Article Figures & Data
Figures
- FIG. 1.
Model of the σB signal transduction network. (A) Two independent signaling pathways converge on the RsbV anti-anti-σ and the RsbW anti-σ, the direct regulators of σB activity. The energy pathway terminates with the RsbP phosphatase (Energy PP2C), which contains a PAS domain implicated in energy sensing; the environmental pathway terminates with the RsbU phosphatase (Environmental PP2C), which is activated by upstream signaling elements. Phosphorylated RsbV (RsbV-P) is the antagonist form found in unstressed cells. When activated by stress, either RsbP or RsbU can dephosphorylate RsbV-P, allowing it to bind and inactivate the RsbW anti-σ. (B) In the environmental signaling pathway, 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 function with RsbS to bind the RsbT kinase in an inactive complex. Following environmental stress, RsbT phosphorylates RsbRA and RsbS, releasing RsbT to bind and 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. (C) RsbR coantagonist proteins share a carboxyl-terminal domain (shaded) with the smaller RsbS antagonist (1, 25). In the RsbR family, this domain contains two conserved threonine (T) residues, and RsbT is known to phosphorylate RsbRA on T171 and T205 in vitro (12). In contrast, RsbS bears an aspartate (D) and a serine (S) at these corresponding positions. Genetic evidence suggests that phosphorylation of S59 is required to relieve RsbS antagonist function (16, 30).
- FIG. 2.
Identification of the RsbS and RsbRA isoforms separated by IEF. Wild-type and mutant cell extracts were analyzed by IEF, and the RsbS or RsbRA signals were detected with specific antibodies, as described in Materials and Methods. In all panels, gel images are oriented with their alkaline regions uppermost, toward the cathode, and the numbered lines to the right indicate the approximate positions of unmodified, singly modified, and doubly modified isoforms. (A) Lane 1, wild-type strain (PB2) (wt); lane 2, the rsbS deletion mutant (PB422) (ΔS). Wild-type extracts were also used for the λPP assays and were incubated for 18 h at 30°C (lanes 3 to 5). Lane 3, cell extract (CE) alone; lane 4, addition of λ reaction buffer and MnCl2 (+λB); lane 5, further addition of λPP (+λPP). (B) Wild-type extracts are in lanes 1 and 4; mutant extracts are in lanes 2 and 3. Lane 2, strain with the RsbS S59A alteration (PB465); lane 3, RsbS S59D (PB477). (C) Lane 1, the wild-type strain (PB2) (wt); lane 2, the rsbRA deletion mutant (PB427) (ΔRA). Wild-type extracts were also used for the λPP assays shown in lanes 3 to 5, labeled as described for panel A. (D) Wild-type extracts are in lanes 1 and 8, and mutant extracts are in lanes 2 to 7. Lane 2, strain with the RsbRA T171A alteration (PB829); lane 3, RsbRA T171D (PB557); lane 4, RsbRA T205A (PB505); lane 5, RsbRA T205D (PB502); lane 6, RsbRA T171A-T205A (PB556); lane 7, RsbRA T171D-T205D (PB558).
- FIG. 3.
Isoform balance of RsbS and RsbRA changes after stress. Wild-type (PB2) cells were stressed by either salt or ethanol addition and then rapidly harvested by filtration at the indicated times (minutes). Samples were analyzed by IEF and immunoblotting as described in Materials and Methods. (A and D) Gel images, oriented with their alkaline regions uppermost and with isoform positions indicated on the right. (B and E) Quantification of the digitized images from panels A and D. Note the discontinuity (≈) in the y axis, designating the fraction of phosphorylated RsbS and RsbRA. ▪, RsbS-P/total RsbS; ▴, RsbRA-P/total RsbRA; ○, β-galactosidase activity of a σB-dependent lacZ reporter fusion carried by strain PB198, cultured in parallel with the cells used for the IEF assay. (C and F) RsbS-P/total RsbS (dark grey bars) and RsbRA-P/total RsbRA (light grey bars) found in three independent stress experiments (average ± standard deviation).
- FIG. 4.
