Instituto de Biotecnología de León INBIOTEC, Parque Científico de León, Avda del Real no. 1, 24006 León, Spain1
Area de Microbioloía, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain2
Author for correspondence: Paloma Liras. Tel: +34 987 291504. Fax: +34 987 291506. e-mail: degplp{at}unileon.es
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ABSTRACT |
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Keywords: phytases, exocellular enzymes, protein purification, gene cloning
Abbreviations: AP, alkaline phosphatase; PNPP, p-nitrophenyl phosphate; X-phosphate, 5-bromo-4-chloro-3-indolyl phosphate
The GenBank accession number for the sequence reported in this paper is AJ278740.
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INTRODUCTION |
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Streptomyces spp. are filamentous Gram-positive bacteria that contribute to the degradation of decaying plant and animal material in soil. Several extracellular Streptomyces enzymes have been studied at the molecular level, including amylases (García-González et al., 1991 ; Vigal et al., 1991
), xylanases (Blanco et al., 1997
), agarase (Bibb et al., 1987
), cellulases (Schlochtermeier et al., 1992
), glucanases (Fernández-Abalos et al., 1992
) and ß-lactamases (Urabe et al., 1990
). However, very little is known about the extracellular phosphatases of Streptomyces and none of them has been purified or cloned.
Inorganic phosphate regulates negatively the biosynthesis of many antibiotics and other secondary metabolites (Liras et al., 1977 ; Martín et al., 1994
). Phosphate regulation of the biosynthesis of candicidin (a polyene macrolide antifungal antibiotic) by Streptomyces griseus IMRU 3570 has been extensively studied (reviewed by Martín, 1989
; Martín et al., 1994
). This strain produces high levels of alkaline phosphatase (AP) activity (Daza et al., 1990
) in parallel with candicidin biosynthesis, under phosphate-starvation conditions. Preliminary evidence indicated that phosphate represses transcription of candicidin biosynthesis genes (Asturias et al., 1990
) by a mechanism similar to that of phosphate repression of AP. Some mutants of S. griseus 3570 deregulated in phosphate control of candicidin biosynthesis (Martín et al., 1979
) are also derepressed in AP.
Characterization of the S. griseus AP was important as the first step to clone the phoA gene by reverse genetics. Availability of the phoA gene will allow us to study the phosphate regulation of this extracellular enzyme at the transcriptional level in order to compare it with the existing knowledge on phosphate regulation of secondary metabolism in this strain (Asturias et al., 1990 ). In this work we describe the purification to homogeneity and substrate kinetics of the extracellular S. griseus AP and the cloning and analysis of the phoA gene.
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METHODS |
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To study production of AP in defined conditions, asparagine-minimal medium was used (asparagine 25 g, D-glucose 5·5 g, MgSO4 . 7H2O 0·123 g, FeSO4 . 7H2O 1·39 µg, ZnSO4.7H2O 1·43 µg, distilled water 1000 ml; pH 7·6) (Martín & McDaniel, 1975 ).
Alkaline phosphatase assay and phosphate determination.
AP activity (EC 3 . 1 . 3 . 1) was measured as follows. Ten microlitres of enzyme solution was added to 50 µl 25 mM Tris/HCl buffer pH 9·5 containing 10 mM p-nitrophenyl phosphate (PNPP) and 0·4 mM CaCl2, and incubated at 30 °C for different times. The reaction was stopped by adding 2 ml 0·5 M Na2CO3 and the absorbance of the p-nitrophenol formed was measured at 410 nm. One unit of enzyme is defined as the activity that forms 1 µmol p-nitrophenol min-1. The specific activity is given as units (mg protein)-1. Phosphate was determined by the FiskeSubbaRow method (Leloir & Cardini, 1957 ) using sodium phosphate buffer as standard. Protein concentrations were measured with the Bradford reagent (Bio-Rad).
Hydrolysis of different substrates.
The phosphatase-mediated release of inorganic phosphate from different substrates was determined by incubating each substrate at 10 mM concentration for 10 min at 30 °C with pure AP; the reaction was stopped by boiling the samples. Inorganic phosphate released by the phosphatase was quantified by the FiskeSubbaRow method as the difference in phosphate present in the samples at zero time and after 10 min reaction.
Chemicals.
