Dipartimento di Scienze di Sanità Pubblica, Università di Roma La Sapienza, Piazzale A. Moro, 5, 00185 Rome, Italy1
Dipartimento di Biologia, Università di Roma Tor Vergata, 00133 Rome, Italy2
Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, 53100 Siena, Italy3
Author for correspondence: Francesca Berlutti. Tel: +39 06 4451324. Fax: +39 06 49914638. e-mail: francesca.berlutti{at}mail.uniroma1.it
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ABSTRACT |
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Keywords: flavobacteria, PhoV, PhoD, Cda, Ca2+-dependent ATPase
Abbreviations: AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; PDP-MG, phenolphthalein diphosphate-methyl green medium; pNPP; p-nitrophenyl phosphate; pNP, p-nitrophenol; TBU, Terrific broth supplemented with 1% glucose; TBUXP, TBU agar containing 50µg BCIP ml-1
The GenBank accession number for pafA reported in this paper is AF157621.
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INTRODUCTION |
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In addition to this major lineage of PhoA-like enzymes, a highly divergent lineage of prokaryotic APs has also been identified, including the PhoD enzyme from Zymomonas mobilis (Gomez & Ingram, 1995 ) and the PhoV enzyme from Synechococcus sp. (Wagner et al., 1995
). These proteins are more closely related to each other (24·2% sequence identity) than to any other of the PhoA-like enzymes, and are produced in a Pi-irrepressible pattern (Gomez & Ingram, 1995
; Wagner et al., 1995
). Both PhoD and PhoV are essentially non-specific enzymes that exhibit broad substrate specificity, with PhoD apparently being more active on ATP than on sugar phosphates. The recombinant PhoD was described as membrane-bound (Gomez & Ingram, 1995
) and the recombinant PhoV as associated with the periplasmic side of the inner membrane (Wagner et al., 1995
). The function of these enzymes remains to be elucidated. However, Gomez & Ingram (1995)
suggested that PhoD could be involved in nucleotide turnover during primary metabolism. Interestingly, the Cda Ca2+-dependent ATPase from Myroides odoratus (formerly Flavobacterium odoratum) (Bernardet et al., 1996
) also belongs to this lineage; it has not been specifically reported as an AP but as a P-type ATPase, functionally similar to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (Desrosiers et al., 1996
; Gambel et al., 1992
), and involved in calcium transport for calcium homeostasis.
In this work we report evidence for the existence of a new member of the latter lineage of prokaryotic APs and describe the sequencing, partial purification and preliminary characterization of PafA enzyme from Chryseobacterium meningosepticum, which exhibits a different behaviour as compared both to PhoD of Z. mobilis and PhoV of Synechococcus, and to Ca2+-ATPase of M. odoratus.
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METHODS |
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Preparation of crude cell extracts or cell fractions.
Crude cell extracts were prepared as follows. Cells were washed twice in normal saline, suspended in normal saline at OD600 20 and disrupted by sonication (three times for 30 s each time at 14 µm amplitude in a MSE Soniprep 150, while kept on ice). Cell debris was then removed by centrifugation (10000 g for 10 min at 4 °C). Periplasmic proteins were extracted from exponentially growing cells by the method of Ferro-Luzzi Ames et al. (1984) .
Protein determination.
Protein concentration in solution was determined by the method of Bradford using a commercial kit (Bio-Rad), with bovine serum albumin as the standard.
Recombinant DNA methodology.
Basic recombinant DNA techniques were essentially as described by Sambrook et al. (1989) . Construction of the genomic library of C. meningosepticum CCUG 4310 in the E. coli plasmid vector pACYC184 was described previously (Rossolini et al., 1998
). The pBluescript SK vector (Stratagene) was used for subcloning procedures. Restriction and modification enzymes were purchased from Boehringer.
Detection of recombinant AP-producing clones.
To detect AP activity in clones of the library, cells were plated onto TBUXP with chloramphenicol and incubated at 37 °C for 2448 h; the plates were then exposed to ammonia vapour for 23 h at room temperature; clones expressing an AP-encoding gene were then identified by their blue colour.
