Research Article | Spotlight

Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli

Krithika Rajagopalan, Elizabeth Nagle, Jonathan Dworkin
Ann M. Stock, Editor
DOI: 10.1128/JB.00225-18

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ABSTRACT

In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys over the past decade in phylogenetically diverse bacteria have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes, such as that of Escherichia coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases, which have extensive sequence and structural homology to eukaryotic Ser/Thr protein phosphatase 2C (PP2C) phosphatases. These proteins, called eukaryote-like Ser/Thr phosphatases (eSTPs), have been identified in a number of bacteria but not in E. coli. Here, we describe a previously unknown eSTP encoded by an E. coli open reading frame (ORF), yegK, and characterize its biochemical properties, including its kinetics, substrate specificity, and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely related E. coli strains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase that is encoded in the same operon.

IMPORTANCE Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacterium Escherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported in E. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

INTRODUCTION

Reversible protein phosphorylation is an important regulatory mechanism in eukaryotes and prokaryotes (1). In eukaryotes, signaling phosphorylation typically occurs on serine, threonine, or tyrosine residues, and it is mediated by the combined action of kinases and phosphatases. In prokaryotes, signaling phosphorylation has been thought to occur largely on histidine and aspartate residues, mediated by histidine kinases of two-component systems (2). However, mass spectrometry-based phosphoproteomic analyses over the past decade have identified numerous Ser/Thr/Tyr-phosphorylated proteins in many bacteria (3–5), including Escherichia coli (6–9). Some of these phosphoproteins and the specific phosphosites are conserved in divergent species (7), suggesting that this regulation may be physiologically relevant.

Ser/Thr kinases from phylogenetically diverse bacteria have been described (5). For example, in E. coli, YeaG plays a role in nitrogen starvation (10), YihE is involved in the Cpx stress response (11) and cell death pathways (12), and HipA regulates bacterial persister formation by phosphorylating a tRNA synthetase (13, 14). However, both the authentic in vivo substrates of these kinases and/or their proximal activating stimuli are largely uncharacterized, complicating efforts to understand their precise physiological roles.

Phosphorylation on serine or threonine residues is more stable than phosphorylation on histidine or aspartate residues, and it is subject to additional regulation by Ser/Thr phosphatases. Analysis of phylogenetically diverse bacterial genomes revealed the presence of genes encoding proteins (15–17) that bear significant resemblance to eukaryotic Ser/Thr protein phosphatase 2C (PP2C) phosphatases (17, 18); hence, they are referred to as eukaryote-like Ser/Thr phosphatases (eSTPs). Some of these proteins have been characterized biochemically and structurally, with these studies confirming their general similarity to their eukaryotic counterparts. Eukaryotic Ser/Thr protein phosphatases are divided into two classes, phosphoprotein phosphatases (PPPs) and metal-dependent protein phosphatases (PPMs), according to structure, presence of signature motifs, metal ion dependence, and sensitivity to inhibitors (19). PPMs require Mg2+/Mn2+ to mediate dephosphorylation of phosphoserine or phosphothreonine residues. A well-studied member of the PPM family is human PP2C (20) which bears a striking resemblance to bacterial PPMs. PPM/PP2C phosphatases are characterized by the presence of 11 signature motifs with 8 absolutely conserved residues (17, 19).

While several bacterial PPM-type phosphatases have been biochemically characterized (21–28), PPM-type phosphatases have not been identified in E. coli, despite strong evidence of Ser/Thr phosphorylation. Here, we characterize a previously undescribed open reading frame (ORF), yegK, which is present in both E. coli B and K strains. This ORF encodes an ∼28-kDa protein that bears sequence similarity to PP2C-type phosphatases. We designate this gene pphC and its protein product as PphC. Recombinant PphC was purified and its enzymatic properties characterized. Despite some differences in sequence conservation compared to other bacterial eSTPs, PphC resembles PP2C-type phosphatases in various biochemical assays. Furthermore, we show that PphC dephosphorylates autophosphorylated YegI (a previously undescribed Ser/Thr kinase), which is encoded in the same operon as that containing pphC. To our knowledge, this is the first report of the identification and biochemical characterization of an E. coli PP2C-like phosphatase.

RESULTS

YegK is an atypical PP2C-like phosphatase.To identify PP2C-like phosphatases in E. coli, we performed a homology-based BLAST search using Bacillus subtilis PrpC, a well-characterized PP2C-like phosphatase (26), as the query. This analysis revealed a previously uncharacterized 762-bp ORF, yegK, which encodes a putative 253-amino acid polypeptide. Sequence analysis and domain prediction of YegK revealed that amino acids 11 to 232 have homology to a PP2C domain (see Fig. 2A). PP2C-like phosphatases include 11 conserved signature motifs (17, 29). Multiple-sequence alignment of YegK with known PP2C-like phosphatases from other Gram-negative bacteria shows presence of these 11 motifs but with low overall sequence similarity. However, unlike other bacterial PP2C homologs (22, 23, 25, 28, 30), YegK contains only six of the eight absolutely conserved residues that are involved in metal binding, coordination, and catalysis (Fig. 1). In particular, the amino acid sequence alignment clearly shows that YegK lacks the conserved glycine residue in motif VI and the aspartic acid residue in motif VIII (Fig. 1).

