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.

An Atypical Phr Peptide Regulates the Developmental Switch Protein RapH

“…In order for pro-PhrH to conform to the pro-Phr peptide consensus sequence, a one-amino-acid gap was introduced in its alignment, and the authors stated that it was difficult at the time to accurately predict the sequence of the mature PhrH peptide (17). Our data suggest that the threonine residue occupying the gap in the alignment is in fact the first residue of the mature PhrH hexapeptide.…”

“…At the highest and probably nonphysiological concentration used (100 M), DRNTT counteracted the effect of rapH overexpression, restoring the PsrfA activity to a level comparable to that obtained in the absence of Phs-rapH induction. As described below, existing data suggest that B. subtilis may not in fact produce DRNTT (17).…”

“…In addition, cleavage of pro-PhrH to generate the hexapeptide TDRNTT places phenylalanine at the Ϫ4 position (see Table S3 in the supplemental material), which was reported to be highly favorable for pro-PhrC cleavage (17). However, cleavage of pro-PhrH to generate the pentapeptide DRNTT places lysine in the Ϫ4 position, and cleavage was found to be most inefficient when lysine occupied the pro-PhrC Ϫ4 position (17).…”

“…However, cleavage of pro-PhrH to generate the pentapeptide DRNTT places lysine in the Ϫ4 position, and cleavage was found to be most inefficient when lysine occupied the pro-PhrC Ϫ4 position (17). Thus, it is unlikely that B. subtilis produces meaningful quantities of DRNTT; rather, mature PhrH is the hexapeptide TDRNTT.…”

“…The sequences of known or predicted mature Phr pentapeptides were previously aligned (27), and the identity of the residues in positions Ϫ1 to Ϫ5 N-terminal to the Phr cleavage site were shown to affect pro-PhrC cleavage (17). In order for pro-PhrH to conform to the pro-Phr peptide consensus sequence, a one-amino-acid gap was introduced in its alignment, and the authors stated that it was difficult at the time to accurately predict the sequence of the mature PhrH peptide (17).…”