A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis

Abstract

Spore formation by Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of intercellular communication. One pathway, which couples the activation of the forespore transcription factor sigma(G) to the action of sigma(E) in the mother cell, has remained mysterious. Traditional models hold that sigma(E) initiates a signal transduction pathway that specifically activates sigma(G) in the forespore. Recent experiments indicating that the mother cell and forespore are joined by a channel have led to the suggestion that a specific regulator of sigma(G) is transported from the mother cell into the forespore. As we report here, however, the requirement for the channel is not limited to sigma(G). Rather, it is also required for the persistent activity of the early-acting forespore transcription factor sigma(F) as well as that of a heterologous RNA polymerase (that of phage T7). We infer that macromolecular synthesis in the forespore becomes dependent on the channel at intermediate stages of development. We propose that the channel is a gap junction-like feeding tube through which the mother cell nurtures the developing spore by providing small molecules needed for biosynthetic activity, including sigma(G)-directed gene activation.

Figure 1.

The sporangium, σ^G, and the functional dissection of SpoIIQ. (A) Shown is a cartoon of the sporangium at the stage of engulfment when σ^G becomes active in the forespore. Also shown are channels composed of AA–AH and Q that are herein proposed to serve as feeding tubes between the mother cell and the forespore. (B) Full-length Q and its truncated variants. Q harbors a short cytoplasmic N terminus (∼20 residues), followed by a transmembrane domain (depicted as a black box), and a large extracellular C-terminal domain (∼240 residues). The region (residues 117–222) that displays similarity to the M23 family of lysostaphin-like peptidases is shaded dark gray. Two highly conserved amino acids, Tyr28 and His202, are depicted as Y and H, respectively. Shown below is the N-terminal region of the E. coli protein MalF used to replace the N-terminal region of Q to create the MalF^TMD-Q chimera; the MalF transmembrane domain is shown as a black box. (C) Heat-resistant spore formation by cells harboring mutant alleles of Q. With the exception of the parental strain harboring wild-type Q (WT), cells were deleted for the Q gene (ΔQ) at its normal location or were deleted for Q and harbored at sacA either wild-type Q (Q⁺) or the mutant alleles Q^ΔC50, Q^ΔC100, Q^ΔC230, Q^Δ202–216, Q^H202A, Q^Δ2–42, malF^TMD-Q, or malF^TMD-Q^Δ202–216 (strains PY79, AHB141, AHB1361, AHB1362, AHB1363, AHB1372, AHB1375, AHB1369, AHB1374, AHB1418, and AHB1533, respectively).

Figure 2.

σ^G activity and AH localization in cells producing mutant Q proteins. (A,B) σ^G activity in strains harboring mutant Q alleles. σ^G-dependent expression of a P_sspB-lacZ fusion inserted at the ywrK locus was monitored during sporulation of strains deleted for Q (ΔQ; closed squares) or deleted for Q and harboring either Q⁺ (closed circles), Q^Δ202–216 (open circles), Q^H202A (open diamonds), malF^TMD-Q (open squares), or malF^TMD-Q^Δ202–216 (open triangles) at the sacA locus (strains AHB1399, AHB1401, AHB1408, AHB1405, AHB1537, and AHB1538, respectively). For clarity, Q^Δ202–216 and Q^H202A data are shown in A, while malF^TMD-Q and malF^TMD-Q^Δ202–216 data are shown in B. β-Galactosidase production in Q deletion and Q⁺ control strains measured for each experiment is shown for comparison. (C,D) Localization of AH in cells producing mutant Q proteins. In each strain, the native AH gene was deleted and a functional gfp-AH fusion was inserted at the amyE locus. (C) GFP-AH fluorescence in wild-type cells (WT; strain AHB1508) or cells deleted for Q (ΔQ; strain AHB1516) at hour 3 of sporulation. Arrowheads indicate wild-type cells that display uneven or punctate GFP-AH localization. Inset cell exemplifies the punctate GFP-AH localization seen in a subset of cells. Bar, 2 μm. Membrane fluorescence from FM 4-64 staining for each corresponding field is also shown below. Membranes of engulfed forespores are not visible due to the membrane impermeability of the FM 4-64 dye. (D) GFP-AH localization at sporulation hour 3 in cells deleted for the endogenous Q gene and expressing either Q⁺, Q^Δ202–216, Q^H202A, malF^TMD-Q, or malF^TMD-Q^Δ202–216 from the sacA locus (strains AHB1527, AHB1531, AHB1552, and AHB1532, AHB1555, respectively).

Figure 3.