FIG 1
FIG 1

YegK is an atypical PP2C-like phosphatase. Amino acid sequence alignment of YegK with bacterial PP2C-like phosphatases. YegK (Escherichia coli) was aligned to PP2C homologs from Gram-negative bacteria using T-Coffee (51) and BoxShade. Identical residues are shaded in black, and similar residues are shaded in gray. The eight absolutely conserved residues found in bacterial PP2Cs are depicted with asterisks. The conserved residues absent in YegK are indicated by arrows. Signature motifs seen in most bacterial PP2Cs are denoted as roman numerals, based on the work of Shi et al. (17). The aspartic acid residue involved in metal binding is depicted with a triangle. Ec, Escherichia coli YegK; Te, Thermosynechococcus elongatus tPphA; Pa, Pseudomonas aeruginosa Stp1; Mx, Myxococcus xanthus Pph1; Se, Salmonella enterica serovar Typhi PrpZ (aa1-260); Ct, Chlamydia trachomatis CTL0511 (Cpp1).

A predicted structure of YegK using the Phyre2 algorithm (see Fig. S1 in the supplemental material) closely resembles the published structure of the bacterial PP2C-like phosphatase PphA from Thermosynechococcus elongatus (31), consisting of β sheets and a catalytic core in the center, surrounded by exterior alpha helices. In addition, yegK is located immediately upstream of yegI, an ORF which encodes a protein with homology to eukaryote-like Ser/Thr kinases. This is consistent with the observation that bacterial Ser/Thr kinases and phosphatases are often located in operons (5). Taken together, these observations suggest that, despite the absence of two highly conserved residues, yegK likely encodes a PP2C-like phosphatase.

Biochemical characterization of YegK.To demonstrate that yegK encodes a functional protein phosphatase, the 762-bp fragment was cloned in frame with the coding sequence for an N-terminal six-histidine tag into the pBAD24 vector (32). The plasmid was transformed into E. coli C43 (DE3), and following protein expression, the cell lysate was subjected to affinity purification using Ni2+-nitrilotriacetic acid (Ni-NTA) resin and subsequent analysis by SDS-PAGE. The protein migrated at an apparent molecular mass of ∼25 kDa, similar to the calculated molecular mass of 28.18 kDa (see Fig. S2 in the supplemental material). The phosphatase activity of purified YegK was determined using an absorbance-based assay that measures the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. Formation of p-nitrophenol detected at an absorbance of 405 nm is directly proportional to the phosphatase activity, which is expressed as nanomoles of p-nitrophenol (pNP) formed per microgram protein. Consistent with the alignment (Fig. 1), YegK displayed a time-dependent increase in phosphatase activity (Fig. 2A), suggesting that it is an active phosphatase. We have therefore renamed YegK PphC (protein phosphatase C), following the nomenclature of two previously characterized E. coli protein phosphatases, PphA and PphB (33), and the bacterial PP2C-like phosphatase PphA from T. elongatus (31).

FIG 2
FIG 2

YegK is an active phosphatase. (A) Domain architecture of YegK. Metal-binding site D46 is indicated. (B) Assessment of phosphatase activity of YegK using pNPP as the substrate. Reactions were performed at 37°C for 60 min with 350 nM phosphatase (WT/D46N mutant) and 5 mM pNPP substrate in phosphatase assay buffer (20 mM Tris [pH 8.0] and 5 mM MnCl2). (C) Size exclusion chromatography profile of WT and D46N YegK. His6-tagged protein (10 nmol) was loaded on a Superdex 75 size exclusion column and eluted in 20 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT, and 1 mM MnCl2.

To further confirm the bioinformatic identification of PphC as a PP2C-like phosphatase, the aspartic acid residue (D46) in motif II was mutated to asparagine. A similar approach was used in the analysis of the PP2C-like phosphatase Cpp1 from Chlamydia trachomatis (22). The mutant protein (PphC-D46N) was purified as above, and phosphatase activity was compared to that of wild-type PphC. PphC-D46N displays no hydrolysis of p-nitrophenol phosphate (pNPP), suggesting that the aspartic acid residue is essential for catalytic activity (Fig. 2B). To ensure that the loss of activity of PphC-D46N was not a consequence of improper folding, PphC and PphC-D46N were subjected to size exclusion chromatography. The gel filtration elution profile shows that PphC-D46N eluted as a single peak and at the same retention volume as PphC, indicating that the loss of phosphatase activity observed with the PphC-D46N mutant is most likely due to a loss of catalytic activity (Fig. 2C).

