Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein

Abstract

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.

FIG. 1.

Substitution of the five residues preceding the CSF cleavage site affects CSF production. (A) Amino acid sequence of the C-terminal 15 residues of PhrC. The five residues preceding the cleavage site are indicated by bold italics, and the mature signaling peptide (CSF) is underlined. The amino acid substitutions in PhrC are indicated below the PhrC sequence. The putative consensus sequence is indicated above the PhrC sequence; “a” indicates an acidic residue, “p” indicates a polar residue, “h” indicates a hydrophobic residue, and “c” indicates a charged residue. (B) Levels of CSF that accumulated in culture medium for strains BAL1191 (WT), BAL1192 (ΔphrC), BAL1187 (D31A), BAL1186 (F32K), BAL1185 (H33A), BAL1184 (V34E), BAL1183 (T35K), and BAL1182 (T35A). For each of three independent experiments, the CSF levels were normalized to the level produced by strain BAL1191. The bars indicate the means of the normalized values, and the error bars indicate the standard errors of the means.

FIG. 2.

Mutant PhrC proteins are secreted at the same level as wild-type PhrC as measured by alkaline phosphatase activity for strains carrying PhrC-PhoA fusions. PhoA activity was measured using culture supernatants of strains BAL2199 (Δss-PhoA), BAL2200 (WT), BAL2201 (T35A), BAL2202 (T35K), BAL2203 (V34E), BAL2204 (H33A), BAL2205 (F32K), and BAL2206 (D31A). The normalized, mean levels of alkaline phosphatase activity from three independent experiments (indicated by bars) are plotted versus the strains assayed. The error bars indicate the standard errors of the means.

FIG. 3.

Synthetic pro-CSF peptides with amino acid substitutions at the −4, −3, and −2 positions are cleaved less efficiently to mature CSF. (A) Sequence of synthetic pro-CSF peptides. (B) Synthetic pro-CSF peptides were incubated with cells of strain BAL950 (Δopp ΔcomQ ΔphrC), and the amount of CSF produced was determined using the biological assay. The normalized mean amounts of CSF produced in three independent experiments are indicated by bars, and the error bars indicate the standard errors of the means. (C) The pro-CSF peptides were incubated with cell wall material, and the ERGMT was quantified by LC-MS/MS-MRM. The intensity of the signal for the parent-to-fragment ion (m/z 386.5) transition is plotted versus the elution time for C₁₈ columns. WT, pro-CSF-WT.

FIG. 4.

Cleavage of mutant pro-CSF substrates by subtilisin and Vpr. Purified subtilisin or Vpr was incubated with the pro-CSF substrates indicated. The level of CSF produced was normalized to the level of CSF produced after incubation with the wild-type pro-CSF substrate (WT). The bars indicate the averages of at least three independent experiments, and the error bars indicate the standard errors of the means. Under these conditions, incubation of subtilisin with pro-CSF and incubation of Vpr with pro-CSF resulted in statistically indistinguishable levels of CSF production.

FIG. 5.

Amino acid substitutions at the −4 position relative to the cleavage site of PhrC. The Phe at position 32 of PhrC was replaced by the other 19 canonical amino acids. The levels of endogenous CSF production by cells expressing the mutant PhrC proteins were determined and normalized to the level of CSF production by the strain expressing wild-type PhrC (amino acid F). The bars indicate the averages for at least three independent experiments, and the error bars indicate the standard errors of the means. The asterisks indicate mutant PhrC strains that were determined to be significantly different from the wild-type strain by the Student t test (P < 0.05).