Characterization of PrpC from Bacillus subtilis, a Member of the PPM Phosphatase Family

Abstract

We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins. The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.

Reversible phosphorylation of proteins plays an important role in a wide variety of cellular processes, including regulation of metabolic pathways, cell differentiation, and signal transduction (5, 14, 15). Signal transduction, involving phosphorylation of histidine and aspartate, is well established in bacteria; however, serine, threonine, and tyrosine residues are also major targets for reversible phosphorylation in bacteria as well as in eukaryotes (7). In eukaryotes, serine/threonine protein phosphatases can be classified into two major families, PPP and PPM, according to their structure, metal ion dependence, and sensitivity to inhibitors (6). Sequence comparison of the primary structures indicates that the minimal catalytic domain in the two families comprises a common core of about 220 and 290 amino acids, respectively (3, 17). PPM phosphatases are characterized by up to 11 motifs conserved in sequence and spacing, with 8 absolutely conserved motifs. A prominent member of the PPM family is the human enzyme protein phosphatase 2C (PP2C), which is implicated in reversing protein kinase cascades and is activated by environmental stress. This protein has recently been crystallized (8).

PP2C-like enzymes are known to catalyze the Mg²⁺- or Mn²⁺-dependent dephosphorylation of phosphoserine and phosphothreonine residues (4), and several bacterial proteins have previously been identified as members of this PPM family. In Bacillus subtilis, the PPM phosphatase SpoIIE plays a key role in the sporulation process, which is initiated by a combination of nutrient starvation, the state of the DNA replication cycle, and the population cell density. SpoIIE is a Mn²⁺-dependent PP2C-like serine phosphatase linked to the cytoplasmic membrane (1) and drives a partner-switching mechanism. This ultimately activates the ς^F transcription factor, once the asymmetrical sporulation septum has formed (9, 10). RsbU and RsbX are also PPM phosphatases which in vitro have been shown to dephosphorylate RsbV-P and RsbS-P. RsbV and RsbS each combine with a cognate Thr/Ser kinase in a module which controls the activity of transcription factor ςB, dependent upon the phosphorylation state of the module and therefore the opposing phosphatase/kinase activities (20). Recent studies by Vijay et al. (19) have also shown that the PPM phosphatase RsbP, previously designated YvfP, is involved in the energy stress response, through the dephosphorylation of the anti-anti-sigma factor RsbV, ultimately leading to the activation of ς^B.

Shi et al. (17) have identified an additional gene, yloO, encoding an apparent member of the PPM family. In this study, we have shown that the gene product of yloO, which we now designate PrpC, does indeed have a phosphatase activity in vitro, and we provide evidence that PrpC has properties characteristic of the PPM family.

Sequence analysis indicates that PrpC belongs to the PPM family.

Analysis of the amino acid sequence of PrpC deduced from the nucleotide sequence of the yloO gene (GenBank accession number Y13937) revealed similarity with prokaryotic and eukaryotic PPM phosphatases, of which a prominent member is the human phosphatase, PP2C (2). For instance, PrpC displays 36% identity with a phosphatase from Chlamydia trachomatis and 26% identity with a phosphatase from Schizosaccharomyces pombe (Fig. 1). The catalytic domain of the PPMs spans about 290 amino acid residues. The size of PrpC, calculated to be 27.5 kDa, suggests that it consists only of the catalytic domain, in contrast to other bacterial PPM family members such as, for example, the SpoIIE phosphatase or the RsbP phosphatase, which have more complex structures (see reference 17). Thus, SpoIIE contains an N-terminal membrane domain, whilst RsbP (YvfP), postulated to regulate phosphatase activity in response to energy stress (21), contains an N-terminal PAS domain, probably involved in protein interactions. The catalytic domain of all PPM molecules can be subdivided into 11 domains, including 8 that are absolutely conserved in all members of the family (17). All eight of these are present in PrpC and in other B. subtilis phosphatases, SpoIIE, RsbX, RbsU, and RsbP. Two additional motifs, Va and Vb, are also present in PrpC, but the role of these domains in the PPM phosphatases is not clear. Importantly, the positions of residues involved in binding metal ions and the phosphate group of substrates are perfectly conserved between PrpC and the human PP2C (see Fig. 1). These observations strongly suggest that PrpC is a serine/threonine protein phosphatase.