Phenylmethylsulfonyl fluoride (PMSF) and the substrates PNPP (disodium hexahydrate), phytic acid (myo-inositol hexaphosphate), umbelliferyl phosphate, -naphthyl phosphate, ß-glycerol phosphate, 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate), glucose 6-phosphate, fluorescein phosphate, tripolyphosphate, ATP, UMP and cAMP used to test enzyme specificity were obtained from Sigma.
Purification of the alkaline phosphatase.
SPG culture supernatants (1000 ml) were concentrated in a dialysis bag by treatment with PEG 20000 for 24 h at 4 °C to a final volume of 430 ml without appreciable loss of activity. Ammonium sulfate solution was added to the concentrated enzyme preparation and the activity precipitated in the range of 4070% saturation. The protein pellet was collected by centrifugation and resuspended in 17·5 ml 25 mM Tris/HCl buffer pH 9·5 containing 0·4 mM CaCl2, applied to a Sephacryl S-300 column (980x26 mm) equilibrated with 100 mM Tris/HCl buffer pH 9·0 and eluted with the same buffer. The active fractions of the eluate were concentrated through P-30 membranes in an Amicon ultrafiltration apparatus. This partially insoluble preparation was then centrifuged at 10000 g for 10 min. The active protein pellet was solubilized in 3% Triton X-100 and mixed with the previous supernatant. The enzyme preparation was adjusted to pH 7·5 and applied to a cation-exchange CM-Sephadex column (160x15 mm) equilibrated with 10 mM Tris/HCl buffer pH 7·5. The phosphatase was eluted with a 01 M linear NaCl gradient. The active fractions were pooled, and concentrated by filtration through Amicon P-30 membranes; the pH was adjusted to 9·4 with 25 mM ethanolamine pH 9·4 and the pooled eluate applied to a PBE94 (Pharmacia) chromatofocusing column (200x20 mm) equilibrated with 25 mM ethanolamine pH 9·4. A pH gradient of 200 ml polybuffer 96/distilled water (1:10, v/v) adjusted to pH 4·0 with acetic acid eluted most of the proteins of the preparation except the phosphatase, which was eluted with 1 M NaCl at pH 4·0.
SDS-PAGE.
Denaturing SDS-PAGE was performed as described by Laemmli (1970) . Non-denaturing PAGE was carried out in the same system but omitting SDS, ß-mercaptoethanol and the boiling treatment of the samples.
Determination of N-terminal amino acid sequence.
The phosphatase protein obtained after chromatofocusing was purified to homogeneity by filtration through a preparative Sphaerogel TSK 3000SW HPLC column (300x21·5 mm) equilibrated with 0·5 M Tris/acetate buffer pH 7·3 with a flow of 0·2 ml min-1. To avoid N-terminal blockage the pure protein was subjected to SDS-PAGE and blotted as indicated by Moos et al. (1988) . The N-terminal amino acid sequence was determined with an Applied Biosystems 477A protein sequencer.
Antibodies and immunoblotting.
Rabbit antisera were obtained against two different AP preparations. Native enzyme purified through a Sphaerogel TSK3000SW column (250 µg) in Freunds complete adjuvant was injected subcutaneously into female New Zealand White rabbits. Three subsequent injections of 125 µg of the protein were administered every 2 weeks in Freunds incomplete adjuvant. Alternatively, AP (about 200 µg protein) denatured by boiling for 3 min in the presence of 2% SDS and 0·1% ß-mercaptoethanol was resolved by SDS-PAGE, the 62 kDa band was cut, equilibrated for 30 min in distilled water, homogenized in saline solution, mixed with Freunds complete adjuvant and injected into the rabbits. Additional inoculations of 100 µg of protein in Freunds incomplete adjuvant were administered every 2 weeks. The behaviour of both anti-phosphatase sera in immunoblotting experiments was identical.
Immunoblottings were performed as described by Towbin et al. (1992) . The blotted protein was detected with the antiserum using a double-antibody enzyme-conjugate immunodetection method and the colour was developed with nitro blue tetrazolium and X-phosphate.
DNA manipulations.
Escherichia coli DH5 was used as host (Sambrook et al., 1989
) and plasmids pUC19 (New England Biolabs) and pBluescript KS(+) (Stratagene) were used as vectors for genetic manipulation. Streptomyces DNA was isolated as described by Hopwood et al. (1985)
. Southern hybridizations were carried out on Hybond-N nylon membranes as indicated by the manufacturer. Oligonucleotides labelled with the Dig-oligonucleotide tailing kit or plasmids labelled by the random-priming method with the Dig DNA labelling kit (Boehringer Mannheim) were used as probes.