AP-positive colonies were subjected to plasmid extraction (Sambrook et al., 1989 ) and appropriate restriction fragments were obtained and subcloned into pBluescript SK. The AP activity of recombinant subclones was detected on PDP-MG plates.
DNA sequencing and computer analysis of sequence data.
All sequences were determined on denatured double-stranded DNA templates by the dideoxy-chain termination method (Sanger et al., 1977 ). The nucleotide sequence was determined for both strands. Sequences were compared using the FASTA 3 algorithm (http://www2.ebi.ac.uk/fasta3/) and aligned using the CLUSTAL W program (http://www2.ebi.ac.uk/clustalw/). Analysis of conserved blocks in sequences was done with the help of the program Block Maker (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html). A phylogenetic tree was generated using the CLUSTAL W 1.75 program (http://www.genebee.msu.su/genebee.html).
Phosphatase assays.
The phosphatase activity of crude cell extracts and of cell fractions was assayed with p-nitrophenyl phosphate (pNPP) as substrate by measuring the released p-nitrophenol (pNP) at 414·5 nm at pH >12 (M=18000 cm-1). The concentration of pNPP in the assays was 1 mM unless otherwise specified. Assays were carried out at 37 °C in a 0·3 ml volume, in 50 mM Tris/HCl buffer (pH 8·5) (assay buffer) unless otherwise specified and were initiated by addition of the substrate. The reaction was stopped by adding 0·7 ml 2 M NaOH. One unit was defined as the amount of enzyme able to release 1 µmol pNP min-1 under the assay conditions. The activity of the partially purified PafA enzyme as a function of the pH was assayed in a similar manner, using the following 50 mM buffer systems: sodium acetate (pH 4, 5, 6 and 6·5); Tris/HCl (pH 6·5, 7, 7·5, 8, 8·5, 9, 9·5 and 10). Significant differences were not observed using different buffer systems at overlapping pH values. The activity of the partially purified PafA enzyme against various substrates was tested in assay buffer by measuring the concentration of released Pi, after incubation for 2 min at 37 °C, by the method of Chifflet et al. (1988)
with minor modifications (the initial reaction volume was 0·1 ml and the volume of all the reagents was doubled). The substrate concentration was 1 mM. Substrate solutions were prepared as 100 mM stock solutions in double-distilled water, stored at -20 °C in small aliquots and discarded after the first thawing. Thermal stability was studied by incubating the enzyme at various temperatures in assay buffer for different periods of time. Thermal inactivation was stopped by cooling the aliquots on ice and the residual AP activity was measured at 37 °C. Reactivation was evaluated by measuring the activity of heated aliquots after 1, 24 and 48 h incubation both at 37 °C and at room temperature. The effect of inhibitors was determined by pre-incubating the enzyme in 0·3 ml assay buffer containing suitable inhibitor concentrations for 20 min at 25 °C and then starting the assay by the addition of pNPP (1 mM final concentration). Reactions were stopped after 5 min incubation at 37 °C and the released pNP was measured as described above. Control assays without the inhibitor were always run in parallel. Kinetic parameters for ATP, ADP and AMP were determined by measuring substrate hydrolysis under initial rate conditions, using the Lineweaver and Burk plot (Segel, 1976
).
Protein electrophoretic techniques.
SDS-PAGE was performed as described by Laemmli (1970) . Zymograms were performed after SDS-PAGE using partially denaturing conditions (the sample was not heated before loading; Thaller et al., 1995
) and Tris/borate-SDS as the running buffer. Following separation, the gels were washed several times with 50 mM Tris/HCl, 1% Triton X-100, pH 8·5, and AP activity was developed after overnight incubation at 37 °C in 50 mM Tris/HCl pH 8·5 with 50 µg BCIP ml-1. Bands with phosphatase activity were revealed by intense blue coloration.
Partial purification of the PafA enzyme.
