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Keywords:

  • Acid phosphatase;
  • Phosphotransferase;
  • Escherichia coli;
  • aphA gene

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

An open reading frame located in the tyrB-uvrA intergenic region of the Escherichia coli MG1655 chromosome was identified as encoding the class B acid phosphatase of this species on the basis of cloning and expression experiments. A protocol for purification of the enzyme (named AphA) was developed, and its properties were analyzed. The enzyme is a 100-kDa homotetrameric protein which apparently requires a metal co-factor for activity. Similarly to other bacterial class B acid phosphatases, it is able to dephosphorylate several organic phosphomonoesters as well as to catalyze the transfer of low-energy phosphate groups from phosphomonoesters to hydroxyl groups of various organic compounds.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Escherichia coli is able to produce a periplasmic acid phosphatase containing a 27-kDa polypeptide component [1]. Further investigation indicated that this enzyme is apparently a member of a family of bacterial acid phosphatases indicated as class B enzymes, which are widespread among different species of enteric bacteria [2]. The class B acid phosphatases thus far purified and characterized in some detail are the nonspecific acid phosphatase II (AphA) enzyme of Salmonella enterica serovar typhimurium[3, 4] and the NapA enzyme of Morganella morganii[5]. Both enzymes are 100-kDa homotetrameric proteins active as phosphomonoesterases on various nucleotide and non-nucleotide organic substrates, and also as phosphotransferases able to catalyze the transfer of low-energy phosphate groups from phosphomonoesters to free hydroxyl groups of various organic compounds. The activity of these enzymes is inhibited by EDTA, suggesting their nature of metallo-proteins.

Searching for homologs of the M. morganii NapA protein in molecular sequences databases, we previously identified an E. coli hypothetical protein encoded by an unknown open reading frame (ORF), located in the tyrB-uvrA intergenic region (at approx. 92 min of the genetic map) of the E. coli chromosome [6], which showed significant sequence similarity to the Morganella protein [5]. This finding suggested that the above ORF could correspond to the gene encoding the E. coli class B acid phosphatase.

In this work we have confirmed this hypothesis by cloning and expression of the putative class B acid phosphatase-encoding gene of E. coli MG1655. We have also developed a simple purification protocol for the E. coli class B enzyme and analyzed its functional properties.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains and genetic vector

E. coli MG1655 (CGSC 6300) was used as the source for sequencing and cloning the E. coli aphA gene. E. coli DH5α[7] was used as the host for genetic vectors and recombinant plasmids. Plasmid pBTac1 (Boehringer Mannheim), which is an E. coli expression vector carrying a multiple cloning site downstream of the strong hybrid tac promoter [8] and upstream of the E. coli rrnB transcription terminator, was used for cloning and expression of the E. coli aphA gene.

2.2Recombinant DNA methodology and DNA sequencing

Basic recombinant DNA methodology was performed essentially as described by Sambrook et al. [7]. Bacterial genomic DNA was extracted as previously described [9]. PCR was performed using the high-fidelity Pfu DNA polymerase (Stratagene). DNA sequencing was performed by the dideoxy-chain termination method [7] and custom oligonucleotides as sequencing primers. Direct sequencing of PCR amplimers was performed as previously described [10]. Sequencing was always performed on both strands.

2.3Protein analysis techniques

Whole cell proteins from bacteria were prepared as previously described [5]. Protein concentration in solution was determined according to the method of Bradford, using a commercial kit (Bio-Rad Protein Assay; Bio-Rad). Bovine serum albumin was used as the standard. SDS-PAGE was performed as described by Laemmli [11]. The acrylamide concentration in the separating gel was 15%. Zymogram detection of the E. coli AphA enzyme following SDS-PAGE was performed as previously described using the phenolphthalein diphosphate-methyl green detection system [2]. Zymogram evaluation of properties typical of class B acid phosphatases (including retention of activity in the presence of 0.01% (w/v) SDS, loss of activity in the presence of 20 mM EDTA, lack of activity toward 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and migration as high-Mr multimers in SDS-PAGE performed under partially denaturing conditions) was performed as previously described [2]. Gel-permeation chromatography for determination of the molecular mass of the purified enzyme was performed as described [12] using Sephacryl S-300 (Pharmacia) as a gel-permeation matrix. The buffer used for column equilibration and elution was 100 mM sodium acetate pH 6 containing 20 mM NaCl.

