- Research article
- Open Access
Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli
BMC Research Notesvolume 7, Article number: 221 (2014)
The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications.
Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 μM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol.
The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops.
The genus Burkholderia is widespread in diverse ecological niches; however, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants [1, 2]. Following the pioneering work of Yabuuchi et al., which described the Burkholderia genus, several investigators have studied Burkholderia species that are phylogenetically distant from the Burkholderia cepacia complex (Bcc species), which are promising candidates for biotechnological applications [4, 2], although their environmental distribution and relevant characteristics for agro-biotechnological applications are not well known.
Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant [5–7]. Interactions between plant roots and mineral phosphate solubilizing (MPS) microorganisms can play an important role in phosphorus nutrition and growth of most plants, microorganisms and crop production. As far as we know, the present report is the first systematic study to show that the membrane-bound acid phosphatase expressed by Burkholderia is strictly regulated by phosphorus. In addition, little is known about the enzyme’s potential applications to improve plant growth by association with the bacteria.
Phosphorus is an essential nutrient that is required in large amounts to maintain levels of key cell molecules, including ATP, nucleic acids, and phospholipids; phosphorus is also a pivotal mediator in the regulation of many metabolic processes, such as energy transfer, protein activation, regulation of enzyme activities, gene activity control, as so in carbon and amino acid metabolic processes .
The uptake of nutrients from different natural environments depends on the secretion of an enormous variety of hydrolytic enzymes, which demonstrate catalytic activity that is specific for the cleavage of a particular substrate. This uptake process is tightly regulated and contains a variety of biochemical reactions that involve the acquisition, storage, and release of enzymes . The study of these processes may provide new insights for the elucidation of gene expression that controls not only the synthesis but also the secretion of enzymes by eukaryotic cells in response to environmental factors, such as pH and levels of carbon, nitrogen, sulfur and phosphorus [10, 11].
In this paper, we report the expression and kinetic characterization of a membrane-bound acid phosphatase produced by Burkholderia gladioli that was isolated from the rhizosphere of Zea mays, which was collected from an agricultural soil in Ponta Grossa-PR-Brazil.
Isolation and identification of Burkholderia sp
The isolation of Burkholderia sp. bacteria from surface-sterilized roots of Zea mays, which were collected from agricultural soil in Ponta Grossa-PR-Brazil, was described by Pedrinho et al., and the bacteria was identified through partial 16S rRNA gene sequencing, using the specific oligonucleotides fD1 and rD1 .
The partial sequencing of the 16S rRNA gene was performed by the use of 1,0 μL of DNA Sequencing-Big Dye Terminator Cycle Sequencing-Ready ABI Prism (Version 3); 3.2 pmols of fD1/rD1 oligonucleotide, 60 ng of DNA, 4.6 μL of buffer (400 mM Tris–HCl, pH 9; 10 mM (MgCl2); and mili-Q (Millipore) H2O for a 10 mL volume.
The amplicons were sequenced using the model ABI 3100 capillary sequencer (Applied Biosystems, Foster City, CA, USA). The fasta sequence was analyzed by comparison using a local tool BLASTN  from NCBI (National Center of Biotechnology Information) and classified by RDP (Ribosomal Database Project).
The evolutionary history was inferred using the Neighbor-Joining method . The optimal tree with the sum of branch length = 0.19803035 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches . The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method  and are in the units of the number of base differences per site. The analysis involved 11 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 733 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 . The 16S rDNA sequence obtained is registered at the International Gene Bank (GenBank), having the access number: JN 700991.
Growth conditions and membrane-bound enzyme isolation
Burkholderia sp. was grown in a liquid medium containing 2% glucose, 0.1% magnesium chloride, 0.025% magnesium sulfate, 0.1% ammonium sulfate, 0.02% potassium chloride, and with or without potassium phosphate. The liquid growth medium, prepared in 250-ml conical flasks, was incubated for 72 h at 30°C under constant rotary shaking at 140 rpm. Actively growing cells were collected by centrifugation, washed twice with 50 mM sodium acetate buffer at pH 6.0, resuspended in 8 mL of the same buffer and then disrupted by sonication at 50 microtips/second with cycles of 30 seconds with a Branson Sonifier model 250. The integral cells were removed by centrifugation at 5,000 g for 15 min. The supernatant was subjected to a two-step differential centrifugation, first at 12,000 g and then for 1 h at 160,000 g to obtain soluble proteins and membrane bound enzyme. The pellet, which corresponds to the membrane-bound enzyme, was resuspended in the same buffer. Aliquots (1.0 ml) were frozen in liquid nitrogen and stored at -20°C without appreciable loss of activity when stored for less than 2 months.
Enzymatic activity measurements
Acid phosphatase activity was determined discontinuously at 37°C, 50 mM acetate buffer, pH 6.0, through the formation of p-nitrophenolate (ϵ = 17600 M-1 cm-1, pH 13) at 410 nm from the hydrolysis of 1 mM p-nitrophenylphosphate (SIGMA®). The enzymatic reaction was initiated by the addition of the enzyme extract to the reaction medium, interrupted by adding 1 ml of 1 M NaOH, and the absorbance was determined at 410 nm.
The determinations were performed in triplicates and the initial velocities remained constant during the incubation time to ensure that substrate hydrolysis was inferior to 5%. In each determination standards were included to estimate the non-enzymatic hydrolysis of substrate.