Purified AP was obtained from crude extracts of phosphatase-producing strain E. coli DH5(pSpessi) grown overnight in TBU with ampicillin. After sonication, the crude extracts were centrifuged at 45000 g for 150 min and the supernatant, containing the phosphatase activity, was applied to a Sepharose DEAE-Fast Flow anion-exchange column (7 cmx2·6 cm, Pharmacia) equilibrated with 15 mM Tris/HCl (pH 7·5) buffer at a constant flow rate of 1 ml min-1. The column was then washed with three column volumes of the equilibration buffer and the bound proteins were eluted with a linear gradient of NaCl (0200 mM in 60 min). Fractions containing the enzymic activity were pooled, dialysed against 50 mM sodium acetate buffer (pH 5·0) clarified by centrifugation (20000 g for 30 min at 4 °C) to remove the precipitate formed following dialysis, and applied to a CM Sepharose-Fast Flow cation-exchange column (13·5 cmx1·6 cm, Pharmacia) equilibrated with 50 mM sodium acetate buffer (pH 5·0) at a constant flow rate of 0·5 ml min-1. The column was then washed with three column volumes of the equilibration buffer and the bound proteins were eluted with a linear gradient of NaCl (0300 mM in 60 min). The active fractions were collected, dialysed against 10 mM Tris/HCl buffer (pH 7·0), concentrated by dialysis against Spectra Gel Absorbent (Spectrum) to a final volume of 2 ml, and further purified through a HiLoad 16/60 Superdex 200 Prep Grade gel filtration column (Pharmacia), equilibrated and eluted with 100 mM sodium sulfate (pH 7·0) in 100 mM NaCl at a constant flow rate of 0·5 ml min-1. To monitor phosphatase elution, phosphatase assays were performed as described above using 1 mM pNPP as substrate.
Molecular mass determination.
The apparent molecular mass of the partially purified enzyme was determined by size-exclusion HPLC, using an Ultraspherogel SEC 3000 column (7·5 mmx30 cm) (Beckman), protected by an Ultraspherogel SEC Guard column (7·5 mmx4 cm) (Beckman), with 100 mM sodium sulfate (pH 7·0) in 100 mM NaCl as the mobile phase, at a constant flow rate of 0·5 ml min-1. Protein elution was monitored at 215 nm and phosphatase activity was measured as described above. Molecular size markers (range 12400200000, Sigma) were used to calibrate the column.
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RESULTS |
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The recombinant plasmid (pCme4) carried by clone Cme4 contained an insert of 12·8 kb (Fig. 3). Subcloning analysis revealed that the AP determinant was apparently located within a 2·5 kb HindIIISpeI fragment (Fig. 3
).
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The pafA coding sequence encodes a 546 aa polypeptide whose amino-terminus exhibits features typical of signal peptides targeting protein secretion into the periplasmic space. A putative terminator region was present 18 bp downstream of the TAA stop codon of pafA gene.
Analysis of the sequence with the program SIGCLEAVE (Nielsen et al., 1997 ) predicted the cleavage of the signal peptide after the Ala residue at position 20. This would yield a mature protein of 526 aa, with calculated molecular mass of 58682 Da and pI of 6·14.
In the sequenced region, the pafA ORF was preceded by an ORF truncated at the 5' end by the NdeI site located at the boundary of the sequenced subclone, which encodes a polypeptide showing significant sequence similarity with the C-terminal moiety of conserved eubacterial and mammalian proteins associated with proton/sodium-glutamate symport (for example from Bacillus stearothermophilus and Bacillus caldotenax) or with glutamate/aspartate transport (for example from Bos taurus). Interestingly, this ORF is in the same orientation as pafA and its termination codon is separated by only 13 bp from the ATG initiation codon of pafA, suggesting that the two genes could be part of the same transcriptional unit.
The origin of the cloned determinant from a single chromosomal region of C. meningosepticum CCUG 4310 was confirmed by PCR experiments. Primers were designed to amplify the 1·9 kb NdeIDraI fragment; a consistent amplimer was obtained using C. meningosepticum CCUG 4310 genomic DNA as template. The amplimer was restricted with EcoRV and PstI; the resulting fragments were consistent with the sequenced region. The G+C content of the cloned region was 37·8 mol%.
