New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis

Abstract

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

FIG. 1

Model of the ς^B signal transduction network. (A) Two signaling pathways converge on phosphorylated RsbV (RsbV-P), the antagonist form found in unstressed cells. The energy-signaling pathway terminates with the RsbP PP2C phosphatase, which may employ its PAS domain to sense energy stress (41). In contrast, the environmental signaling pathway terminates with the RsbU PP2C phosphatase, which is activated by upstream elements (23, 47). When activated by stress, either the RsbP or the RsbU phosphatase removes the serine phosphate from RsbV-P (41, 44, 47). Dephosphorylated RsbV then binds the RsbW anti-ς factor, forcing it to release ς^B, which can then activate its target general stress genes (2, 4, 10). (B) The upstream signaling elements which activate RsbU include the RsbS antagonist and the RsbT kinase, which are homologs of RsbV and RsbW, respectively (23, 47). Unphosphorylated RsbS is thought to be the antagonist form found in unstressed cells, and this form binds the RsbT kinase. Following environmental stress, RsbS is phosphorylated by RsbT, which is then released to bind and activate the RsbU phosphatase by direct protein-protein interaction (24, 47). Genetic and biochemical analyses indicate that RsbR (shaded) acts as a positive regulator of ς^B activity by magnifying the activity of the RsbT kinase (1, 15). In contrast, the RsbX PP2C phosphatase appears to fulfill a feedback role by indirectly communicating the level of ς^B protein in the cell (37, 43, 47). We show here that the four RsbR paralogs (shaded box) also act via the environmental signaling pathway.

FIG. 2

Sequence similarities between RsbR, its paralogs, and RsbS. The percent identical residues and the number of residues over which the similarity extends are shown in the first two columns, and the statistical significance of the Blast alignment is shown in the final column. E values of <1e-10 are considered probably significant, and those of <1e-20 are considered significant. The shaded regions indicate the sequences most highly conserved among the RsbR paralogs and the smaller RsbS antagonist protein. For RsbR, residues T171 and T205 are phosphorylated in vitro by the RsbT kinase and are important for RsbR function in vivo (15). These residues are conserved in YkoB, YojH, and YqhA, whereas the split paralogs YetI and YezB each carry one residue. In contrast, YtvA lacks the conserved threonines and instead bears a PAS domain in its N-terminal region. PAS domains are often found in proteins that monitor changes in redox potential, oxygen tension, or energy levels (40). The analysis reported here indicates that YtvA acts in the environmental stress-signaling branch.

FIG. 3

Effects of single null mutations on β-galactosidase activity of a ς^B-dependent transcriptional fusion. ς^B activity was measured indirectly, using a single-copy transcriptional fusion between the well-characterized ctc promoter (21, 30) and a lacZ reporter, with the PB198 wild-type control indicated by ○. At time zero, cells were subjected to either salt stress (A and D) or ethanol stress (B and E) in early logarithmic growth or to energy stress elicited by entry into stationary phase (C and F). (A to C) Assays using PB565 (ytvAΔ1::ery, ■); (D to F) assays using PB528 (ykoBΔ1::kan, ▴).

FIG. 4

Double-mutant analyses indicate that rsbR is dependent on ykoB, whereas ytvA acts independently. A ctc-lacZ transcriptional fusion was used to measure ς^B activity, with the PB198 wild-type control indicated by ○. At time zero, cells were subjected to salt stress in early logarithmic growth. (A) Effects of the ytvA null allele (PB565; ytvAΔ1::ery, □) and the rsbR null allele (PB491; rsbRΔ1, ◊) are additive in the double mutant (PB566; rsbRΔ1 ytvA1::ery, ■). (B) The ytvA null allele (PB565; ytvAΔ1::ery, □) and the ykoB null allele (PB528; ykoBΔ1::kan, ▵) have opposite effects on response to salt stress, and in the double mutant (PB578; ykoBΔ1::kan ytvAΔ1::ery, ●) the response is a blend of both phenotypes. (C) The ykoB null allele (PB528; ykoBΔ1::kan, ▵) and the rsbR null allele (PB491; rsbRΔ1, ◊) have opposite effects on response to salt stress, and in the double mutant (PB531; rsbRΔ1 ykoBΔ1::kan, ▴) the ykoB null allele fully masks the positive regulatory phenotype of the rsbR null allele. The seven strains shown were assayed together, and the results were distributed into the appropriate panels.

FIG. 5

Analyses of triple and quadruple mutants indicate that YojH and YqhA act in concert with RsbR and YkoB in the environmental signaling pathway. A ctc-lacZ transcriptional fusion was used to measure ς^B activity in unstressed cells during logarithmic growth and also during entry into stationary phase. (A) PB198 (wild type, ○); PB531 (rsbRΔ1 ykoBΔ1::kan, ▵); PB653 (rsbRΔ1 ykoBΔ1::kan yojHΔ1::ery, ●); PB530 (rsbRΔ1 ykoBΔ1::kan yqhAΔ1::spc, ■); and quadruple mutant PB629 (rsbRΔ1 ykoBΔ1::kan yojHΔ1::ery yqhAΔ1::spc, ▴). (B) PB198 (wild type, ○); quadruple mutant PB629 (rsbRΔ1 ykoBΔ1::kan yojHΔ1::ery yqhAΔ1::spc, ▴); PB639 (rsbRΔ1 yojHΔ1::ery yqhAΔ1::spc, ▾); and PB654 (rsbRΔ1 rsbTΔ1 ykoBΔ1::kan yojHΔ1::ery yqhAΔ1::spc, ⧫)

FIG. 6

Purified RsbR, YkoB, YojH, and YqhA are phosphorylated by the RsbT environmental signaling kinase. Purified proteins were incubated with [γ-³²P]ATP in kinase assay buffer, as described in the text. After 60 min at 37°C, the reaction mixtures were subjected to SDS-PAGE and autoradiography. Reactions included the kinase of interest, either RsbT (A), RsbW (B), or SpoIIAB (C), and a target protein: lane 1, the cognate antagonist protein of the kinase, either RsbS (A), RsbV (B), or SpoIIAA (C); lane 2, RsbR; lane 3, YkoB; lane 4, YqhA; lane 5, YojH; lane 6, YtvA; lane 7, YetI; lane 8, YezB. In lanes 1 to 6, the faint bands of lower mobility than the main band are presumed to be dimers and multimers of the target protein.

FIG. 7

Cartoon summarizing the interactions among RsbR, its paralogs, and RsbS. As shown in Table 2, RsbR, YkoB, YojH, YqhA, and the fused YetI-YezB proteins interacted with each other and with the environmental signaling antagonist protein RsbS when paired in the yeast two-hybrid system. In contrast, YtvA interacted only with RsbR and YqhA. None of the newly characterized RsbR paralogs interacted with the downstream RsbT or RsbU regulator from the environmental signaling pathway, with RsbV or RsbW from the common part of the ς^B regulatory pathway, or with SpoIIAA or SpoIIAB from the ς^F regulatory pathway.