A late phase of σ^F activity is unmasked in the absence of σ^G and requires channel proteins. (A) σ^F-dependent P_Q-lacZ expression during sporulation of wild-type cells (WT; closed circles), cells deleted for the gene encoding σ^F (ΔsigF; closed triangles), deleted for the gene encoding σ^G (ΔsigG; closed squares), or deleted for sigG and harboring a wild-type copy of the sigG gene inserted at the ywrK locus (ΔsigG + ywrK∷sigG⁺; open circles) (strains AHB881, AHB938, AHB882, and AHB1305, respectively). In each of these strains and those described below, the P_Q-lacZ reporter gene was inserted at the amyE locus. (B,C) σ^F-dependent β-galactosidase production in cells deleted for sigF, AA–AH, or Q in the presence (B) or absence (C) of the sigG gene (sigG⁺ vs. ΔsigG). Cells expressing the P_Q-lacZ reporter gene either harbored the wild-type sigF, AA–AH and Q genes (sigF⁺ AA–AH⁺ Q⁺; closed circles) or were deleted for sigF (ΔsigF; closed triangles), AA–AH (ΔAA–AH; open triangles) or Q (ΔQ; open diamonds) (sigG⁺ strains: AHB881, AHB938, AHB1134, and AHB939, respectively; sigG deletion strains: AHB882, AHB915, AHB1017, and AHB916, respectively). (D) σ^F-dependent P_Q-lacZ activity during sporulation of strains lacking σ^G and producing mutant Q protein lacking residues His202–Pro216. Each strain harbored a deletion of the sigG gene. Additionally, the strains were deleted for Q (ΔQ; closed squares) or were deleted for Q and harbored at the sacA locus Q⁺ (closed circles) or the Q^Δ202–216 mutant (open diamonds) (strains AHB1453, AHB1463, and AHB1470, respectively).

Figure 4.

Late activity of a heterologous RNA polymerase requires the AA–AH and Q channel proteins. (A) Cartoon of the construct used to direct synthesis of T7 RNAP (T7 RNA polymerase) in the forespore during sporulation. The gene encoding T7 RNAP was placed under the control of the σ^F-dependent P_Q promoter and was integrated into the chromosome at either the origin-proximal amyE locus or at an origin-distal location downstream from the ylnF gene. The proximity of the P_Q-T7 RNAP construct to the origin of replication (which is anchored to the forespore pole of the sporangium) determines whether it will be present in the forespore immediately following asymmetric septation or following a delay. For simplicity, only the forespore chromosome is shown. (B,C) T7 RNAP-directed P_T7-lacZ expression during sporulation of wild-type cells (WT; black squares), cells deleted for sigF (open circles) or AA–AH (open triangles), or cells engineered to express Q^Δ202–216 (open diamonds). In B, all strains harbor the P_Q-T7 RNAP construct integrated at an origin-proximal chromosome position (the amyE locus), whereas C shows the results from corresponding strains with the P_Q-T7 RNAP construct inserted at an origin-distal position (downstream from ylnF). In all strains, the P_T7-lacZ reporter was located at an origin-proximal position (the ywrK locus). Arrows highlight the difference in β-galactosidase levels at an early time of sporulation (hour 2) that results from switching the P_Q-T7 RNAP chromosome position. Origin proximal T7 RNAP strains: AHB1125 (WT), AHB1131 (ΔsigF), AHB1132 (ΔAA–AH), and AHB1382 (Q^Δ202–216). Origin distal T7 RNAP strains: AHB1449 (WT), AHB1474 (ΔsigF), AHB1475 (ΔAA–AH), and AHB1545 (Q^Δ202–216). (D) Immunoblot analysis of whole-cell extracts from sporulating wild-type cells (WT; left panel) or cells deleted for sigF (center panel), or AA–AH (right panel), using antibodies to T7 RNAP, β-galactosidase, or, as a loading control, σ^A. An asterisk marks a protein that displays cross-reactivity with the anti-β-galactosidase antibody. Strains were identical to those used in C, with P_Q-T7 RNAP integrated at an origin-distal chromosome position: AHB1449 (WT), AHB1474 (ΔsigF), and AHB1475 (ΔAA–AH).

Figure 5.

Q is a bifunctional protein. (A) Forespore-specific T7 RNAP-directed P_T7-lacZ activation during sporulation of strains expressing Q⁺ (solid squares), malF^TMD-Q (solid triangles), or Q^Y28A (solid diamonds) (strains AHB1542, AHB1547, and AHB1543, respectively). In each of these strains and those described below, the construct expressing T7 RNAP (P_Q-T7 RNAP) was inserted at the origin-distal ylnF locus, whereas the P_T7-lacZ reporter gene was integrated at the origin-proximal ywrK gene. (B) T7 RNAP-directed β-galactosidase production in strains harboring the P_T7-lacZ reporter and coexpressing the alleles malF^TMD-Q and Q^ΔC230 (open triangles; strain AHB1571) or malF^TMD-Q and Q^Y28A,ΔC230 (closed triangles; strain AHB1579). Data for control strains expressing wild-type Q (Q⁺; closed squares), malF^TMD-Q (open circles) or Q^ΔC230 (open diamonds) alone are also shown (strains AHB1542, AHB1567, and AHB1544, respectively). (C) T7 RNAP-dependent P_T7-lacZ reporter gene activity was measured during sporulation of strains expressing Q^Y28A (closed diamonds), Q^Y28A and deleted for AA–AH (open diamonds), Q^{Y28A,Δ202–216} (open triangles), or deleted for Q altogether (ΔQ; closed circles) (strains AHB1543, AHB1562, AHB1546, and AHB1476, respectively).