2.4Purification of the AphA enzyme

The AphA enzyme was purified from E. coli DH5α(pATac) grown aerobically at 37°C in TB medium [7] containing 60 mM glucose instead of glycerol (TBD) and ampicillin (250 μg ml−1) for plasmid selection. This glucose-containing medium was used to avoid production of the endogenous DH5α class B enzyme [1] while allowing a relatively high-level expression of the cloned MG1655 aphA gene. Isopropyl β-d-thiogalactopyranoside (IPTG) (1 mM final concentration) was added to the culture when it reached an OD600 of 0.8. 6 h after induction cells were collected by centrifugation and the enzyme was extracted from the periplasmic space by a modified spheroplasting technique [13]. Briefly, cells were resuspended in 200 mM Tris-HCl, pH 8, (2.5 ml g−1 of cell wet weight), an equal volume of 200 mM Tris-HCl, pH 8, containing 1 M sucrose was added, EDTA and lysozyme were then added to achieve a final concentration of 10 mM and 4 mg g−1 of cell wet weight, respectively, and an equal volume of bidistilled water was finally added, and the mixture was incubated at room temperature for 30 min. After addition of MgCl2 to a final concentration of 10 mM, the suspension was centrifuged at 32.000×g for 30 min, and the cleared supernatant represented the crude enzyme extract. PEG 6000 was added to the crude extract to a 10% (w/v) concentration and the precipitate was removed by centrifugation (10.000×g for 20 min at 4°C). The enzyme remained in the supernatant from which it was subsequently precipitated by increasing the PEG concentration to 25%. The pellet obtained after 25% PEG precipitation was solubilized in 10 mM Tris-HCl, pH 7.45, containing 1 mM MgCl2 (approx. 1/50 of the original culture volume), the solution was centrifuged at 10.000×g for 5 min to remove any remaining insoluble material, and the cleared supernatant was loaded onto a DEAE-Sepharose FF (Pharmacia) column equilibrated with 10 mM Tris-HCl pH 7.45 containing 1 mM MgCl2. The flowthrough fractions containing the enzymatic activity were pooled, dialyzed against 10 mM MES-NaOH, pH 6, containing 1 mM MgCl2 and 1% PEG 6000, and loaded onto a CM-Sepharose FF (Pharmacia) column equilibrated with the dialysis buffer. Under these conditions the enzyme adsorbed to the cation exchanger, from which was subsequently eluted with 25 mM NaCl in the same buffer. The fractions containing the enzymatic activity were pooled and concentrated by ultrafiltration. At this stage the enzyme can be stored at −70°C for at least 1 year without measurable loss of activity.

2.5Phosphatase assays

Phosphatase assays were performed in 50 mM Tris-maleate buffer, pH 6.5 at 37°C, in a volume of 0.3 ml, using each substrate at 1 mM concentration and 0.04 μg of the purified enzyme, unless otherwise specified. Activity was determined by measuring the released inorganic phosphate (Pi) [14], unless otherwise specified. The pH optimum was assayed in buffers of pH ranging from 4.5 to 8.5. Dephosphorylation of various organic phosphoesters was assayed as previously described [15, 16] at pH 6.5. Cleavage of bis-p-nitrophenyl phosphate (bis-pNPP) was also assayed by measurement of the released p-nitrophenol (pNP) at 414.5 nm and pH 12. Susceptibility of the purified enzyme to various substances was assayed at pH 6.5 using p-nitrophenyl phosphate (pNPP) as substrate and measuring the released pNP at 414.5 nm and pH 12. In these assays the enzyme was incubated with each substance for 15 min at 25°C before addition of the substrate. Low-energy phosphotransferase activity was assayed as previously described [4, 5] at pH 6.5, using 2 mM pNPP as a phosphate donor and various acceptors carrying free hydroxyl groups.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Identification of the E. coli gene encoding class B acid phosphatase

The nucleotide sequence of an E. coli MG1655 gene, previously identified as putatively encoding the class B acid phosphatase on the basis of sequence similarity to the M. morganii napA gene [5], was determined by means of direct sequencing of corresponding PCR amplimers to resolve some ambiguities and a frameshift reported at that level in the database entry [6]. According to sequencing results, the putative E. coli class B acid phosphatase-encoding gene (named aphA after the name assigned to the corresponding Salmonella allele [3]) appeared to be 711 bp long, starting from an ATG codon located 1.1 kb downstream of the tyrB termination codon, and terminating at a TGA codon located 1.3 kb downstream of the uvrA termination codon (Fig. 1). The initiation codon is preceded by a recognizable ribosomal-binding sequence (Fig. 1a). The E. coli aphA ORF has the potential to code for a polypeptide of 237 amino acids (Fig. 1A) with a predicted molecular mass of 26.103 Da, which shows an amino terminal sequence pattern typical of signal peptides targeting protein secretion to the periplasmic space via the general secretory pathway [17].

