A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

Abstract

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

Figure 1

Specificity of Rap phosphatase inhibition by Phr pentapeptides. Inhibition of Rap phosphatase activity by the Phr peptides results in decreased dephosphorylation of Spo0F∼P. Reactions in lane 1 are the control containing KinA (0.1 μM) and Spo0F (10 μM) only, whereas lane 2 shows the level of dephosphorylation obtained by RapA (5 μM) (A and C) or RapB (5 μM) (B and D). Reactions were carried out as described. (A) Increasing concentrations of PhrApep5 result in decreased dephosphorylation of Spo0F∼P by RapA. PhrApep6 also shows some inhibition but at extremely reduced efficacy. PhrApep5 was added at 10, 20, 30, 40, 50, 100, and 200 μM, in lanes 3–9, respectively; PhrApep6 was added at 10, 50, 100, and 200 μM in lanes 10–13, respectively. (B) PhrApep5 does not inhibit RapB activity. PhrApep5 was added at 10, 30, 50, 100, and 200 μM in lanes 3–7, respectively. (C) PhrCpep5 does not inhibit RapA activity. PhrCpep5 was added at increasing concentrations as in B. (D) PhrCpep5 moderately inhibits Spo0F∼P dephosphorylation by RapB. Increasing concentrations of PhrCpep5 (lanes 3–9) and PhrCpep6 (lanes 10–13) were added as in A.

Figure 2

In vivo complementation of a phrA mutant (JH12954) (open symbols) by exogenously provided Phr peptides. Sporulation assays were performed by the Sterlini and Mandelstam resuspension method (23). Peptides were added at the concentration indicated. □, PhrApep5; ○, PhrApep6; ▪, JH642.

Figure 3

The spo0L892 mutant form of RapA (P259L) is insensitive to PhrA peptide inhibition. Phosphorylation of Spo0F (10 μM) was carried out as in Fig. 1 (lane 1) and as described in Materials and Methods. Purified RapA892 was used at 5 μM final concentration and added to the reaction in the absence (lane 2) or presence of pentapeptides at 10, 20, 30, 40, 50, 100, and 200 μM final concentration (lanes 3–9, respectively) or exapeptide at 10, 50, 100, and 200 μM (lanes 10–13, respectively). (A) PhrApep5 (lanes 3–9) or PhrApep6 (lanes 10–13). (B) PhrCpep5 (lanes 3–9) or PhrCpep6 (lanes 10–13).

Figure 4

Phr peptide sequence-dependent specificity for target recognition. (A) Amino acid sequence of PhrA and PhrC pentapeptides and their modified forms obtained by single amino acid replacement at position 1, 3, and 4 of PhrApep5 with the corresponding residue of PhrCpep5 and vice versa. The ability of the mutant peptides to inhibit RapA (B) or RapB (C) phosphatase activity (5 μM) on Spo0F∼P (10 μM) was tested in the standard reaction conditions described in Materials and Methods. Peptides were all used at 200 μM final concentration. The control level of Spo0F phosphorylation is shown in lane 1 whereas the dephosphorylation of Spo0F∼P by RapA or RapB is in lane 2. Lanes 3–10 contain the peptides in the order indicated by the numbers in parentheses.

Figure 5

In vivo assay of modified pentapeptides. Sporulation assays were carried out with the resuspension method of Sterlini and Mandelstam (23). (A) Strain JH12954 carrying a deletion of the PhrA coding gene was resuspended in the sporulation medium in the presence of peptides at 10 μM final concentration. The wild-type level of sporulation is given by strain JH642 (lane 1), whereas strain JH12954 grown in the absence of peptides is shown in lane 2. PhrApep5, PhrCpep5, pep 878, pep 879, pep 880, pep 881, pep 882, and pep 883 were added to the cultures represented in lanes 3–10, respectively, and the number of spores per ml is shown by the bars. (B) Strain JH642 carrying the rapB multicopy plasmid pIPB213A was grown in the presence of 5 μg/ml chloramphenicol (Cm). The wild-type control strain was JH642 carrying the multicopy vector pBS19 (lane 1). Lane 2 shows the level of sporulation of JH642/pIPB213A in the absence of peptides, whereas lanes 3–10 show the number of spores per ml obtained by the addition of peptides (10 μM) as in A.

Figure 6

Modulation of the phosphorelay by the PhrA peptide export–import circuit that regulates the RapA phosphatase activity. RapA is induced by ComA∼P and prevents sporulation during competence development by dephosphorylating Spo0F∼P. PhrA is first exported, processed, and then imported by the oligopeptide transport system as a pentapeptide. The PhrA pentapeptide directly inhibits RapA activity, thereby allowing sporulation to initiate.