore and an arrowhead a mother cell. A 3-μm scale bar is shown in panel E; all images are on the same scale.

FIG. 6.

Effect on σ^G activity of relocating spoIIIG to an ectopic locus in a strain with the spoIIA locus deleted. The activity of σ^G is assessed as β-galactosidase activity expressed from an sspA-lacZ transcriptional fusion in the following strains with the spoIIA locus deleted: open triangles, SL12436; open circles, SL11727, spoIIIG::neo; filled circles, SL11763, spoIIIG::neo amyE::spoIIIG.

FIG. 7.

Effect of deletions of the spoIIG operon on σ^G activity in strains with the spoIIA locus deleted. The activity of σ^G is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains with the spoIIA locus deleted: open squares, SL12436; closed circles, SL12137 (the spoIIGA and spoIIGB structural genes are deleted, but the spoIIG promoter is retained); open circles, SL12426 (the spoIIGA and spoIIGB structural genes and the spoIIG promoter are deleted).

FIG. 8.

Effect of ectopic expression of spoIIGB on σ^G activity in a strain with the spoIIA locus deleted. The activity of σ^G is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains with the spoIIA locus deleted: open triangles, SL12436; open circles, SL11758 (spoIIGBΔ::spc thrC::P_spac(hy)-spoIIGB) in the absence of IPTG; filled circles, SL11758 in the presence of IPTG.

FIG. 9.

Effect of ectopic expression of spoIIGA on σ^G activity in strains with the spoIIG and spoIIA loci deleted. The activity of σ^G is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains with the spoIIA locus deleted: filled circles, SL12359 (spoIIGΔ::cat thrC::P_spac(hy)-spoIIGA) in the absence of IPTG; open circles, SL12359 in the presence of 1 mM IPTG.

FIG. 10.

Effect of deletion of spoIIA and spoIIGB on the accumulation of σ^G during sporulation. Protein samples (300 μg) were obtained at the indicated time (h) after the start of spore formation in MSSM and fractionated by electrophoresis. They were analyzed for σ^G by Western blotting using a polyclonal antiserum to σ^G. The strains used were SL10369 (spo⁺), lanes 1 to 4; SL12436 (spoIIAΔ), lanes 9 to 12; SL11671 (spoIIAΔ spoIIGBΔ), lanes 5 to 8; SL12042 (spoIIAΔ spoIIGBΔ thrC::sigE), lanes 13 to 16; SL11727 (spoIIAΔ spoIIIG::neo), lane 17. Samples were taken at the end of exponential growth (lanes 1, 5, 9, and 13) and 2 h (lanes 2, 6, 10, and 14), 4 h (lanes 3, 7, 11, and 15), and 6 h after the end of exponential growth (lanes 4, 8, 12, 16, and 17). Lanes 1 to 8 and 9 to 17 are from two separate gels.