Sequence analysis.
The cloned DNA fragments were sequenced in a Perkin Elmer ABI Prism 310 automatic sequencer. Comparison of proteins was made using the FASTA-3 program and the SWALL Database. The nucleotide sequence of the phoA gene has been deposited in the EMBL/GenBank/DDBJ database under accession number AJ278740.
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RESULTS |
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To confirm inhibition of phosphatase formation, supernatants of 24, 36 and 40 h cultures of S. griseus grown in SPG medium and in SPG supplemented with 10 mM phosphate were analysed by SDS-PAGE (Fig. 1a) and immunoblotted with anti-phosphatase antibodies. Two immunoreacting bands were found in the control cultures (Fig. 1b
). One of them corresponds to a protein of 62 kDa (in agreement with the molecular mass determined for the AP monomer: see below) which was found in broths from cultures grown in SPG medium but not in phosphate-supplemented cultures (Fig. 1b
, lanes 3 and 4). A second immunoreactive band of 33 kDa was also inhibited by phosphate; it is probably a degradation product of the 62 kDa AP monomer since the 33 kDa form appears with increasing intensity during the fermentation, coinciding with the loss of the 62 kDa band (Fig. 1c
).
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The enzyme preparation treated with 0·5% Triton X-100 was further purified by cation exchange through CM-Sephadex and eluted with a NaCl gradient [specific activity 7·45 units (mg protein)-1 (Table 1)]. The active samples were chromatofocused in a PBE94 column as indicated in Methods. Most of the activity (99·3%) eluted from the column as a single peak with 1 M NaCl solution (Fig. 2a
), giving an enzyme preparation with a specific activity of 15·6 units (mg protein)-1 (Table 1
) and a final recovery of 12%.
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The enzyme preparation obtained after HPLC filtration was assayed after non-denaturing 10% PAGE by soaking the gel in buffer containing PNPP. A protein with AP activity unable to penetrate the 10% acrylamide-bisacrylamide was observed in the boundary with the stacking gel. This band was excised from the gel, homogenized in distilled water and applied to: (i) a denaturing SDS-PAGE gel and (ii) a 7·5% non-denaturing gel. A single band of 62 kDa was found in the SDS-PAGE gel after Coomassie blue staining whereas in the native gel AP activity migrated slowly in the gel close to the 440 kDa marker.
Optimal parameters for alkaline phosphatase activity
Using a pure phosphatase preparation [36 units (mg protein)-1] the reaction on PNPP as substrate was linear for at least 90 min. The activity was not strictly dependent on CaCl2 addition but increased when Ca2+ was added to the assay, to a maximum value of 234% in the presence of 0·4 mM CaCl2 with respect to the enzyme preparation without CaCl2 added. This result indicates that the S. griseus AP might be functionally similar to the calcium-requiring Bacillus phytases (Kerovuo et al., 1998 ).
The optimal pH for the enzyme on PNPP was 9·5, with a sharp decrease in the range of 7·59·5 and 11·012·5. At pH 11 the activity was less than 85%. The activity was nil at pH 7·0.
The enzyme activity increased with temperature to a maximum at 50 °C. At 1 mM concentration, Zn2+, Cd2+, Hg2+ and Sn2+ ions inhibited the activity 95100% and Mn2+ and Co2+ exerted a 75% inhibition, but Fe2+ and Fe3+ were not inhibitory. EDTA produced 100% inhibition, probably by chelating Ca2+ ions. Arsenate ions (1 mM) did not affect the phosphatase activity. PMSF at 1 mM and 5 mM caused 40% and 85% inhibition, respectively.
Substrate specificity
The S. griseus AP showed a Km for PNPP of 130 µM. The activity on PNPP was competitively inhibited by inorganic phosphate (Ki 220 µM) and by tripolyphosphate. If the activity on PNPP is taken as 100%, the activities on sodium pyrophosphate and phytic acid, under the same assay conditions, were 120 and 187% (however these substrates release two and three phosphate groups respectively). Umbelliferyl phosphate, -naphthyl phosphate, ATP, UMP and ß-glycerol phosphate were used with 54, 39, 23 and 19% efficiency as compared to PNPP. Activities in the range of 710% were found with fluorescein phosphate, glucose 6-phosphate, tripolyphosphate, cAMP and X-phosphate as substrates. These results indicate that the S. griseus AP uses a wide range of inorganic and organic phosphorylated substrates.
