Molecular analysis of Phr peptide processing in Bacillus subtilis

Abstract

In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

FIG. 1.

Schematic representation of the rapA-phrA (A), rapC-phrC (B), and rapE-phrE (C) loci. The thick arrows indicate the extents of the open reading frames. Thin arrows indicate the positions of the transcriptional promoters present in each locus with the corresponding sigma factors. The positions of the consensus sequences for binding of the ComA response regulator are indicated by boxes. Relevant restriction sites are indicated in parentheses if they were generated through a PCR amplification reaction and without parentheses if they exist on the B. subtilis chromosome. Restriction site symbols: E, EcoRI; B, BamHI; Bg, BglII; H, HindIII; D, DraI; Bc, BclI. The lengths of the fragments used in this study for site-directed mutagenesis are shown by the lines labeled with a number in parentheses. Transcription terminators are indicated by the symbol Ω.

FIG. 2.

Phr peptide cleavage sites. The amino acid sequences of the wild-type (WT) PhrA, PhrC, and PhrE peptides are shown with those of their mutant derivatives generated by site-directed mutagenesis. The positions of residues relevant to this study are indicated by subscript numbers. The mutated residues are boxed. The active pentapeptide inhibitors are underlined. The domain organization of Phr proteins includes the positively charged amino-terminal residues (+++) and the hydrophobic and the hydrophilic regions. Asterisks indicate the putative SPase cleavage sites as identified by the program SignalP (28).

FIG. 3.

Transcription analysis of the rapA-lacZ fusion in phrC mutants. β-galactosidase assays were carried out in Schaeffer's sporulation medium, and β-galactosidase activity was time point analyzed at hourly intervals before and after the transition phase (T0). Strains: JH12981 (wild type), •; JH23059 (phrC mutant 1 [T35P]), ▴; JH23060 (phrC mutant 2 [T35A]), ▪.

FIG. 4.

Domain requirement for the processing of PhrA. The wild-type (WT) sequence of the 44-amino-acid PhrA peptide is shown with the deletion mutant proteins and the His tag extension mutant protein (PhrA-His). The A-to-P change in the PhrAΔ14-6 protein is indicated by a box. The active pentapeptide inhibitors are underlined. The domain organization is indicated as described in the legend to Fig. 2. The positions of two relevant residues are given by subscript numbers for reference. +++ indicates positively charged amino-terminal residues.

FIG. 5.

Processing of the PONA substrate with the six-His tag into the nuclease A (NA) mature protein by the B. subtilis SipS, SipV, and SipT SPases. The reactions were carried out at pH 8.0 as described in Materials and Methods, and reaction products were analyzed on SDS-10% PAGE in Tris-HCl-Tricine buffer, pH 8.45. The reaction timesare expressed in minutes. The sizes of the molecular weight markers (lane 1) are indicated. (A) PONA (18 μM) was incubated alone (lanes 2 and 5) or with SipS (0.13 μM; lanes 3 and 4) for the times indicated. SipS alone is shown in lanes 6 and 7. Shown are the results for reaction mixtures containing phosphatidylethanolamine, cardiolipin, and lipid extracts. (B) PONA (18 μM) was incubated alone (lanes 2 and 5) or with SipV (3 μM; lanes 3 and 4) for the times indicated. SipV alone is shown in lanes 6 and 7. Shown are the results for reaction mixtures containing lipids, as in panel A. (C) PONA (14 μM) was incubated alone (lanes 2 and 5) or with SipT (1.4 μM; lanes 3 and 4) for the times indicated. SipT alone is shown in lanes 6 and 7. Shown are the results for reaction mixtures containing only the B. subtilis lipid extract.

FIG. 6.

Analysis of PhrA processing by SPases in vitro. Samples were analyzed by SDS-PAGE electrophoresis using 18% acrylamide in Tris-HCl-Tricine buffer, pH 8.45. A synthetic PhrA peptide (44 amino acids; 57 μM) was incubated with SipS (0.57 μM), SipT (5.7 μM), and SipV (5.7 μM) singly (data not shown) or in combination in reaction buffer at pH 10.0. Lane 1, molecular size markers; lanes 2 and 5, PhrA peptide alone; lanes 3 and 4, PhrA peptide and SipS, SipT, and SipV; lanes 6 and 7, SipS, SipT, and SipV.