PP2C phosphatases belong to the PPM family of metal-dependent Ser/Thr phosphatases that require either Mg2+ or Mn2+ for activity (19, 29). The requirement for a divalent cation for PphC phosphatase activity was assessed by measuring hydrolysis of pNPP in the presence of MgCl2, MnCl2, CaCl2, ZnCl2, or NiCl2. Since pNPP hydrolysis was observed only in the presence of MnCl2 but not in that of MgCl2, CaCl2, ZnCl2, or NiCl2, PphC is a Mn2+-dependent phosphatase (Fig. 3A; see Fig. S3 in the supplemental material). This result is consistent with the requirement for Mn2+ ions for previously characterized bacterial PP2Cs (21–25, 27). The concentration dependence on Mn2+ of PphC phosphatase activity was measured, and the optimal MnCl2 concentration was determined to be between 1 to 2 mM (Fig. 3B).

FIG 3
FIG 3

PphC (YegK) is a Mn2+-dependent PP2C-like phosphatase. (A) Metal dependency of PphC phosphatase was tested using pNPP as the substrate. Reactions were carried out at 37°C for 60 min in buffer containing 350 nM phosphatase and 5 mM MgCl2, 5 mM MnCl2, or both, with 5 mM pNPP substrate. (B) Effect of Mn2+ concentration on PphC catalytic activity using pNPP as a substrate. Reactions were carried out at 37°C for 30 min in assay buffer containing 350 nM phosphatase, 5 mM pNPP substrate, and different concentrations of MnCl2.

The effect of different classes of protein phosphatase inhibitors on PphC phosphatase activity was tested using pNPP as a substrate (Table 1). PphC activity dramatically decreased in the presence of the general protein phosphatase inhibitor sodium phosphate, and it was slightly affected by sodium fluoride (∼30% decrease at 100 mM). Okadaic acid, a known inhibitor of protein phosphatase 1 (PP1) family of phosphatases (34), did not inhibit PphC activity. PphC activity was also unaffected by sodium orthovanadate, a known tyrosine phosphatase inhibitor. Aurin tricarboxylic acid and 5,5′ methylene disalycilic acid, which were previously reported to inhibit Staphylococcus aureus Stp1 (35, 36), had little effect on PphC phosphatase activity. Similarly, sanguinarine chloride, an inhibitor of human PP2Cα (37), did not affect phosphatase activity. However, an ∼60% decrease in activity was detected in the presence of the bivalent metal chelator EDTA, confirming that PphC is a metal-dependent phosphatase. Together, these data are consistent with the characterization of PphC as a PP2C-like phosphatase.

View this table:
TABLE 1

Inhibitor effects on PphC activitya

PphC phosphatase activity is different in closely related E. coli strains.We identified striking differences in the genetic architecture surrounding the pphC (yegK) locus in E. coli K and B strains. Specifically, in the E. coli B strain REL606, pphC (yegK) is immediately upstream of yegI, an ORF encoding a putative Ser/Thr kinase, whereas in the E. coli K strain MG1655, yegJ, a putative ORF, is located between the pphC (yegK) and yegI genes, facing the opposite direction (Fig. 4A). This different genomic organization is conserved in other K and B strains, suggesting that it predates the divergence of these lineages (38). A reasonable prediction is that pphC and/or yegI expression would be affected by the presence of the divergently oriented yegJ, but as we have not identified conditions under which we can robustly detect pphC expression, it has not been possible to evaluate this prediction.

FIG 4
FIG 4

PphC phosphatase activity differs in closely related E. coli strains. (A) Genetic map of yegK/yegI operon from E. coli B strain REL606 and K strain MG1655; arrows denote ORFs. The genes yegL, yegK, and yegI encode a von Willebrand factor type A, a PP2C-like phosphatase, and a eukaryote-like Ser-Thr kinase, respectively. The gene mdtA encodes a subunit of a multidrug efflux pump, and yegD encodes an actin family protein. The yegJ gene encodes a protein of unknown function. (B) Amino acid sequence alignment of PphC from E. coli B strain REL606 and K strain MG1655. Identical residues are shaded in black, and similar residues are shaded in gray. Signature motifs seen in bacterial PP2C phosphatases are depicted as Roman numerals based on the work of Shi et al. (17). The aspartic acid residue involved in metal binding is conserved and is indicated by a triangle. The conserved residues found in bacterial PP2Cs are depicted with arrows. (C and D) Enzyme kinetics of PphC. Substrate-dependent activity was assessed using different concentrations of pNPP (0.1 to 6.4 mM) in assay buffer containing 350 nM phosphatase and 5 mM MnCl2. Data were fitted to a Michaelis-Menten curve (C) and a Lineweaver-Burk plot (D). (E) The Lineweaver-Burk plot was used to determine Km, Vmax and kcat values.