FIG. 1.

Comparison of PrpC with other PPM family members. PrpC was compared with eukaryotic members of the PPM family. Following the convention of Shi et al. (17), highly conserved amino acid residues are boldfaced. Residues involved in binding metal and phosphate ions are indicated by small filled and open arrows, respectively. BS, B. subtilis; SC, Saccharomyces cerevisiae ptc1 or tpd1; SP, S. pombe ptc1; HS, Homo sapiens PP2C; CT, C. trachomatis ct259. At the bottom of the figure, the long arrows connect motifs which are conserved.

Cloning the gene; overproduction and purification of PrpC.

For cloning prpC, chromosomal DNA from B. subtilis was used as template in PCR amplification to prepare the prpC gene with appropriate restriction sites at both ends. The sequences of primers 1 and 2, used for this construct, are shown in Table 1. The amplified fragment was digested with AflIII and BamHI and was ligated into the pET302 vector (kindly provided by Chris van der Does), encoding a six-residue His tag, immediately downstream of the ATG, previously opened with NcoI and BamHI. The AflIII and NcoI restriction enzymes in this case have compatible ends. The resulting plasmid, encoding an in-frame His-tagged version of PrpC plus an enterokinase cleavage site downstream of the six His residues, was designated pOMG700. Subsequent sequencing of the construct with an ABI Prism 310 automatic sequencer confirmed the nucleotide sequence of the inserted prpC gene and the additional 39 nucleotides at the 5′ end. This plasmid was used to transform competent cells from Escherichia coli DH5α. Transformants carrying pOMG700 were grown in Luria-Bertani (LB) medium at 30°C until an A₅₇₀ of ≈0.5 was reached. Isopropyl-β-d-thiogalactopyranoside (IPTG) (0.5 mM final concentration) was added, and incubation continued for 2.5 h. High levels of a protein migrating with a mobility equivalent to 37 kDa were detected when cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (Fig. 2). This is higher than the predicted molecular size (29 kDa), indicating that the protein runs aberrantly.

TABLE 1.

Sequences of the oligonucleotides used in this study

No.	Sequencea	Restriction site
1	5′-ATAATAacatgtTAACAGCCTTAAAAACAGATAC-3′	AflIII
2	5′-AAAAAAggatccCCCGCCGCCTATGACG-3′	BamHI
3	5′-TTGTccatggTAGCTTCTTGGGGTATC-3′	NcoI
4	5′-GCTTggtaccCATCTAAATCTAGG-3′	KpnI
5	5′-CCCGaagcttAAATGTCACAATCCG-3′	HindIII
6	5′-TGAGccatggTTAACTTTCCGAGGGCGC-3′	NcoI
7	5′-GCTTggtaccCAAGTTGAAGAGGG-3′	KpnI
8	5′-GTTTgaattcGCATGAGCGATGGC-3′	EcoRI
9	5′-AAATGGCaagcttAACAGATTCTATCGGTAAACAGCA-3′	HindIII
10	5′-CATAGAAggatccTGTCTCCGGGTTCTATATCAAAGG-3′	BamHI

^aRestriction sites in oligonucleotides are in boldface italics and are lowercased. The second codon of PrpC in oligonucleotide 1 is underlined. 

FIG. 2.

Overproduction and purification of PrpC. Proteins from E. coli DH5a(pOMG700) and purified PrpC were analyzed by SDS-PAGE and stained with Coomassie blue. Lane 1, proteins from E. coli DH5α(pOMG700) in the absence of IPTG; lane 2, proteins from E. coli DH5α(pOMG700) 2.5 h after induction by IPTG; lane 3, purified PrpC protein.