Dependence of RsbS and RsbRA phosphorylation on the RsbT environmental signaling kinase and the T171 residue of RsbRA. Wild-type and mutant cells were stressed by ethanol addition and then rapidly harvested by filtration at the indicated times (minutes). Gel images are oriented with their alkaline regions uppermost and with isoform positions indicated on the right. For both panels A (RsbS) and B (RsbRA), lanes 1 to 3 show extracts from the wild type (strain PB2), lanes 4 to 6 show extracts from the rsbT deletion mutant (PB421), lanes 7 to 9 show extracts from the rsbRAT171A substitution mutant (PB829), and lane 10 shows an additional extract from unstressed wild-type cells.
- FIG. 5.
New model for environmental stress signaling. The phosphorylation states of the RsbR coantagonist and RsbS antagonist proteins are controlled by the opposing activities of the RsbT kinase and the RsbX feedback phosphatase. In unstressed cells RsbRA is already partially phosphorylated on T171, and from the genetic analysis reported elsewhere (18) we propose that this phosphorylation is required for signaling. Following an environmental stress, RsbS becomes phosphorylated on S59, triggering the release of RsbT and the activation of the RsbU phosphatase. Phosphorylation of RsbRB, RsbRC, and RsbRD is not shown but is presumed to resemble that of RsbRA.
Tables
- TABLE 1.
B. subtilis strains
Strain Genotype Reference or construction PB2 trpC2 168 Marburg strain PB198 amyE::ctc-lacZ trpC2 8 PB421 rsbTΔ1 trpC2 pCK1(16) → PB2a PB422 rsbSΔ1 trpC2 16 PB427 rsbRAΔ1 trpC2 2 PB465 rsbSS59A trpC2 16 PB477 rsbSS59D Pspac(rsbV+rsbW+sigB+rsbX+) trpC2 16 PB502 rsbRAT205D trpC2 2 PB505 rsbRAT205A trpC2 2 PB556 rsbRAT171A-T205A trpC2 18 PB557 rsbRAT171D trpC2 12 PB558 rsbRAT171D-T205D trpC2 12 PB829 rsbRAT171A trpC2 18 ↵ a Arrow indicates transformation of donor plasmid into recipient.
- TABLE 2.
Adjacent RsbRA and RsbS ortholog genes in diverse prokaryotic genomes
Organism RsbRA orthologa E valueb RsbS orthologa E valueb Bacillus subtilis RsbRA 1e-149 RsbS 6e-52 Listeria monocytogenes NP_464415 7e-68 NP_464416 1e-36 Nostoc punctiforme ZP_00107852 1e-32 ZP_00107851 2e-20 Burkholderia fungorum ZP_00034596 2e-32 ZP_00034597 2e-19 Chloroflexus aurantiacus ZP_00018003 4e-32 Nonec Pseudomonas fluorescens ZP_00084339 2e-31 ZP_00084338 4e-16 Cytophaga hutchinsonii ZP_00117139 8e-30 ZP_00117138 4e-20 Xanthomonas campestris NP_636545 2e-29 NP_636546 1e-18 Streptomyces coelicolor NP_631378 1e-27 NP_631377 2e-16 Vibrio vulnificus NP_762059 2e-26 NP_762060 1e-09 Thermoanaerobacter tengcongensis NP_622693 1e-24 NP_622694 5e-17 Chromobacterium violaceum NP_900548 2e-23 NP_900549 1e-07 ↵ a For organisms other than B. subtilis, each ortholog is indicated by its accession number in the NCBI Protein Database. Organisms are listed by similarity of their RsbRA orthologs to B. subtilis RsbRA.
↵ b E value for the alignment resulting from a BLASTP search (5) of proteins available in the NCBI Microbial Genomes Database, using the default parameters of the on-site program. Only the most similar ortholog for a given bacterium is shown.
↵ c Gene encoding the most similar C. aurantiacus RsbRA ortholog (ZP_00018003) is flanked by genes for additional RsbRA paralogs, from a total of 18 such genes in the organism. The gene for an RsbS ortholog (ZP_00018452; 5e-17) is not adjacent to any of these RsbRA-like genes.