In addition to this difference in the genetic architecture around the pphC locus, multiple-sequence alignment of pphC from a K strain (MG1655) and a B strain (REL606) revealed that only ∼92% of the PphC sequence is conserved (Fig. 4B). This is in contrast to the extremely high degree of sequence identity typically observed for the E. coli proteome; that is, more than half of the proteins in MG1655 have 100% sequence identity with the corresponding proteins in REL606 (38). To determine whether these differences in amino acid sequence affect phosphatase activity, the gene product of yegK from E. coli MG1655 was overexpressed and purified by the same method used to purify REL606 PphC. The enzyme kinetics of the two proteins was compared using the pNPP assay (Fig. 4C). Phosphatase activity (picomoles of pNP per minute per microgram) was determined with increasing concentrations of pNPP and Km and Vmax values were calculated (Fig. 4D). Since the Km and Vmax of Rel606 PphC is lower than those of MG1655 PphC (Fig. 4E), it reaches a maximum velocity at a lower substrate concentration. Similarly, the kcat values were calculated to be 0.2089 s−1 and 0.093 s−1 for REL606 and MG1655 PphC, respectively. Previously reported kinetic values for known bacterial PP2Cs range from 0.35 mM to 5.7 mM pNPP for Km, 0.1 to 4.5 μmol/min/mg for Vmax and 0.1 to 7.4 s−1 for kcat (22–24, 26, 27, 39–43), indicating that PphC has relatively low phosphatase activity in vitro compared to that of previously characterized bacterial PP2C-like phosphatases.

Substrate specificity of PphC.The results of the pNPP assay suggested that PphC has phosphatase activity. Therefore, we investigated whether PphC is capable of dephosphorylating a protein substrate. β-Casein is phosphorylated on five serine residues at the N terminus (44) and was used as a model protein in our assay. Using Mn2+-Phos-tag–SDS-PAGE to monitor phosphorylation state (45), untreated β-casein migrated at an apparent molecular mass of 30 kDa (Fig. 5A, lane 1), but β-casein that had been preincubated with PphC in MnCl2 buffer exhibited a mobility shift (Fig. 5A, lane 2). Since such a change is indicative of a loss in phosphorylation, PphC likely dephosphorylated the serine residues of β-casein. Furthermore, this mobility shift was not detected following incubation of β-casein with the catalytic mutant PphC-D46N (Fig. 5A, lane 4). Dephosphorylation of β-casein by PphC and not PphC-D46N was also observed in MgCl2 buffer (see Fig. S5 in the supplemental material). However, the extent of dephosphorylation was 2-fold lower in comparison to that of MnCl2 buffer. These results indicate that PphC is capable of acting as a serine phosphatase in a Mn2+- or a Mg2+-dependent manner.

FIG 5
FIG 5

Substrate specificity of PphC. (A) Effect of PphC on phospho-β-casein as a substrate. Dephosphorylation reactions were carried out at 37°C for 60 min in buffer containing 50 mM Tris (pH 8), 10 mM MnCl2, 4 μg phosphorylated β-casein, and 1.5 μg (9 μM) phosphatase. Molecular weights are indicated on the left in kilodaltons, (B) Effect of PphC on YegI kinase. Dephosphorylation reactions were carried out at 37°C with 2 μM phosphorylated YegI and 4 μM PphC in reaction buffer, as described in Materials and Methods. Reactions were stopped at t = 45 min and run on 12% SDS-PAGE followed by autoradiography. (C) Percent relative 32P incorporation was calculated by densitometry analysis using FIJI software. Data represent the mean ± standard error (SE) from five independent experiments. Statistical analysis used an unpaired t test (***, P value < 0.0001).

The substrate specificity of PphC was further examined using commercially available phosphopeptides. Previously characterized PP2Cs have demonstrated preferential specificity to phosphoserine/threonine peptides over phosphotyrosine peptides (22, 23, 30, 40). Phosphatase assays were performed with phosphoserine RRA(pS)VA, phosphothreonine KR(pT)IRR and phosphotyrosine RRLIEDAE(pY)AARG peptide substrates (see Fig. S4A in the supplemental material). In MnCl2 buffer, PphC released 2-fold and 6-fold more phosphate in the presence of the phosphotyrosine peptide compared to that released by the phosphothreonine peptide and phosphoserine peptide, respectively. In comparison to activity in MnCl2 buffer, PphC activity in MgCl2 buffer was substantially lower toward all phosphopeptides, further confirming that PphC is a Mn2+-dependent phosphatase. To confirm that the phosphopeptides are capable of being dephosphorylated, we used a known PP2C-like phosphatase (B. subtilis PrpC) as a positive control. As expected, PrpC dephosphorylated both serine and threonine residues and had minimal activity to tyrosine (Fig. S4B). Thus, since PphC had overall minimal activity on phosphopeptides in comparison to PrpC, they may not be ideal substrates for PphC.

Identification of a PphC substrate.Bacterial PP2C-like phosphatases are often present in the same operon as a Ser/Thr kinase (5) and the kinase is often itself a substrate of the phosphatase (25–27, 46). As noted above, pphC (yegK) is located immediately adjacent to yegI, a 1,947-bp ORF encoding a 648-amino-acid protein with homology to eukaryote-like Ser/Thr kinases (Fig. 4A) (5). Thus, to examine if YegI could serve as a substrate for PphC, autophosphorylated YegI kinase (Fig. 5B, lane 1) was incubated in the presence of wild-type or D46N mutant PphC and assayed for loss of phosphorylation. While wild-type PphC dephosphorylates YegI kinase, as indicated by the loss of the radiolabeled band (Fig. 5B, lane 2, and C), this effect is not seen following incubation with the catalytic mutant PphC-D46N (Fig. 5B, lane 3, and C).

