The growth-promoting and stress response activities of the Bacillus subtilis GTP binding protein Obg are separable by mutation

Abstract

Bacillus subtilis Obg is a ribosome-associating GTP binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (GSR). It is unclear whether the roles of Obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. The present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for Obg in each process. The first allele [obg(G92D)] encodes a missense change in the protein's highly conserved "obg fold" region. This mutation impairs cell growth and the ability of Obg to associate with ribosomes but fails to block sporulation or the induction of the GSR. The second obg mutation [obg(Delta22)] replaces the 22-amino-acid carboxy-terminal sequence of Obg with an alternative 26-amino-acid sequence. This Obg variant cofractionates with ribosomes and allows normal growth but blocks sporulation and impairs the induction of the GSR. Additional experiments revealed that the block on sporulation occurs early, preventing the activation of the essential sporulation transcription factor Spo0A, while inhibition of the GSR appears to involve a failure of the protein cascade that normally activates the GSR to effectively catalyze the reactions needed to activate the GSR transcription factor (sigma(B)).

FIG. 1.

Mutations of B. subtilis Obg protein sequence. The 428-amino-acid B. subtilis Obg protein has three domains (11, 54): (i) a glycine-rich N-terminal region. the “Obg fold” (amino acids 1 to 158), which is of unknown function but is conserved among members of the Obg subfamily, (ii) a GTP binding domain consisting of amino acids 159 to 342, and (iii) a TCS domain (amino acids 343 to 428), unique to B. subtilis Obg and named for the three protein families in which the domain is found (ThrRS, GTPase, and SpoT). The Obg92GD mutation is a missense change (GGC to GAC) at nucleotide 92. ObgΔ22 is a substitution of 22 amino acids (NH-RERGAKDGDIIRLLEFEFEFID-COOH) at the C-terminal end of Obg for the 26-amino-acid sequence (NH-SCRRASRIPAHWRPLLVDPSSVPSLA-COOH).

FIG. 2.

Sedimentation analysis of ObgG92D and Obg22/26. Crude extracts of wild-type BSA46, ObgG92D, and Obg22/26 were prepared as described and subjected to centrifugation through a 10 to 30% sucrose gradient containing 10 μM GIDP. Fractions were collected from the bottom of the tube (fraction 1) and analyzed by SDS-PAGE. (A) The protein profile of a representative extract was visualized by Coomassie blue staining. The characteristic cluster of low-molecular-mass ribosomal proteins in fast-sedimenting fractions is bracketed in the Coomassie blue-stained gels. (B) Western blot analyses using polyclonal antibodies against Obg are presented for BSA46 (panel 1), ObgΔ22 (panel 2), and ObgG92D (panel 3). Fraction numbers are indicated at the top of the figure.

FIG. 3.

Ribosome sedimentation in obg mutant strains. Wild-type and obg mutant extracts were sedimented at 4°C and 12,000 rpm in a Beckman XLA analytical ultracentrifuge. The histograms illustrate the percentage of the protein (OD₂₈₀) in wild-type (A), obg(G92D) mutant (B), and obg(Δ22) mutant (C) extracts that sedimented with the indicated sedimentation coefficients (Svedberg).

FIG. 4.

spoIIG transcriptional activation of Obg mutants. BSH113 (spoIIGB::lacZ) (□), BSK100 [spoIIGB::lacZ obg(G92D)] (▵), and BSK101 [spoIIGB::lacZ obg(Δ22)] (○) were grown in DSM until logarithmic growth ceased. Samples were then collected at hourly intervals and analyzed for P_spoIIGA-dependent β-galactosidase, as described in Materials and Methods.

FIG. 5.

FM4-64 staining of Obg mutants in σ^E mutant cells. (A) BSH113, (B) BSK100 [obg(G92D)], and (C) BSK101 [obg(Δ22)] cells were cultured for 18 h on Difco sporulation agar at 37°C. Bacteria were suspended in water, stained with FM4-64, and observed by fluorescence microscopy. Arrows indicate the double septa in both BSH113 and BSK100.

FIG. 6.

Model of σ^B regulation and σ^B operon. (A) σ^B is present in prestressed cells but held inactive in a complex with an anti-σ^B protein, RsbW (6, 18). σ^B is released from its anti-sigma factor, RsbW, by the binding of the anti-sigma factor antagonist, RsbV, to RsbW (18). In unstressed cells RsbV is phosphorylated by RsbW and inactive (3, 18, 51). Physical and nutritional stress activate novel phosphatases to dephosphorylate and reactivate RsbV-P. RsbU, the phosphatase that responds to physical stress, requires an additional protein, RsbT, for activity. RsbT is both a kinase and an RsbU activator. In unstressed B. subtilis cells, RsbT is held inactive by RsbS (56) in a complex with RsbR and a family of homologous proteins (RsbRB, RsbRC, and RsbRD) that facilitate the RsbT-RsbS interactions (1, 2, 23, 30). Upon exposure to physical stress (e.g., ethanol or osmotic shock), RsbT becomes empowered to phosphorylate RsbR and RsbS, freeing itself to interact with RsbU, to induce the dephosphorylation of RsbV-P. Once dephosphorylated, RsbV can sequester RsbW, freeing σ^B to activate the GSR (14, 30, 56). Negative regulation is reestablished by RsbX, a phosphatase that dephosphorylates RsbR-P and RsbS-P, allowing RsbR/S to again sequester RsbT (14, 49, 56). Nutritional stress (e.g., glucose or phosphate limitation) generates an unknown signal to activate a separate phosphatase (RsbP/Q) that is able to dephosphorylate RsbV-P (10, 28, 47, 51). (B) σ^B (sigB) is coexpressed in an eight-gene operon (rsbR, rsbS, rsbT, rsbU, rsbV, rsbW, and rsbX) from a promoter (P^A) likely recognized by σ^A-containing RNA polymerase (27, 41, 53). A σ^B-dependent promoter (P^B) within the operon upregulates the distal four genes upon σ^B activation (5, 9).

FIG. 7.

Activation of σ^B in Obg mutants. (A) BSA46 (□), BSKG92D (▵), and BSK22/26 (○) were grown in LB medium until reaching an OD₅₄₀ of ∼0.4 and then treated with ethanol. Samples were collected at 10-min intervals and assayed for σ^B-dependent β-galactosidase activity. (B) BSA419 (P_SPAC rsbT) (squares) and the obg variants of this strain, BSK102 [obg(G92D)] (triangles) and BSK103 [obg(Δ22)] (circles), were grown in LB medium until reaching an OD₅₄₀ of 0.4. Cultures were then split, and 1 mM IPTG was added to half of the cultures (open symbols), with the other half of the cultures uninduced (solid symbols). Samples were taken and assayed as for panel A. (C) BSK115 (P_B28::P_SPAC rsbW313) (diamonds) and the congenic obg mutant strains BSK104 [obg(G92D)] (triangles) and obg(Δ22) (squares) were grown in LB medium to an OD₅₄₀ of 0.4. Cultures were split, induced, and analyzed as for panel B.