Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control

Abstract

The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression required spo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducing srf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of srf-lacZ at neutral pH. The results suggest that srf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.

FIG. 1

Diagram showing the relationships among the regulatory factors governing srf transcription initiation. The srf operon as well as the regions of the srf genes encoding the amino-acid-activating modules of the peptide synthesis enzyme surfactin synthetase is shown. The comS gene, encoding the regulator of competence development, lies within the srfB gene of srf. Two pheromone-dependent pathways are shown. One pathway requires the ComX peptide which is processed by ComQ and which activates srf transcription through its interaction with ComP. ComP activates ComA by donating a phosphate, but it might also exert negative control by dephosphorylating ComA in the absence of ComX (see Discussion). The pheromone CSF activates srf transcription by inhibiting the ComA-phosphate phosphatase RapC. Import of processed, active CSF [CSF(act.)] requires the oligopeptide permease Spo0K. The inactive form of CSF [CSF(inact.)] is encoded by phrC, transcription of which requires the ς^H form of RNA polymerase. ς^H is encoded by spo0H, the transcription of which is regulated by Spo0A. Spo0A represses the abrB gene, which encodes a negative regulator of spo0H transcription. Cyto. Mem., cytoplasmic membrane.

FIG. 2

Expression of srfA-lacZ in LAB452 grown in DSM-GG (squares), DSM-GGTris (triangles), and DSM without GG (circles). T₀ denotes the onset of stationary phase. The arrow indicates the time of Tris-HCl addition. β-Gal, β-galactosidase.

FIG. 3

Effect of Tris-HCl addition on surfactin production during B. subtilis growth. A TLC analysis showing the amounts of surfactin initially in the medium (lane 1), in the negative control at 3 h after addition of water (lane 2), and at 3 h after addition of Tris-HCl (lane 3) is presented. Lane 4 contains a surfactin standard. Sample volumes analyzed were specific to the total amount of cell protein in each sample.

FIG. 4

(A) Expression of srfA-lacZ in LAB2693 (srfA-lacZ ΔcomP) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (B) Expression of srfA-lacZ in LAB452 (srfA-lacZ) (squares) and in LAB2694 (srfA-lacZ ΔcodY) (circles) grown in DSM-GG (open symbols) and DSM-GGTris (filled symbols). (C) Expression of srfA-lacZ in LAB2691 (srf-lacZ ΔphrC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 (SPβsrfA-lacZ) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (D) Expression of srfA-lacZ in LAB2690 (srfA-lacZ ΔrapC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (E) Expression of srfA-lacZ in LAB2692 (srfA-lacZ Δspo0K) grown in DSM-GG (circles) and DSM-GGTris (triangles) and in LAB452 (srfA-lacZ) grown in DSM-GG (squares). Refer to Fig. 1 for details.

FIG. 4

(A) Expression of srfA-lacZ in LAB2693 (srfA-lacZ ΔcomP) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (B) Expression of srfA-lacZ in LAB452 (srfA-lacZ) (squares) and in LAB2694 (srfA-lacZ ΔcodY) (circles) grown in DSM-GG (open symbols) and DSM-GGTris (filled symbols). (C) Expression of srfA-lacZ in LAB2691 (srf-lacZ ΔphrC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 (SPβsrfA-lacZ) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (D) Expression of srfA-lacZ in LAB2690 (srfA-lacZ ΔrapC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (E) Expression of srfA-lacZ in LAB2692 (srfA-lacZ Δspo0K) grown in DSM-GG (circles) and DSM-GGTris (triangles) and in LAB452 (srfA-lacZ) grown in DSM-GG (squares). Refer to Fig. 1 for details.

FIG. 4

(A) Expression of srfA-lacZ in LAB2693 (srfA-lacZ ΔcomP) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (B) Expression of srfA-lacZ in LAB452 (srfA-lacZ) (squares) and in LAB2694 (srfA-lacZ ΔcodY) (circles) grown in DSM-GG (open symbols) and DSM-GGTris (filled symbols). (C) Expression of srfA-lacZ in LAB2691 (srf-lacZ ΔphrC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 (SPβsrfA-lacZ) grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (D) Expression of srfA-lacZ in LAB2690 (srfA-lacZ ΔrapC) grown in DSM-GG (open circles) and DSM-GGTris (filled circles) and in LAB2583 grown in DSM-GG (open squares) and DSM-GGTris (filled squares). (E) Expression of srfA-lacZ in LAB2692 (srfA-lacZ Δspo0K) grown in DSM-GG (circles) and DSM-GGTris (triangles) and in LAB452 (srfA-lacZ) grown in DSM-GG (squares). Refer to Fig. 1 for details.

FIG. 5

Effects of DSM-GG and DSM-GGTris culture supernatants on expression of srfA-lacZ. LAB452 (srfA-lacZ), LAB2692 (srfA-lacZ Δspo0K), and LAB2693 (srfA-lacZ ΔcomP) were grown in DSM-GG to a low density and then mixed 1:1 with pH-adjusted supernatants as described in Materials and Methods. Samples were collected every 20 min for β-galactosidase activity assays. Figures are representative of at least two experiments run under identical conditions. (A) Recipient cultures contained cells of strain LAB452 (srfA-lacZ). (B) Culture medium supernatants were collected from late-growth cultures of strains OKB192 (ΔcomX), LAB2691 (ΔphrC), and LAB452 (wild type). All supernatants were adjusted to pH 6.1. Recipient cultures contained cells of strain LAB2692 (srfA-lacZ Δspo0K). (C) All supernatants were adjusted to pH 6.1. Recipient cultures contained cells of strain LAB2693 (srfA-lacZ ΔcomP). (D) Supernatants of late-growth JMS377 (ΔcomQ ΔphrC) culture, JH642 (wild-type parent) late-growth culture, and JH642 early-growth culture adjusted to pH 6.1. Recipient early-growth cultures contained cells of LAB452 (srfA-lacZ).