Structural relationships between the PafA enzyme and other bacterial APs
As already mentioned above, at the level of primary structure, the PafA AP of C. meningosepticum exhibited the closest similarity with the Cda Ca2+-ATPase of M. odoratus (51·9% identity; 73·1% similarity) (Peiffer et al., 1996 ) and with the PhoV AP of Synechococcus PCC 7942 (49·9% identity; 72·2% similarity) (Wagner et al., 1995
), whereas lower degrees of similarity were observed with the PhoD AP of Z. mobilis (22·1% identity; 44·4% similarity) (Gomez & Ingram, 1995
) and with the PhoA AP of E. coli (14·0% identity; 30·1% similarity) (Bradshaw et al., 1981
).
The analysis of the deduced amino acid sequence of pafA did not reveal outstanding homologies with the putative sites of phosphorylation of either PhoV (55% identity) (Wagner et al., 1995 ) or Cda (55% identity) (Peiffer et al., 1996
). However, the Asp-158 residue of the Ca2+-ATPase, which is implicated in the autophosphorylation process of the P-type ATPases (Peiffer et al., 1996
), is retained in the PafA sequence. As far as Z. mobilis PhoD is concerned, the Thr-107 residue, indicated as probably being involved in the formation of covalent phosphoryl-enzyme intermediate (Gomez & Ingram, 1995
), was conserved in PafA, PhoV (Wagner et al., 1995
) and Cda sequences (Peiffer et al., 1996
).
Comparison of PafA and of the related PhoV, Cda and PhoD enzymes with the E. coli PhoA AP at the level of conserved blocks in the aligned sequences (Fig. 4) revealed notable similarities in some of the regions that in PhoA are known to be involved in metal binding and phosphorylation (Kim & Wyckoff, 1991
). In particular, all the residues involved in Zn2 binding (Asp-51, Asp-369 and His-370) are conserved in PafA and related enzymes, while of those involved in Zn1 binding only Asp-327 and His-331 are conserved and no clear counterpart is detectable for His-412 (a conserved histidine is actually present in the C-terminal region of PafA and related enzymes, but not within a sequence block conserved with PhoA). Concerning the residues involved in Mg binding, only Asp-51 is conserved, while Thr-155 and Glu-322 are not conserved in PafA and related enzymes. Finally, the Ser-102 residue that in PhoA is involved in phosphorylation, is not conserved in PafA and related enzymes (Fig. 4
).
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Characterization of the PafA enzyme
The molecular mass of the polypeptide component, estimated by SDS-PAGE, was approximately 60 kDa and that of the native protein, estimated by size-exclusion chromatography, was approximately 180 kDa. Experiments using ultrafiltration were confirmatory. In fact, the partially purified enzyme was apparently completely retained by ultrafiltration membranes with a 100 kDa cut-off.
Zymograms revealed that the partially purified protein exhibited a pattern identical to that shown by the enzyme extracted from the parental strain (data not shown).
The optimum pH was studied with pNPP as substrate. Phosphatase activity was observed between pH 7·0 and pH 9·5, with optimal activity at pH 8·5. The enzyme lost 50% of its activity at pH 7·5 and 70% at pH 9·0, being virtually inactive at pH 10 and at pH 5 or lower.
Orthovanadate and EDTA were greatly inhibitory to the PafA activity (IC50 0·5 µM and 30 µM, respectively). Reactivation of enzymic activity after dialysis was not successful. Also, Pi inhibited the PafA activity (IC50 340 µM).
The enzyme was heat-labile. Incubation of PafA for 10 min at 50 or 60 °C caused 40 and 60% inactivation, respectively. Heating at 100 °C for 2 min caused total loss of activity. No restoration of phosphatase activity was observed even upon prolonged incubation of the heat-treated enzyme at room temperature or at 37 °C.