The N-terminal sequence of the purified protein shows similarity to alkaline phosphatases
The semipreparative HPLC Sphaerogel TSK 3000SW filtration step was important to obtain homogeneous AP to sequence the N-terminal end of the protein since it removed two minor contaminating proteins (Fig. 2b) barely visible in Fig. 2(c)
, lane 6. Two separate sequencing experiments in different laboratories gave a clear sequence of 18 amino acids: RLREDPFTLGVASGDPHP with a single ambiguity in the aspartic acid at position 15. This amino acid sequence was found to be 61% similar to the N-terminal sequence of the PhoD phosphatase of Bacillus subtilis (Eder et al., 1996
).
Cloning of the phoA gene
SmaI-, SalI-, SacI- or PvuII-digested DNA from S. griseus was transferred to nylon membranes and probed with a 33 nucleotide degenerate oligomer based on the N-terminal amino acid sequence of the APs from S. griseus and B. subtilis. Several strong bands of hybridization were detected. SalI-digested DNA was extracted from the agarose gel in the region giving strong hybridization and used to construct a mini-library in the E. coli vector pUC19. When this plasmid library was probed again with the 33-mer probe, one clone gave a strong hybridization signal and was found to contain a 1·2 kb SalI DNA insert. The nucleotide sequence of the fragment showed that it contained a truncated ORF1 encoding a protein with high similarity to the phosphodiesterase/AP encoded by phoD of B. subtilis and to the sequence of the unpublished AP of Streptomyces tendae (Q9RCK5). To clone the entire ORF1 a 450 bp NotIEco72I DNA fragment internal to the cloned ORF was used to screen SmaI-, NotI-, NcoI-or KpnI-digested S. griseus DNA. A 2·3 kb NotI DNA fragment, giving positive hybridization, was found to overlap partially with the 1·2 SalI fragment. This fragment was extracted from the gel and subcloned in pBSKS(+). Four transformants were found by hybridization to contain the 2·3 kb NotI DNA insert. This insert was isolated and completely sequenced.
The complete ORF1 contains 1695 nucleotides and showed a clear preference for codons containing C or G in the third position. It was preceded by a putative ribosome-binding site GAGGAG complementary to the 3' end of Streptomyces lividans 16S rRNA, located 814 nt upstream of the GTG start codon. The ORF encodes a 565 amino acid protein with a deduced molecular mass of 62678 Da. When compared with the proteins of the SWALL database the deduced protein showed a high similarity with the putative AP of S. tendae (72·7% identical amino acids), with the APs of Streptomyces coelicolor (71% identity with Q9RKP2, 65% with Q9XA97 and 37% with CAB51460), and with phoD of B. subtilis (40·5% identity) (Fig. 3a) throughout the entire sequence of the protein. Therefore, we have named the S. griseus cloned gene phoA and propose the designations phoA, phoB and phoC for the homologous genes of S. coelicolor (Q9RKP2, Q9XA97 and CAB51460, respectively), following the order of decreasing identity. A fourth ORF (PhoD) encoding a putative phosphatase present in the S. coelicolor genome (Q9XAN2) shows much lower identity (27%).
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N-terminal region, active site and motifs of interest in PhoA of S. griseus
The amino acid sequence determined from the N-terminal end of the pure protein corresponds to amino acids 7590 of the protein encoded by phoA. This region is conserved, especially amino acids 8090, in the phosphatases of S. tendae, B. subtilis PhoD, and PhoA, PhoB and PhoC from S. coelicolor (motif A in Fig. 3a) but it is absent from the PhoD phosphatase of S. coelicolor.
Amino acid sequences located in the ß-sheets A, C, D, F, G, H and I, corresponding to the ligands for metals and the phosphorylation site in the enzyme (Hulett et al., 1991 ), occur in the APs PhoA and PhoB of B. subtilis but interestingly they are not well conserved in the PhoD protein of B. subtilis that is the more closely related to the S. griseus AP.
Comparison of the S. griseus amino acid sequence with those of E. coli AP, B. subtilis PhoA and PhoB and other putative Streptomyces phosphatases suggests that the active centre of the S. griseus AP corresponds to amino acids 144167 with S147 (Fig. 3b) as phosphate-binding site. Serine147 is replaced by threonine in the AP of S. tendae and in S. coelicolor PhoA.