In these assays, a phosphorylation product of ∼25 kDa, corresponding to PphC-D46N, was observed (Fig. 5B, lane 3), suggesting that YegI could also phosphorylate PphC. However, we have been unable to demonstrate any effect of this modification on PphC activity using either the pNPP or the β-casein dephosphorylation assays (data not shown). Interestingly, while PphC-D46N is phosphorylated, phosphorylation of wild-type (WT) PphC is not observed (Fig. 5B, lane 2), suggesting that either PphC can dephosphorylate itself or that inactivation of the YegI kinase by PphC could result in decreased phosphorylation of PphC-WT.

DISCUSSION

Mass spectrometry-based phosphoproteomic analysis has revealed that many Ser/Thr/Tyr residues undergo phosphorylation in phylogenetically diverse species. In most cases, the kinases and phosphatases responsible for these modifications have not been identified, although the presence of homologs of eukaryotic Ser/Thr kinases and phosphatases in most (if not all) bacterial species suggests that they could play a role. These so-called eukaryote-like Ser/Thr kinases and their partner PP2C-like Ser/Thr phosphatases have extensive structural and biochemical similarity with their eukaryotic counterparts (5). In E. coli, extensive Ser/Thr phosphorylation has been observed, with several studies reporting >75 phosphorylated proteins (6–9). However, the kinases and phosphatases responsible for making or removing these modifications are not known. Here, we describe biochemical analysis of PphC, a protein product of a previously undescribed E. coli ORF, yegK, which encodes the first reported PP2C-like Ser/Thr phosphatase in E. coli.

PphC contains all of the 11 conserved motifs present in PPM/PP2C phosphatases (16, 17, 19, 29). However, unlike bacterial PP2C-like phosphatases from other Gram-negative bacteria, PphC has only 6 out of the 8 absolutely conserved residues involved in metal coordination and catalysis (31, 43, 47). Specifically, PphC lacks a conserved glycine residue in motif VI and a conserved aspartate residue in motif VIII (Fig. 1A). The aspartate residue in motif VIII is important for metal ion coordination in bacterial PP2C-like phosphatases (31, 43, 47). Despite these differences in amino acid sequence, PphC effectively hydrolyzed the chromogenic substrate pNPP, suggesting that the requirement of all 8 residues as a criterion for assessing the likelihood that an ORF encodes a PP2C-like phosphatase may be too stringent.

The regulation of PP2C-like phosphatases has been studied in a number of contexts, including Mycobacterium tuberculosis PstP, whose activity is stimulated by phosphorylation on multiple Thr residues by two eukaryote-like Ser/Thr kinases (48). Although we observed that PphC is phosphorylated by its partner Ser/Thr kinase, YegI (Fig. 5B), we were unable to detect any effect on PphC activity. Another regulatory mechanism occurs in the B. subtilis SpoIIE protein, a PP2C-like phosphatase that plays an essential role during sporulation. A single residue in SpoIIE (Val-697) mediates an alpha-helical switch that changes the coordination of a metal ion in the active site and thereby activates the phosphatase (49). However, since this residue is not conserved in PphC, a similar regulatory mechanism is probably not operating in the context of PphC function.

Bacterial PP2C-like phosphatases are known to dephosphorylate phosphorylated serine/threonine (pSer/pThr)-containing peptides (22–24). PphC was initially identified by a homology search using B. subtilis PrpC, but unlike PrpC (Fig. S4B), PphC displayed low preference for pSer/pThr/pTyr peptides (Fig. S4A). This minimal activity against phosphopeptides is in contrast to the ability of PphC to dephosphorylate the pSer-containing protein substrate β-casein. A possible explanation is that PphC may require additional residues for substrate recognition and/or binding that are not present in the phosphopeptides. However, this is not likely to be a sufficient explanation, as β-casein is a generic substrate that would not be expected to contain specificity determinants for PphC. Alternatively, this result suggests that there may be limits to using phosphopeptide dephosphorylation as an accurate assay of PP2C-like phosphatase function.

Bacterial eukaryote-like Ser/Thr kinases and PP2C-like phosphatases are often encoded in the same operon, and in many cases the kinase is a substrate for the phosphatase (21, 25–27). Similarly, in the E. coli B strain REL606, the ORF yegI encodes a putative eukaryote-like Ser/Thr kinase and is located immediately downstream of pphC. Our data demonstrate that YegI is a substrate of PphC (Fig. 5C). However, in E. coli K strain MG1655, there is a putative intervening ORF, yegJ, that is located between the yegI and pphC genes and is transcribed divergently (Fig. 4A), suggesting potential regulatory differences between the two strains. We have been unable to observe transcription of the pphC locus under any conditions, so we do not know if yegJ has an effect on expression of this locus. Interestingly, mutations in yegI, the gene encoding the partner kinase of PphC, repeatedly emerge in long-term evolution experiments (50), suggesting that this kinase/phosphatase pair may have significant fitness effects. Consistently, the presence of the potentially disruptive yegJ locus in the K lineage may be also reflect these effects. However, at present, in the absence of a deeper understanding of the physiological role of YegI/PphC pair, these effects remain mysterious.