For purification of PrpC, the induced cells were harvested, resuspended in 10 ml of buffer A (20 mM HEPES [pH 8.0], 150 mM NaCl, 10 mM imidazole), and disrupted by sonication, and the cell-free supernatant was loaded onto a column containing immobilized Ni⁺² fast-flow chelating Sepharose (Pharmacia). After a wash with 65 mM imidazole, the protein was eluted by a linear gradient of imidazole (65 to 250 mM) as a single band (Fig. 2). This band was concentrated and stored at −70°C in 20 mM HEPES buffer (pH 8.0) containing 150 mM NaCl, 1 mM Mn²⁺, and 10% (vol/vol) glycerol. Under these conditions, about 1 mg of pure protein was obtained from 1 liter of bacterial culture.

Enzymatic activity of PrpC.

Since the sequence of PrpC indicated a relationship to PPM enzymes, it was of interest to analyze the enzymatic activity of the PrpC protein with regard to substrates that can be dephosphorylated. First, the purified protein was assayed for its ability to cleave p-nitrophenyl phosphate (PNPP). Indeed, PrpC efficiently hydrolyzed this synthetic substrate, with an optimum pH of 8.8, at 30°C, in 50 mM Tris-HCl. A characteristic of the PPM family is the requirement for Mn²⁺ or Mg²⁺. The structure of human PP2C, established from X-ray analysis, indicates that the residues involved in metal ion-catalyzed dephosphorylation are highly conserved and are present in the catalytic site. Thus, it has been proposed that Asp residues 38, 60, 239, and 282 (conserved in motifs I, II, CIII, and XI, respectively), together with Glu-37 and Gly-61, constitute a center for binding a bivalent metal ion. Metal-associated water molecules act as a nucleophile to attack the phosphorus atom by an S_N2 mechanism and also as a donor in order to protonate the seryl leaving group oxygen atom (8). Indeed, the enzyme displayed an optimum Mn²⁺ concentration of 0.5 mM (data not shown). In contrast, 1 mM Mg²⁺, Ca²⁺, K⁺, or Mo was found to inhibit the reaction. The enzymatic parameters K_m and V_max, measured at 37°C, were 2.3 ± 0.2 mM and 1.36 ± 0.06 μmol/min per mg of protein, respectively (using a molar extinction coefficient [ɛ₄₂₀] of 12,500 M⁻¹ cm⁻¹).

The properties of PrpC, as a member of the PPM phosphatase family, were confirmed by analyzing the effects of several inhibitors. Thus, ammonium molybdate, sodium orthovanadate, and okadaic acid, which do not inhibit PPM phosphatases, all failed to inhibit the phosphatase activity of PrpC. In contrast, PrpC was strongly inhibited by Ca²⁺, Zn²⁺, N-ethylmaleimide, and β-glycerolphosphate (50% inhibition at 400 μM and 0.1, 7, and 20 mM, respectively [data not shown]).

The phosphorylated form of PrkC is a substrate for PrpC.

Interestingly, prpC encoding the phosphatase is located immediately upstream of prkC (11). PrkC appears to be a membrane-linked protein kinase (our unpublished data), belonging to the Hanks superfamily (13). A similar organization of genes encoding a putative Ser/Thr phosphatase and a kinase is present in the Mycoplasma genitalium chromosome (12).

In our laboratory, PrkC was able to autophosphorylate in vitro on a threonine residue (unpublished data; not shown). An obvious candidate target for PrpC was therefore PrkC. The protein kinase was therefore first incubated under optimal conditions for autophosphorylation with [γ-³²P]ATP. The radiolabeled enzyme was then mixed with purified PrpC, and dephosphorylation of PrkC was observed (Fig. 3A). In particular, the results presented in Fig. 3B clearly indicate that, under these conditions, PrkC was extensively dephosphorylated by PrpC. Finally, the phosphatase activity of purified PrpC was tested by addition to a cell extract from B. subtilis labeled with [γ-³²P]ATP in vitro. In this case, we also observed dephosphorylation of an endogenous 67-kDa protein (data not shown).

FIG. 3.