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DISCUSSION |
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Production of the S. griseus AP protein is strongly decreased by inorganic phosphate, as shown by immunoblotting of the AP in phosphate-limited and phosphate-supplemented cultures. Both forms of the AP were absent from phosphate-supplemented cultures, suggesting that de novo synthesis of the AP is inhibited by phosphate. Promoter analysis and transcriptional studies of the cloned gene are now in progress and will allow us to study the molecular mechanisms of phosphate control of gene expression in Streptomyces.
As described in this article the S. griseus AP uses a wide range of substrates including inorganic oligophosphates and polyphosphates as well as a variety of organic phosphates. The enzyme is stimulated by Ca2+ ions as are the Bacillus phytases (Kerovuo et al., 1998 ; Powar & Jagannathan, 1982
) but they lack the RHGE/DRXP motif characteristic of phytases and acid phosphatases (Piddington et al., 1993
). Phytases (EC 3 . 1 . 3 . 8) are members of the histidine acid phosphatases subfamily that form phosphorylated protein intermediates during the substrate hydrolysis (Mitchell et al., 1997
). Comparison of the S. griseus PhoA amino acid sequence with those of other phosphatases revealed very little homology (about 10%) to fungal phytases. In E. coli there is a phosphatase, encoded by the appA gene, that shows acid phosphatase and phytase activity (Golovan et al., 2000
), and B. subtilis possesses a phosphatase with optimal pH for phytic acid hydrolysis of 7·5 (Powar et al., 1982
). However, the optimal pH for the S. griseus phosphatase is 9·5 and there are major differences in structure between the S. griseus AP and other phytases.
When the S. griseus PhoA protein was compared with other proteins in the databases a higher similarity was found with the phosphatase of S. tendae and with proteins deduced from the S. coelicolor genome sequence. The similarity to the E. coli AP was surprisingly low (14% identical residues). Indeed the E. coli enzyme is a homodimer that contains two Zn2+ ions and one Mg2+ ion in each active centre (Kantrowitz, 1994 ), whereas the S. griseus AP was stimulated by Ca2+ but was inhibited by Zn2+. The S. griseus PhoA is more similar to the B. subtilis Ca2+-dependent phosphatase (Kerovuo et al., 1998
; Powar & Jagannathan, 1982
), to the phosphatase of Bacillus amyloliquefaciens which has been crystallized with Ca2+ (Ha et al., 1999
, 2000
) and to the thermostable phosphatase of Thermus caldophilus (Park et al., 1999
) that is also inhibited by Zn2+.
The N-terminal sequence of the mature AP corresponds to amino acids 75RLREDPFTLGVASGDPHP92. This N-terminal region is well conserved in the PhoD protein of B. subtilis, the PhoA, PhoB and PhoC proteins of S. coelicolor and the APs of S. tendae but not in the PhoD of S. coelicolor. The processing site of the S. griseus AP does not correspond to the canonical AXA proposed by von Heijne (1986) . However, there are two conserved sequences 50AAA53 and 63AGA65 that may be cleavage sites for signal peptidases and the N-terminal end of the mature AP may originate from additional processing of a pro-AP as has been described for the phytase of Aspergillus niger (van Hartingsveldt et al., 1993
).
AP signature sequences are conserved in the S. griseus AP. The ß-sheet A of the S. griseus AP corresponds to amino acids 81100 (Fig. 3b), where the conserved D89 is involved in metal binding in the E. coli enzyme. The sequence of the ß-sheet G for several APs is shown in Fig. 3(b)
. The E388 and D393 (asparagine in S. coelicolor PhoA and B. subtilis PhoD) are metal-binding sites in the E. coli enzyme and are conserved in S. griseus AP. Additional ß-sheets as well as the T206 in sheet C, H450 in ß-sheet H and H509 in ß-sheet I (which does not exist in E. coli) have been described as metal-binding sites in B. subtilis and E. coli APs.
No genes encoding phosphatases with similarity to the E. coli AP have been found in the genome of S. coelicolor. All this evidence indicates that the extracellular APs of S. griseus and other Streptomyces species belong to a family of APs only distantly related to the E. coli enzyme (Lim et al., 2000 ). No APs similar to the S. griseus AP have been found in the M. tuberculosis genome or the available M. leprae, M. bovis or C. diphtheriae sequences, suggesting that the PhoA-encoded AP is specific for soil-living actinomycetes.
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ACKNOWLEDGEMENTS |
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Received 16 October 2000;
revised 29 January 2001;
accepted 14 March 2001.