In addition to the differences in the genome organization around pphC in the E. coli B and K lineages, the amino acid sequence of PphC differs between the two strains, with several nonconservative substitutions. This observation is intriguing, given that half of the proteome is identical between these two strains (38). Although these differences are not in residues that are absolutely conserved among bacterial PP2C-like phosphatases (31, 43, 47), they may have functional consequences, since the two proteins exhibited modest differences in enzyme kinetics (Fig. 4C to E). Future work will be aimed at identifying the impact of specific substitutions in the residues that differ in the relative activity of the different PphC alleles.

Finally, PP2C phosphatases play important roles in cellular regulation in more complex systems, including those of mammals (19). One issue in investigating PP2C function in these in vivo contexts is that specific chemical inhibitors do not exist. Thus, bacterial homologs, such as PphC, may be useful in identifying cell-permeable inhibitors, both because of the technical tractability of the organism and the presence of only a single PP2C phosphatase gene in the genome.

In summary, our study provides the first evidence for the existence of a PP2C-like phosphatase in E. coli. Despite some differences in sequence conservation compared to other PP2Cs, PphC is an active PP2C-like phosphatase, albeit with lower Km and Vmax values than those of other bacterial PP2Cs. Future studies will be required to identify physiological substrates of PphC and to ascertain its physiological role in vivo. We expect that further characterization of PphC's partner kinase, YegI, will greatly facilitate these efforts.

MATERIALS AND METHODS

Bacterial strains and growth conditions.E. coli DH5α cells were used for regular cloning, and C43(DE3) and LOBSTR (BL21-DE3) strains were used for expression of recombinant proteins. E. coli cells were grown in LB Lennox broth supplemented with ampicillin (100 μg/ml) at 37°C with shaking (220 rpm) unless otherwise specified. Details of strains, plasmids, and primers used in the study are described in Tables S1, S2, and S3, respectively, in the supplemental material.

Cloning and expression of YegK (PphC).Genomic DNA from E. coli REL606 and MG1655 was isolated using a Wizard genomic DNA purification kit (Promega) following the manufacturer's instructions. The E. coli yegK (pphC) gene was PCR amplified using Phusion polymerase (Thermo Scientific) from REL606 genomic DNA using primers KR38/KR39. The sequence for an N-terminal His6 tag was included in the primer. The PCR product was digested with NcoI/PstI and ligated into a similarly digested pBAD24 plasmid backbone. Ligation products were transformed into DH5α cells and selected on LB-ampicillin plates. The resulting plasmid generated an N-terminal His6-tagged YegK (PphC) fusion protein.

For protein expression, all plasmids were transformed into C43DE3 cells and plated on LB ampicillin (100 μg/ml) agar plates. Single colonies were inoculated into 3 ml of LB supplemented with ampicillin (100 μg/ml) for overnight cultures. The next day, 400-ml cultures were diluted 1:250 and grown to an optical density at 600 nm (OD600) of 0.6 to 0.8. Recombinant protein was induced by addition of arabinose (0.2%, wt/vol) for 3 h at 25°C. Cells were harvested at 6,000 × g for 15 min at 4°C. Pellets were washed with ice-cold 50 mM EDTA and centrifuged at 7,000 rpm for 15 min at 4°C. Washed pellets were saved at −80°C until use.

Oligonucleotide site-directed mutagenesis.Point mutation of aspartic acid residue D46 was generated by two-step overlap PCR mutagenesis using primer pairs KR38/KR41 and KR40/KR39 with Phusion polymerase. A second PCR was performed with primers KR38/KR39, using primary PCR products as a template, and the subsequent PCR products were digested as above and ligated into pBAD24 to generate an N-terminal His6 tagged D46N YegK(PphC) fusion protein. Plasmid cloning was subsequently verified by restriction enzyme digest and DNA sequencing (Operon).