image

Figure 1. (a) Nucleotide sequence of the E. coli MG1655 aphA ORF and flanking regions. Nucleotide number 1 represents the first nucleotide of the aphA start codon. The putative ribosomal binding site of the aphA ORF is underlined. The deduced amino acid sequence of the protein potentially encoded by the aphA ORF is reported below the nucleotide sequence. The amino-terminal region of the protein (amino acids 1–23) showing a sequence pattern typical of signal peptides targeting secretion to the periplasmic space [17] is underlined. Primers HD16 and HD17 used for PCR amplification and cloning of the aphA ORF in the E. coli expression vector pBTac1 are reported in lowercase letters above the sequence, at corresponding positions. The HD16 primer included a BamHI linker to facilitate subcloning of the amplimer in plasmid pBTac1. (b) Physical map of the E. coli MG1655 chromosomal region containing the aphA ORF. Distances between aphA and tyrB or uvrA are indicated according to Blattner et al. [6]. The nucleotide sequence of the E. coli aphA gene reported in this paper has been submitted to the GenBank/EMBL/DDBJ database and assigned accession no. X86971.

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The aphA ORF was amplified from the genomic DNA of E. coli MG1655 using primers HD16 and HD17 (Fig. 1A) and the amplimer, digested with BamHI, was cloned into the E. coli expression vector pBTac1 digested with SmaI and BamHI. In the resulting plasmid, named pATac, the aphA ORF along with its ribosomal-binding site was located downstream of the tac promoter and followed by the transcription terminator present in the vector. The identity and fidelity of the cloned amplimer carried by pATac was confirmed by sequencing (data not shown). When E. coli DH5α(pATac) was grown under conditions permissive for transcription from the tac promoter but not for production of the endogenous DH5α class B acid phosphatase [1]), it expressed a zymogram-detectable acid phosphatase which showed the same properties reported for the E. coli class B enzyme (Fig. 2), confirming the identity of the E. coli aphA gene.

image

Figure 2. Zymogram detection and analysis of the acid phosphatase activity produced by E. coli DH5α(pATac). Lane pATac was loaded with whole-cell proteins (approx. 20 μg) of DH5α(pATac). Consistently with previous observations [1], under the same experimental conditions no production of the endogenous class B acid phosphatase was detectable in DH5α(pBTac1) (lane pBTac1). The class B acid phosphatase produced by E. coli MG1655 under suitable growth conditions [1], detected by the same zymogram procedure, is shown for comparison (lane MG1655). Similarly to the class B acid phosphatase produced by MG1655 [2], the phosphatase activity produced by DH5α(pATac) migrated as a 100-kDa oligomer when SDS-PAGE was performed under partially denaturing conditions (lane pATac-PD) and appeared to retain its activity in the presence of 0.01% (w/v) SDS (lane pATac-S), while being susceptible to EDTA inhibition (lane pATac-E) and inactive toward BCIP (lane pATac-B). The four rightmost lanes were loaded with half of the protein loaded in lane pATac. Molecular size markers are reported in kDa on the left. Whole-cell proteins of DH5α(pATac) and DH5α(pBTac-1) were prepared from cells grown in TBD medium containing ampicillin (250 μg ml−1) for plasmid selection. IPTG (1 mM final concentration) was added to the cultures when they reached an OD600 of 0.8 and the cells were collected 6 h after induction.