At pH 8·5, the enzyme exhibited a broad substrate specificity, being able to dephosphorylate a great variety of phosphomonoesters with the exception of 2':3'-cyclic AMP and phytic acid. PafA hydrolysed D-glucose 1-phosphate, D-glucose 6-phosphate, ribose 5-phosphate and -naphthyl phosphate at rates of 47, 15, 66 and 99% of that measured with pNPP. Moreover, it was able to dephosphorylate 3'- and 5'-nucleotides such as ADP, ATP, 3'-AMP, 3'-GMP, 3'-UMP, 5'-AMP, 5'-GMP, 5'-CMP and 5'-UMP at rates of 83, 109, 100, 60, 96, 96, 104, 61 and 39%, respectively, of that measured with pNPP. Kinetic parameters towards adenosine mono, di- and triphosphate are reported in Table 1
. The enzyme showed a high activity against all these substrates; the highest affinity was obtained with ATP and the highest Vmax value was recorded for ADP. The Vmax/Km ratio for ATP was twofold higher than that for ADP and 10-fold higher than that for AMP.
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DISCUSSION |
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The pafA product exhibited a relatively high homology with the Cda Ca2+-ATPase of M. odoratus and the PhoV AP of Synecochoccus PCC 7942, a lower homology with the PhoD AP of Z. mobilis, and an even lower homology with the E. coli PhoA AP. Interestingly, the sequence of PafA demonstrates homology with E. coli PhoA in regions near to the metal-binding sites of PhoA, and some important residues involved in the active site of PhoA (Kim & Wyckoff, 1989 ; Murphy & Kantrowitz, 1994
) are conserved in the PafA sequence. It could be suggested that regions important for the function of the enzymes could be derived from a common ancestor and that subsequent evolution has caused differentiation in the biochemical and functional properties of the two lineages.
PafA can be considered a broad-spectrum AP with a preferential activity on ATP. PhoD of Z. mobilis and the Ca2+-ATPase of M. odoratus also demonstrated the highest activity on ATP (Gomez & Ingram, 1995 ; Desrosiers et al., 1996
). However, Cda is a narrow-spectrum enzyme as it is able to hydrolyse essentially only ATP (Desrosiers et al., 1996
), whilst PhoD is active on different substrates (Gomez & Ingram, 1995
). PhoV of Synechococcus, on the other hand, does not show a particularly high activity on ATP, and is able to dephosphorylate several substrates at similar rates (Wagner et al., 1995
). PafA, therefore, apparently shares some functional characteristics with both Cda and PhoV. Like both of them, PafA is partially inhibited by Pi (Gambel et al., 1992
; Wagner et al., 1995
); like Cda, PafA is highly sensitive to o-vanadate (Desrosiers et al., 1996
); like PhoV, PafA is inactivated by temperature (Wagner et al., 1995
) unlike other APs (Garen & Levinthal, 1960
).
The sequenced fragment of the pafA region indicates that the gene is probably part of an operon as it is located downstream of another gene whose product is similar to proton/glutamate symport proteins. This fact, taken together with the high activity on ATP shown by PafA, led us to suppose that this enzyme could be involved in the energy-producing process necessary for the function of the associated transport protein(s).
The analysis of the phylogenetic tree allowed us to recognize a new enzyme family of APs evolutionarily distant from that of PhoA of E. coli and other PhoA-like enzymes. This new enzyme family encompasses enzymes from taxonomically unrelated species; all the enzymes share some features such as molecular mass, Pi-irrepressible expression and periplasmic location. Among the enzymes included in the new family, some (PafA, PhoV and PhoD) are broad-spectrum enzymes and one (Cda) is a narrow-spectrum enzyme. PafA, however, exhibits a relevant activity on ATP, at rates similar to that of Cda, indicating that it could be an evolutionary intermediate between this latter enzyme and the Pi-irrepressible APs. Perhaps in this family could be included also the AP of Prevotella intermedia that was described as a tyrosine-phosphatase enzyme with molecular mass of 54 kDa, showing partial similarity with the PhoD and PhoV sequences (Ansai et al., 1998 ). However, as the complete amino acid sequence is not yet known, this enzyme was not included in the phylogenetic analysis.
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ACKNOWLEDGEMENTS |
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Received 5 March 2001;
revised 4 June 2001;
accepted 15 June 2001.
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