Purification of recombinant PphC.Frozen pellets were suspended in lysis buffer (20 mM Tris [pH 8.0], 250 mM NaCl, 30 mM imidazole, 10 mM β-mercaptoethanol, 0.2% Triton X-100, 10 mg/ml lysozyme, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated on ice for 30 min. Initial lysis was carried out by four cycles of freeze/thaw alternating a dry ice/ethanol bath and 37°C. Lysate was passed through a 22-gauge needle and added to prechilled 2-ml screw-cap tubes with 0.1-mm silica beads. Cells were lysed using a FastPrep-24 5G instrument (MP Biomedical) using 2 rounds of 6.5 m/s intensity for 40 s with 4 min incubation on ice between rounds. Lysates were cleared by centrifugation at 20,000 × g for 30 min at 4°C. Cleared lysates were incubated in 5-ml Pierce columns with Ni-NTA–agarose beads (Qiagen) at 4°C for 1 h. Lysate was allowed to flow through, and beads were washed with 10 column volumes of wash buffer (20 mM Tris [pH 8.0], 250 mM NaCl, 30 mM imidazole, and 10 mM β-mercaptoethanol). His6-tagged protein was eluted using increasing concentrations of imidazole (100 to 500 mM) in 20 mM Tris [pH 8.0] and 250 mM NaCl. Elution fractions were analyzed on a 10% SDS-PAGE gel, and fractions containing protein were pooled and dialyzed overnight at 4°C using a Slide-A-Lyzer mini-dialysis device, with a 10,000 (10K) molecular weight cutoff (MWCO) (Thermo Scientific), in phosphatase storage buffer (20 mM Tris [pH 8.0], 50 mM NaCl, 1 mM dithiothreitol [DTT], and 1 mM MnCl2). Dialyzed samples were concentrated in Amicon Ultra 10K centrifugal filters to 1 ml and then loaded onto a HiLoad 16/60 Superdex 75 prep grade (GE Biosciences) gel filtration column. The column was preequilibrated and run with phosphatase storage buffer. PphC elutes at a retention volume of ∼70 ml. Fractions containing PphC were pooled, concentrated, and assessed for purity using a 12% SDS-PAGE gel. The catalytic variant was purified using the same protocol. Proteins were stored at −80°C.

Cloning and expression of YegI.The yegI gene was PCR amplified using Phusion polymerase (Thermo Scientific) from E. coli REL606 genomic DNA using primers KR58/KR59. The sequence for an N-terminal His6 tag was included in the primer. The PCR product was digested with NcoI/SphI and ligated into a similarly digested pBAD24 plasmid backbone. Ligation products were transformed in DH5α cells and selected on LB-ampicillin plates. The resulting plasmid generated an N-terminal His6-tagged YegI fusion protein. Plasmid cloning was subsequently verified by restriction enzyme digestion and DNA sequencing (Operon). For protein expression, the plasmid was transformed in E. coli LOBSTR (Kerafast) cells and plated on LB-ampicillin (100 μg/ml) agar plates. Single colonies were inoculated into 3 ml of LB supplemented with ampicillin (100 μg/ml) for overnight cultures. The next day, a dilution of 1:250 in 500 ml of LB was grown to an OD600 of 0.6 to 0.8. Recombinant protein was induced by addition of arabinose (0.2%, wt/vol) for 3 h at 18°C. Cells were harvested at 6,000 × g for 15 min at 4°C. Pellets were washed with ice-cold 50 mM EDTA and centrifuged at 7,000 rpm for 15 min at 4°C. Washed pellets were saved at −80°C until use.

Purification of recombinant YegI.Frozen pellets were suspended in lysis buffer (50 mM Tris [pH 8.0], 200 mM NaCl, 10 mM β-mercaptoethanol, 1 mM PMSF, and 2% [wt/vol] Sarkosyl) and incubated at room temperature for overnight lysis. Cells were subsequently lysed using sonication. Lysates were cleared by centrifugation at 15,000 × g for 30 min at 4°C and incubated in 5-ml Pierce columns with Ni-NTA–agarose beads (Qiagen) at 4°C for 1 h. Lysate was allowed to flow through, and beads were washed with 10 column volumes of wash buffer (50 mM Tris [pH 8.0], 200 mM NaCl, 30 mM imidazole, 10 mM β-mercaptoethanol, and 0.05% [wt/vol] Sarkosyl). His tagged protein was eluted using 300 mM imidazole in 50 mM Tris (pH 8.0), 200 mM NaCl, 10 mM β-mercaptoethanol, and 0.05% (wt/vol) Sarkosyl. Elution fractions were tested on 12% SDS-PAGE gel, and fractions containing protein were pooled and dialyzed overnight at 4°C using Snakeskin dialysis tubing, 10K MWCO (Thermo Scientific), in kinase storage buffer (20 mM Tris [pH 8.0], 125 mM NaCl, 10% glycerol, and 1 mM DTT). Dialyzed protein was assessed for purity using 12% SDS-PAGE gel and stored at −80°C.

Phosphatase assays.The phosphatase activity of PphC (YegK) was determined by measuring hydrolysis of p-nitrophenol phosphate (pNPP) to p-nitrophenol using spectrophotometry. Assays were performed in triplicate in a 96-well plate. Each well contained 350 nM purified PphC (WT or mutant) in assay buffer (10 mM Tris [pH 8.0], 5 mM MnCl2). Reactions were initiated by addition of 5 mM pNPP (NEB), and absorbance measurements were recorded every 10 min at 405 nm for 60 min in a Tecan Infinite 200 plate reader. Amount of phosphate released is represented as nanomoles of pNP per microgram of protein, and the amount of pNP was calculated using an extinction coefficient of 18,000 M−1 · cm−1.

