Blocking chromosome translocation during sporulation of Bacillus subtilis can result in prespore-specific activation of sigmaG that is independent of sigmaE and of engulfment

Abstract

Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase sigma factors, which are activated in the order sigma(F)-sigma(E)-sigma(G)-sigma(K). Of these, sigma(G) becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding sigma(G), spoIIIG, is directed in the prespore by RNA polymerase containing sigma(F) but also requires the activity of sigma(E) in the mother cell. When first formed, sigma(G) is not active. Its activation requires expression of additional sigma(E)-directed genes, including the genes required for completion of engulfment. Here we report conditions in which sigma(G) becomes active in the prespore in the absence of sigma(E) activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the sigma(E)-directed regulation appears to be to coordinate sigma(G) activation with the completion of engulfment, not to control the level of sigma(G) activity. It seems plausible that the role of sigma(E) in sigma(G) activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of sigma(G) in the prespore is. Competition for core RNA polymerase between sigma(F) and sigma(G) is unlikely to be of major importance.

FIG. 1.

The P_spoIIIG promoter is expressed in the absence of σ^E when it is located at the origin-proximal amyE locus in a spoIIIE36 mutant. Transcription directed by the P_spoIIIG promoter was assessed by determining the β-galactosidase activity in the following strains containing an amyE::P_spoIIIG-lacZ fusion: SL5430 (spo⁺) (○), SL10117 (spoIIGB::erm) (□), SL10086 (spoIIIE36) (•), and SL11106 (spoIIIE36 spoIIGB::erm) (▪).

FIG. 2.

Activation of σ^G independent of σ^E in a spoIIIE36 mutant with spoIIIG located at amyE. The activity of σ^G was assessed by determining the β-galactosidase activity in the following strains: SL10369 (spo⁺ P_sspA-lacZ@sspA) (•), SL12916 (spoIIIE36 amyE::spoIIIG spoIIR::P_sspA-lacZ) (○), SL12918 (spoIIIE36 spoIIR::P_sspA-lacZ) (▪), SL12936 (spoIIIE36 amyE::spoIIIG spoIIR::P_sspA-lacZ spoIIR@spoIIR) (□), and SL12938 (spoIIIE36 amyE::spoIIIG spoIIR::P_sspA-lacZ spoIIR@spoIIR spoIIAC::neo) (▵).

FIG. 3.

Location of σ^E-independent σ^G activity. (A to C) Examples of bacteria stained with FM4-64 (red) and expressing GFP (green) under control of the σ^G-directed sspA promoter. (A) SL10969 (spo⁺); (B) SL12864 (spoIIIE36 amyE::spoIIIG spoIIR) (the arrows indicate an abortively disporic cell); (C) SL12929 (spoIIIE36 amyE::spoIIIG spoIIR⁺) (the arrow indicates a partly engulfed prespore). (D to I) Strain SL12972 expressing CFP (blue) under control of the σ^F-directed spoIIR promoter (D, F, G, and I) and expressing YFP (yellow) under control of the σ^G-directed sspA promoter (E, F, H, and I). Bacteria were stained with FM4-64 (red). Panel F is an overlay of panels D and E and includes the FM4-64 image. Panel I is an overlay of panels G and H and includes the FM4-64 image. Bar = 3 μm for all images.

FIG. 4.

Effect of σ^G activity on σ^F activity in the preengulfment prespore. The activity of σ^F was assessed by determining the β-galactosidase activity in the following strains: SL12857 (spoIIIE36 spoIIR::P_spoIIR-lacZ) (○) and SL12861 (spoIIIE36 spoIIR::P_spoIIR-lacZ amyE::spoIIIG) (•).

FIG. 5.

Activation of σ^G is independent of SpoIIQ in a spoIIIE36 amyE::spoIIIG background. The activity of σ^G was assessed by determining the β-galactosidase activity in the following strains: SL12916 (spoIIIE36 amyE::spoIIIG spoIIR::P_sspA-lacZ) (○) and SL13225 (spoIIIE36 amyE::spoIIIG spoIIR::P_sspA-lacZ spoIIQ::neo) (•).