The blue-light receptor YtvA acts in the environmental stress signaling pathway of Bacillus subtilis

Abstract

The general stress response of the bacterium Bacillus subtilis is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the ytvA structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.

FIG. 1.

Model of the σ^B signal transduction network. (A) Two independent signaling pathways converge on RsbV anti-anti-σ and RsbW anti-σ, the direct regulators of σ^B activity. Phosphorylated RsbV (RsbV-P) is the antagonist form in unstressed cells. When activated by the upstream elements shown in panel B, the environmental signaling phosphatase RsbU dephosphorylates RsbV-P, allowing it to bind RsbW and induce the release of σ^B. (B) In the environmental signaling branch, RsbS and RsbT are paralogs of RsbV and RsbW, respectively. RsbS is the antagonist form in unstressed cells, and RsbRA, RsbRB, RsbRC, and RsbRD are redundant coantagonists that make up a large environmental signaling complex with RsbS. This complex binds the RsbT phosphatase regulator and holds it inactive. Following environmental stress, RsbT phosphorylates RsbRA and RsbS, releasing RsbT to activate the RsbU phosphatase. The RsbX feedback respectively. RsbS is the antagonist form in unstressed cells, and RsbRA, RsbRB, RsbRC, and RsbRD are redundant coantagonists that make up a large environmental signaling complex with RsbS. This complex binds the RsbT phosphatase regulator and holds it inactive. Following environmental stress, RsbT phosphorylates RsbRA and RsbS, releasing RsbT to activate the RsbU phosphatase. The RsbX feedback phosphatase returns the system to its prestress condition. Phosphorylation of RsbRB, RsbRC, and RsbRD is not shown but is thought to resemble that of RsbRA. Here we present evidence that the blue-light receptor protein YtvA is also a constituent of the large environmental signaling complex. (C) Members of the RsbR family share a C-terminal STAS domain (shaded) with the smaller RsbS antagonist but have different N-terminal domains: either a nonheme globin domain thought to be important for signaling (RsbR coantagonist proteins) or a LOV domain with an FMN chromophore that forms a light-dependent photoadduct with LOV-C62 (YtvA blue-light receptor). Residues at conserved positions within the STAS domain are charged (RsbS D26, YtvA E168 and E202) or are phosphorylated by RsbT (RsbS S59, RsbRA T171 and T205).

FIG. 2.

YtvA overexpression induces σ^B activity. Effects of YtvA overexpression were measured with a σ^B-dependent ctc-lacZ transcriptional fusion present in single copy on the B. subtilis chromosome. IPTG was added to logarithmically growing cultures to induce ytvA expression from the P_spac promoter of multicopy plasmid pTG5659. Samples were taken at the indicated times and assayed for β-galactosidase (β-Gal) activity as described in Materials and Methods. Filled circles, PB801 (wild type with pTG5659); open triangles, PB806 (rsbTΔ1 with pTG5659; filled triangles, PB907 (rsbΔU2 with pTG5659). All strains were grown in BLB supplemented with 10 μg/ml kanamycin to prevent loss of the multicopy plasmid; cells remained in logarithmic growth through the time shown. Values are corrected for the basal activity of the PB743 control (wild type with the pDG148 vector alone).

FIG. 3.

Association of YtvA with RsbRA and RsbRB in cell extracts. (A) Two cell extracts were prepared: one from strain PB593, encoding a His-tagged version of RsbRA (+), and the other from wild-type control strain PB2 with no tag (−). After nickel affinity chromatography, proteins were analyzed by SDS-PAGE and Western blotting with specific antibodies. Lanes 1 and 2, anti-RsbRA (RA); lanes 3 and 4, anti-YtvA (YtvA). Positions of molecular mass standards are shown on the left (sizes are in kilodaltons). (B) RsbRA-containing fractions from the nickel affinity step shown in panel A were applied to a Sephacryl S300 sizing column; the resulting fractions were analyzed by SDS-PAGE and Western blotting with anti-RsbRA, -RsbRB, -RsbS, or -YtvA antibody. Arrows indicate the elution volumes of the molecular mass standards (sizes are in kilodaltons). (C) Proteins were coimmunoprecipitated from cell extracts with anti-RsbRB antibody and a protein G-agarose conjugate. The precipitated proteins were separated by SDS-PAGE and analyzed by Western blotting with antibody specific for the protein of interest. For each antibody, proteins precipitated from two different cell extracts are shown: wt indicates the PB2 wild type, and Δ indicates the PB545 control bearing a null rsbRB allele. Lanes 1 and 2, anti-RsbRB antibody (RB); lanes 3 and 4, anti-YtvA antibody. Each pair of lanes contains one specific signal for the protein detected, shown by the arrows on the right, and also nonspecific signals for protein G and the IgG light and heavy chains. Positions of molecular mass standards are indicated on the left (sizes are in kilodaltons). These cell extracts, column fractions, and immune precipitates are the same as those shown in Fig. 4 and 5 of reference , and elements of those figures are reprinted here (with the permission of the publisher) for comparison with the additional anti-YtvA antibody data in panels A, B, and C.