Metal dependence was determined by incubating 350 nM purified PphC in assay buffer containing either 5 mM MgCl2, 5 mM MnCl2, 5 mM CaCl2, 5 mM ZnCl2, 5 mM NiCl2, or no metal. Absorbance measurements were recorded as above. To determine optimal MnCl2 concentrations, reactions were carried out with 350 nM purified WT PphC in assay buffer with different MnCl2 concentrations (0 to 10 mM), and absorbance was measured at 405 nm.

Sensitivity to phosphatase inhibitors was determined by measuring phosphatase activity of purified PphC in the presence of the following inhibitors: sodium phosphate (Sigma), sodium fluoride (Sigma), okadaic acid (Cell Signaling), sodium orthovanadate (NEB) or EDTA (Macron Chemicals), sanguinarine chloride (Tocris), aurin tricarboxylic acid (Alfa Aesar), and 5,5′ methylene disalycilic acid (Acros Organics). Okadaic acid, sanguinarine chloride, aurin tricarboxylic acid, and 5,5′ methylene disalycilic acid were diluted in dimethyl sulfoxide (DMSO) to get a 1 mM stock. The remaining inhibitors were diluted to stock concentrations in sterile water (pH 8). Purified WT PphC (150 nM) was incubated in assay buffer containing indicated concentrations of inhibitor or DMSO-water for 5 min. Reactions were initiated by addition of 5 mM pNPP, and absorbance was recorded every 5 min for 15 min at 30°C in a Tecan Infinite 200 plate reader.

The kinetic parameters of PphC were determined by varying the pNPP concentration (0.1 to 6.4 mM) in a reaction with 350 nM purified wild-type PphC from either REL606 and MG1655 in assay buffer. Hydrolysis was monitored every 5 min for 30 min in the linear range of the reaction. Initial reaction velocities were calculated for every substrate concentration. To determine Km and Vmax values, the data were fitted to a Michaelis-Menten curve and a Lineweaver-Burk plot was derived using GraphPad Prism 7 software.

Synthetic phosphopeptides.To assess substrate specificity, 200 μM serine (RRApSVA), threonine (KRpTIRR), or tyrosine (RRLIEDAEpYAARG) phosphopeptides (Millipore) were incubated with 350 nM phosphatase in a 50-μl reaction mixture containing 20 mM Tris (pH 8.0) with either 5 mM MnCl2 or 5 mM MgCl2 for 30 min at 37°C. Phosphatase reaction was stopped by addition of Biomol Green reagent (Enzo). The reaction mixture was incubated at room temperature for 25 min to allow color development, and absorbance at OD620 was measured. Phosphate standard (Enzo) was used to calculate the amount of phosphate released.

β-Casein.Phosphorylated β-casein (Sigma) was dissolved in 50 mM Tris (pH 7.5) and 150 mM NaCl to a final concentration of 4 mg/ml. For the phosphatase assay, 4 μg of β-casein was incubated with 1.5 μg WT or D46N PphC in 20 μl of 50 mM Tris (pH 8.0) with either 10 mM MnCl2 or 10 mM MgCl2 at 37°C for 1 h. Reactions were stopped with 3× SDS loading dye, and samples were heat denatured at 95°C for 5 min. Samples were loaded on 10% SDS-PAGE gel containing 50 μM Phos-tag acrylamide according to the manufacturer's instructions (Wako). Gels were run at a constant voltage of 150 V for 75 min at 4°C. Proteins were visualized by Coomassie staining.

Dephosphorylation of autophosphorylated YegI.Autophosphorylation of YegI kinase was performed by addition of 5 μCi of [γ-32P]ATP (PerkinElmer) to 2 μM purified YegI in 10 μl of reaction buffer containing 50 mM Tris (pH 7.5), 50 mM KCl, 0.5 mM DTT, 10 mM MgCl2, 10 mM MnCl2, and 200 μM ATP. Phosphatase storage buffer or PphC or PphC-D46N was added to the above reaction mixture to a final concentration of 4 μM. Reaction mixtures were incubated at 37°C for 45 min, reactions were stopped using 3× Laemmli buffer, and mixtures were boiled for 5 min at 95°C. Samples were resolved on a 12% SDS-PAGE gel and visualized by straining with Coomassie dye. Radioactive dried gel was exposed and visualized by autoradiography.

ACKNOWLEDGMENTS

We thank present and former members of our lab for helpful discussions. We acknowledge Elizabeth Nagle for performing some of the initial characterization of PphC.

This work was supported by an HHMI International Student Fellowship to K.R. and by NIH grant GM114213 to J.D.

We declare no conflict of interest.

K.R. performed all of the experiments, and K.R. and J.D. wrote the manuscript.

FOOTNOTES

    • Received 17 April 2018.
    • Accepted 21 June 2018.
    • Accepted manuscript posted online 2 July 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00225-18.

  • [This article was published on 24 August 2018 with a byline that lacked Elizabeth Nagle. The byline was updated in the current version, posted on 22 October 2019.]

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KEYWORDS

Ser/Thr phosphorylation
metal-dependent phosphatase

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