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3.2Purification of the E. coli AphA enzyme

The AphA enzyme was purified from E. coli DH5α(pATac) as described in Section 2. The modified spheroplasting protocol used for extraction of the enzyme from the periplasmic space allowed an efficient recovery of the enzymatic activity, notwithstanding the use of EDTA during the procedure. The sequential PEG precipitation steps removed part of the contaminating material from the periplasmic extract (Fig. 3), concentrated the enzyme in a smaller volume, and avoided the need for dialysis before the subsequent chromatography step. Anion exchange chromatography was successful in removing most of the residual contaminating proteins, the AphA enzyme being by far the most abundant component in the column eluate (Fig. 3). The final cation exchange chromatography step was able to yield the protein more than 99% pure, as evaluated by SDS-PAGE (Fig. 3). Using this purification procedure a yield of approx. 3–4 mg of purified AphA protein per liter of culture was reproducibly obtained.

image

Figure 3. SDS-PAGE analysis of various stages of purification of the E. coli AphA protein. Lanes: a, crude extract obtained using the modified spheroplasting technique; b, supernatant after the 10% PEG 6000 precipitation step of the crude extract; c, soluble material obtained after resuspension of the 25% PEG 6000 precipitate; d, flowthrough of the DEAE-Sepharose FF column; e, enzyme-containing fractions eluted from the CM-Sepharose FF column (∼20 μg of protein). After electrophoresis the gel was stained with Coomassie brilliant blue R-250 (the detection sensitivity of the procedure was ∼0.2 μg of protein). Molecular size markers are reported in kDa on the left.

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The molecular mass of the purified protein, estimated by gel-permeation chromatography, was 101 kDa. Considering the approx. 25 kDa size of the polypeptide component (Fig. 3 and sequencing data) the native enzyme appeared to have a homotetrameric quaternary structure.

3.3Functional properties of the E. coli AphA enzyme

The AphA enzyme exhibited maximal activity at a pH value around 6–6.5 toward 5′-AMP, and at a slightly lower pH value (5.5–6) toward 3′-AMP and pNPP (data not shown).

At pH 6.5 and 1 mM substrate concentration, the enzyme appeared able to dephosphorylate several organic phosphomonoesters including 5′- and 3′-nucleotides, 2′-deoxy-5′-nucleotides, pNPP, phenyl phosphate, glycerol 2-phosphate, ribose 5-phosphate, O-phospho-l-amino acids and phytic acid, showing the highest reaction velocities with aryl phosphates (pNPP, phenyl phosphate and O-phospho-l-tyrosine) and nucleotides. Under the same experimental conditions no activity was detectable toward ATP, phosphodiesters, glycerol-1-phosphate, glucose 1-phosphate or glucose 6-phosphate (Table 1).

Table 1.  Relative activities of the E. coli AphA enzyme toward various organic phosphoesters
SubstrateRelative activitya
  1. aRelative activities were calculated on mean values of initial reaction velocities obtained from three independent measurements. The standard error of the mean values was always lower than 10%. The initial reaction velocity for 5′-AMP under the above experimental conditions (see Section 2 for details) was 5.20±0.26 μmol min−1 of Pi released per mg of purified enzyme.

5′-AMP1.00
5′-UMP2.12
5′-CMP1.06
5′-GMP2.46
3′-AMP3.51
3′-UMP3.03
3′-CMP2.57
3′-GMP4.21
2′-Deoxy-5′-AMP2.12
2′-Deoxy-5′-UMP1.95
pNPP12.53
Phenyl phosphate11.82
ATP<0.05
2′:3′-Cyclic AMP<0.05
2′:3′-Cyclic UMP<0.05
Bis-pNPP<0.05
Glycerol 1-phosphate<0.05
Glycerol 2-phosphate0.25
Glucose 1-phosphate<0.05
Glucose 6-phosphate<0.05
Ribose 5-phosphate0.53
O-Phospho-l-serine0.49
O-Phospho-l-threonine0.37
O-Phospho-l-tyrosine8.95
Phytic acid0.47

The activity of the AphA enzyme was inhibited by EDTA, suggesting its nature of metallo-protein. The enzyme activity was also inhibited by nucleosides, Pi and Ca2+, while being stimulated by Mg2+ and unaffected by F (Table 2).

Table 2.  Effect of various substances on the activity of the E. coli AphA enzyme
Substance (mM)Relative activitya
  1. aRelative activities were calculated on mean values of initial reaction velocities obtained from three independent measurements. The standard error of the mean values was always lower than 10%. The initial reaction velocity for pNPP under the above experimental conditions (see Section 2 for details) was 66.7±1.7 μmol min−1 of pNP released per mg of purified enzyme.

None1.00
EDTA (5)0.06
EDTA (15)<0.01
Adenosine (0.1)0.34
Uridine (0.1)0.21
Guanosine (0.1)0.29
Cytidine (0.1)0.53
Pi (5)0.86
Pi (20)0.37
Pi (100)0.15
Ca2+ (1)0.77
Ca2+ (5)0.51
Mg2+ (1)1.75
Mg2+ (5)1.88
F (1)0.98

The AphA enzyme also showed a phosphotransferase activity able to catalyze the transfer of low-energy phosphate groups from an organic phosphomonoester donor to organic acceptors carrying free hydroxyl groups (Table 3). From these experiments it was also evident that the activity of the enzyme was stimulated by ethanol (Table 3).

