Loss of compartmentalization of σ(E) activity need not prevent formation of spores by Bacillus subtilis

Abstract

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σ(F) in the smaller cell, the prespore. σ(F) then directs a signal via the membrane protease SpoIIGA to activate σ(E) in the larger mother cell by processing of pro-σ(E). Here, we show that σ(E) can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σ(E) activity is not essential for spore formation. σ(E) activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE*, which encodes a constitutively active form of σ(E), but not of spoIIGB, which encodes pro-σ(E). We infer that σ(E) compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predominantly located in the mother cell and as a consequence confines σ(E) activation to it. In addition, we find that CsfB, previously shown to inhibit σ(G), is independently inhibiting σ(E) activity in the prespore. CsfB thus appears to serve a gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents σ(G) from becoming active before completion of engulfment and helps prevent σ(E) from becoming active at all.

FIG. 1.

Localization of activity of σ^E and other sporulation-associated sigma factors in strains with various modifications to expression of the spoIIG operon. Examples of bacteria stained with FM4-64 (red) and expressing GFP (green) from gfp transcriptional fusions to promoters directed by different sigma factors are shown. Promoters are as follows: σ^E-directed P_spoIID (A to H and L), σ^E-directed P_cotEp1 (I), σ^F-directed P_spoIIQ (J), and σ^G-directed P_sspA (K). Strains are either csfB+ (A to D and I to K) or csfB null (E to H and L). (A) Strain SL14657 at T5 (amyE::P_spoIIQ-sigE* ΔspoIIGB::spc). (B) SL14574 at T2.5 (spoIIG::P_spoIIQ-spoIIG). (C) SL14631 at T2.5 (spoIIG::P_spoIIQ-spoIIG amyE::spoIIG). (D) SL14712 at T2.5 (amyE::P_spoIIQ-spoIIG). (E) SL14656 at T5 (amyE::P_spoIIQ-sigE* ΔspoIIGB::spc ΔcsfB::cat). (F) SL14578 at T2.5 (spoIIG::P_spoIIQ-spoIIG ΔcsfB::cat). (G) SL14613 at T2.5 (spoIIG::P_spoIIQ-spoIIG amyE::spoIIG ΔcsfB::cat). (H) SL14715 at T2.5 (amyE::P_spoIIQ-spoIIG ΔcsfB::spc). (I) SL14868 at T2.5 (amyE::P_spoIIQ-spoIIG). (J) SL14816 at T2.5 (amyE::P_spoIIQ-spoIIG). (K) SL14711 at T5 (amyE::P_spoIIQ-spoIIG). (L) SL15062 at T2.5 (amyE::P_spoIIQ-spoIIGA ΔcsfB::spc). The scale bar shown in panel J is 3 μm and is applicable to all images.

FIG. 2.

Engulfment of a prespore expressing σ^E activity. Successive images from time-lapse microscopy of strain SL14712, amyE::P_spoIIQ-spoIIG thrC::P_spoIID-gfp. Membranes were stained with FM4-64 (red); GFP (green) indicates the location of σ^E activity. Time in each image is the time in minutes (′) from the first frame. Scale bar, 2 μm. Bacteria were placed on agarose pads containing MSSM and incubated at 33°C.

FIG. 3.

Schematic representation of a model for the compartmentalization of σ^E activation. The membrane protein SpoIIGA is indicated by filled circles, and SpoIIR is indicated by a star. SpoIIGA is first synthesized before the spore septum is formed and is distributed randomly throughout the cell membrane. As a consequence, upon septation the large majority of SpoIIGA is located in the mother cell. Some component of the recently completed septum captures SpoIIGA that has diffused from the peripheral membrane. When SpoIIR is synthesized, it is secreted into the space between the septal membranes. There, it can interact with SpoIIGA from either the mother cell or the prespore. (SpoIIR may or may not be the component that captures SpoIIGA in the septum.) Because the preponderance of SpoIIGA is in the mother cell, mother cell SpoIIGA wins out in the competition for the very limited amount of SpoIIR. As a consequence, only SpoIIGA in the mother cell is activated, and so conversion of pro-σ^E to active σ^E occurs only in the mother cell (shown in path A). However, if expression of SpoIIGA in the prespore is artificially enhanced, then activation of σ^E can also occur in the prespore (path B).