PrpC dephosphorylates the phosphorylated form of PrkC. A 1.6-μg portion of purified His-tagged PrkC was autophosphorylated in vitro at 30°C for 10 min in 20 μl of buffer, comprising 10 mM HEPES (pH 8.0), 10 mM MgCl₂, 1 mM MnCl₂, 40 mM KCl, and 325 mM NaCl with 10 μCi of [γ-³²P]ATP (final concentration, 170 μM). Dephosphorylation of PrkC was carried out with 1 μg of purified PrpC at 37°C for appropriate times. The reaction was stopped by addition of 2 volumes of ice-cold acetone, the precipitate was washed, and the dried pellet was mixed with electrophoresis sample buffer, analyzed by SDS-PAGE, and visualized by using a phosphorimager and autoradiography. (A) Lane 1, time zero, no PrpC added; lane 2, 2 h of incubation at 37°C in the presence of 0.5 mg of PrpC; lane 3, 2 h of incubation at 37°C in the presence of 1 mg of PrpC; lane 4, 2 h of incubation at 37°C in the absence of PrpC. (B) Kinetics of dephosphorylation of PrkC by PrpC. Radioactivity indicated in lane 1 was taken as 100%. All values are given in relative units. Results were visualized in both cases with a phosphorimager, and band intensity was quantitated using ImageQuant software.

Expression of PrpC during the growth phase.

In order to obtain an indication of the role of PrpC in vivo, we examined when this gene was expressed. We checked for the presence of PrpC protein in cells during the growth phase, by Western blotting experiments, using a rabbit polyclonal antibody directed against purified PrpC (Fig. 4). An approximately 31-kDa polypeptide was clearly detected in exponentially growing cells in nutrient broth at 37°C, which appeared to remain a constant fraction of cell mass throughout the growth phase, and the protein was stable at least into early-stationary phase. This polypeptide runs at a position higher than the predicted value of 27.5 kDa. However, importantly, in the control in which prpC was specifically deleted, this band was absent. For the preparation of this deletion, we cloned the kanamycin resistance gene (PCR fragment obtained with primers 3 and 4 [Table 1]), surrounded by the two flanking regions upstream (using primers 5 and 6) and downstream (using primers 7 and 8) of prpC, into pMTL20, which was kindly provided by Claude Bruand. B. subtilis W168 was then transformed with the plasmid with selection for Kan^R involving a double crossover.

FIG. 4.

Expression of PrpC during the growth phase. Samples for Western blotting were taken at times indicated by arrows (A) and were blotted against a rabbit polyclonal antibody to purified PrpC and developed with an ECF kit (Amersham-Pharmacia). (B) Lanes 1 to 4, extract from wild-type bacteria in the logarithmic-growth phase; lanes 5 and 6, extract from wild-type bacteria in stationary phase; lane 7, extract from a strain in which prpC was deleted.

Investigating the possible regulation of prpC expression in relation to stress conditions.

The expression of prpC was monitored by use of a transcriptional fusion of lacZ to prpC obtained using PCR primers 9 and 10 (Table 1), by the general procedure described previously (18). The results indicated that, under normal growth conditions, only low levels of PrpC are synthesized as measured by β-galactosidase activity (16). Since some of the previously studied members of the PPM family in B. subtilis were implicated in stress responses, we examined the expression of prpC under different conditions, using the lacZ fusion. Cells grown in nutrient broth at 37°C were exposed to mitomycin (to induce the SOS response), to osmotic or oxidative shock conditions, to ethanol, or to acidic pH, but no significant change in the expression of the lacZ fusion could be detected. Similarly, expression of the fusion was not induced when cells grown in Spizizen medium were starved for glucose. The expression of prpC does not, therefore, appear to be coregulated with these major stress responses. We also examined the expression of prpC after a shift from 30 to 45°C by directly analyzing the amount of PrpC, using Western blotting. The results showed no induction. However, this does not rule out the possibility that PrpC is essential for certain major stress responses, but that the basal level of activity is sufficient, as was found for other PPM phosphatases; this is now under investigation.

Since prpC and prkC are encoded by adjacent genes and the kinase can be dephosphorylated by PrpC, whatever the role of PrpC, we anticipate that this phosphatase and this protein kinase are likely to form a functional couple in vitro.