Table 3.  Phosphotransferase activity of the E. coli AphA enzyme in the presence of pNPP as a donor of low-energy phosphate groups and various acceptors containing free hydroxyl groups
Acceptor (mM)Productiona ofPhosphate transfer (%)
pNPPi  
  1. aExpressed in μmol min−1 mg−1 of purified enzyme. Data represent mean values of three independent measurements. The standard error of the mean values was always lower than 10%.

H2O72.171.80
Ethanol (1000)147.247.168
Adenosine (0.1)23.921.510
Uridine (0.1)14.513.57

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Cloning and expression of an E. coli ORF, putatively identified as the gene encoding the class B acid phosphatase of this species on the basis of sequence similarity of its product with the M. morganii class B acid phosphatase [5], allowed us to confirm its identity and to develop an expression system for protein purification and characterization. It should be noted that overexpression of the aphA gene in strain DH5α(pATac) was not associated to any gross detrimental effect on bacterial growth, suggesting that an increased production of the AphA enzyme is not particularly toxic for the cell.

The protocol developed for purification of the E. coli AphA protein from the overproducing strain is simpler and more rapid in execution than those previously devised for other class B bacterial acid phosphatases [4, 5], and reproducibly yielded consistent amounts of purified enzyme.

Analysis of the purified E. coli class B enzyme showed structural and functional properties overall similar to those previously reported for the AphA enzyme of S. enterica serovar typhimurium[3, 4] and for the NapA enzyme of M. morganii[5], suggesting that enterobacterial class B acid phosphatases be a rather homogeneous protein family. The extension of substrate analysis carried out in this work showed that the E. coli enzyme can actually dephosphorylate a broad array of organic phosphomonoesters, but apparently exhibits a marked preference for aryl phosphoesters and, to a lesser extent, for nucleotides (both 3′- and 5′-). The preference for aryl phosphoesters appeared to be more pronounced than that observed with the Morganella enzyme and could reflect differences in their primary structure [5]. Measurement of kinetic parameters toward various substrates would be useful for a more precise evaluation of the catalytic properties of different class B enzymes and their comparison.

Concerning the possible physiological role of these enzymes, it has been previously proposed, on the basis of comparative data with E. coli and of physiological studies performed with some mutants (although not genetically characterized), that in S. enterica serovar typhimurium the AphA enzyme could represent a physiological equivalent of the periplasmic 5′-nucleotidase (UshA) and, possibly, also of the alkaline phosphatase which are present in E. coli and in several other Enterobacteriaceae but not in Salmonella[3]. However, it has recently been shown that most S. enterica serovars other than typhimurium actually produce a functional UshA homolog [18], and that production of a class B acid phosphatase is not restricted to S. enterica serovar typhimurium but also occurs in E. coli and in several other enterobacterial species [1, 2] which are also able to produce periplasmic 5′-nucleotidase and alkaline phosphatase activities [19, 20]. Moreover, the regulation of class B enzymes appears to be independent of the pho regulon, being also produced in the presence of high Pi concentrations [1, 2]. Current knowledge on enterobacterial phosphatases, therefore, would suggest reconsideration of the above hypothesis. In fact, the relatively broad substrate profile shown by the E. coli AphA enzyme would suggest that it (and likely also the other enterobacterial homologs) might function as a periplasmic broad-spectrum dephosphorylating enzyme able to scavenge both 3′- and 5′-nucleotides and also additional organic phosphomonoesters. Characterization of the kinetic parameters of this enzyme toward selected substrates, along with investigations on strains carrying genetically defined aphA mutations, are warranted to understand the physiological role of this class of highly conserved bacterial enzymes and to ascertain the significance of the phosphotransferase activity shown by these enzymes under laboratory conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported in part by grants no.94.02875.CT04 and 94.02925.CT04 from the C.N.R, and by a grant from the University of Siena `Quota 60%– 1995' to G.M.R. Thanks are due to Tiziana di Maggio for technical assistance. This manuscript is dedicated to the memory of Giuseppe Satta, who initially guided and encouraged us to proceed in this field of investigation.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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