FIG. 4.

Transcriptional organization of the ytvB-ytvA interval. (A) RACE-PCR located the 5′ end of the ytvA message 68 nucleotides upstream from the ATG initiation triplet, at the G residue labeled +1. This residue is preceded by sequences resembling an extended σ^A promoter (underlined). DNA fragments from the region were used to construct single-copy transcriptional fusions to lacZ (shaded), all with the same 3′ end (at +276 within ytvA) but with different 5′ ends, as shown: filled squares, PB967 (−339); filled circles, PB963 (−76); filled triangles, PB965 (−20). (B) β-Galactosidase (β-Gal) accumulation assays for the three fusions shown in panel A. Logarithmically growing cells were allowed to enter stationary phase and sampled at the indicated times.

FIG. 5.

Spx activates trxB and ytvA fusion expression. Strains bearing single-copy fusions of trxB-lacZ (A) or ytvA-lacZ (B) were engineered to express the Spx^DD disulfide stress regulator from a P_spank-hy promoter integrated at amyE (45). Spx^DD expression was induced in logarithmically growing cells by adding IPTG at time zero. Cells entered stationary phase at 50 min, indicated by the vertical dotted line. Panel A, ORB4556 (trxB-lacZ) with (filled circles) or without (open circles) added IPTG. Panel B, PB980 (ytvA-lacZ) with (filled squares) or without (open squares) added IPTG. Both fusions were assayed in the ORB4342 background to ensure that the strains were otherwise isogenic (Table 2). β-Gal, β-galactosidase.

FIG. 6.

The C62 residue is required for the positive effect of YtvA overexpression in the absence of stress. The effects of wild and mutant YtvA proteins were measured with a σ^B-dependent ctc-lacZ transcriptional fusion. IPTG was added to logarithmically growing cultures to induce ytvA expression from the P_spac promoters of the different multicopy plasmids (time zero), and samples were assayed for β-galactosidase (β-Gal) activity. Filled circles, PB801 (with pTG5659: wild ytvA); open triangles, PB1003 (with pMB5876: ytvAΔ1); filled triangles, PB1004 (with pMB5877: ytvAΔ2); open squares, PB1005 (with pMB5878: ytvAC62A); filled squares, PB1006 (with pMB5879: ytvAC62S). Strains were grown in BLB supplemented with 10 μg/ml kanamycin to prevent plasmid loss; values were corrected for the basal activity of the PB743 control (with the pDG148 vector alone). The inset shows YtvA levels in PB801 (filled circle), PB1005 (open square), and PB1006 (filled square) assayed by Western blotting.

FIG. 7.

The C62 residue is not required for the positive effect of YtvA overexpression in the presence of salt stress. The effects of wild and mutant YtvA proteins were measured with a σ^B-dependent ctc-lacZ transcriptional fusion. IPTG was added to logarithmically growing cultures to induce ytvA expression from multicopy plasmids (at time −100 min), and NaCl was added to induce the environmental stress response (at time zero). Samples were assayed for β-galactosidase (β-Gal) activity. PB743 (pDG148 vector alone) with (filled triangles) added NaCl, PB801 (pTG5659: wild ytvA) with (filled circles) or without (open circles) NaCl, and PB1006 (pMB5879: ytvAC62S) with (filled squares) or without (open squares) NaCl are shown. Growth was in BLB supplemented with 2.5 μg/ml neomycin to prevent plasmid loss. Values were corrected for the basal activity of the PB743 control (pDG148 vector alone) with no added salt.