A unit of enzyme activity was defined and expressed as the amount of enzyme that releases one nmol of p-nitrophenolate per minute, per milligram of protein present in the enzymatic extract, under test conditions.
Thermal inactivation of membrane-bound acid phosphatase
Samples of membrane-bound enzyme in 50 mM acetate buffer at pH 6.0 were incubated in a water bath at different temperatures for variable periods of time. Immediately after the water bath treatment, samples were chilled in an ice-water bath to stop the inactivation process, and the remaining PNPPase activity was assayed as described above.
Effect of several compounds on the p-nitrophenylphosphatase activity
Reactions were carried out in 50 mM acetate buffer at pH 6.0, containing 1 mM of PNPP and the following compounds: phosphate (10 mM); EDTA (10 mM); arsenate (1 mM); magnesium (2 mM); calcium (1 mM); zinc (1 mM); cobalt (1 mM); levamisole (10 mM); sodium tartrate (10 mM); bafilomycin A1 (1 mM); oligomycin (1.5 mg/ml); ouabain (1.3 mM); pantoprazol (6 mM); PHMB (1 mM); and vanadate (0.5 mM), in a final volume of 1.0 ml. The reaction was initiated by the addition of the enzyme and stopped by the addition of 1.0 ml of 1.0 M NaOH at the appropriate time. In each determination standards were included to estimate the non-enzymatic hydrolysis of substrate.
Effect of pH on p-nitrophenylphosphate hydrolysis by membrane-bound acid phosphatase
Assays were buffered with 50 mM acetate for the pH range 3.5-6.5, and 50 mM Tris–HCl for the pH range 6.5-8.0; each reaction contained 1 mM of PNPP. There was no significant difference among the two buffers used at pH 6.5. The pH before and after each kinetic determination did not differ by more than 0.05 units. The reaction was initiated by the addition of the membrane-bound enzyme and stopped with 1.0 ml of 1.0 M NaOH at the appropriate time. In each determination standards were included to estimate the non-enzymatic hydrolysis of substrate.
Determination of protein concentrations
Protein concentrations were determined according to the method described by Hartree . Bovine serum albumin was used as the standard in both cases.
Estimation of kinetic parameters
V, v, K0.5 and n obtained from substrate hydrolysis reactions were fit using a microcomputer as described by Pizauro et al.. Data are reported as the mean of triplicate determinations that differed by less than 5%.
Results and discussion
To quickly and reliably determine if the isolate belonged to the Burkholderia species, the amplified 16S rDNA was compared with the most similar found in databases, and phylogenetic analysis showed that the strain belonged to a Burkholderia species (Figure 1). This isolate clustered with a set of Burkholderia strains classified as Burkholderia gladioli (GenBank BankIt accession nº JN 700991).
Figure 2 shows the effect of increasing concentrations of phosphate in the medium used to grow Burkholderia gladioli. The acid phosphatase activity was maximal at 5 mM phosphate, and reduced activity was observed when Burkholderia gladioli was grown in medium containing higher concentrations of phosphate. These results suggest that acid phosphatase in Burkholderia gladioli is synthesized exclusively under Pi-limiting conditions, which is characterized as Pi-repressible activity. The behaviors observed here show that acid phosphatase activity is downregulated by exogenous Pi concentration, suggesting that there is a tight coupling among the utilization of exogenous Pi, mobilization of endogenous reserves, and derepression of acid phosphatase. In fact, several phosphatases from plant and fungal origins were shown to be induced by phosphate deficiency [21–24].
The benefits to use differential centrifugation to obtain the membrane-bound acid phosphatase is that this method is easy to reproduce, fast and highly reproducible. This method resulted in the separation of two fractions. The supernatant containing soluble proteins, which represented less than 5% of total activity and the pellet, corresponding to the membrane-bound enzyme, which represented more than 95% of total activity, and showed specific activity of 103.9 U/mg for the chromogenic substrate; this fraction was used in further studies.
Membrane-bound enzyme was stable after 6 h of incubation at 45°C in 100 mM acetate buffer at pH 6.0, but the enzyme was inactivated at higher temperatures, exhibiting a t1/2 that varied from 23 h at 50°C to 5 min at 70°C. When the temperature was increased from 10°C to 70°C, enzyme inactivation followed first order kinetics, and no break in the inactivation curves was observed from 10°C to 25°C (Figure 3), suggesting that this enzyme is associated with lipids in the membrane but is not an intrinsic membrane-bound enzyme . This behavior is consistent with that reported for alkaline phosphatase from rat bone matrix-induced cartilage , which is anchored to the membrane by phosphatidylinositol [27, 28], and acid phosphatase with phytase activity from Mucor hiemalis. In addition, according to Kondo et al. , acid phosphatase from Burkholderia is a glycoprotein that is translocated during glycosylation from the cytoplasm to the outer membrane and then excreted into the environment.
The rate of hydrolysis of PNPP (Figure 4) by membrane-bound enzyme reached a maximum at pH 6.0, and this pH was selected as the incubation medium for the activity assays of acid phosphatase. This value for the optimum pH is similar to those reported for bacterial nonspecific acid phosphohydrolases , acid phosphatase from Aspergillus ficuum, and Burkholderia cepacia.
The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. The specific activity of the enzyme for the hydrolysis of PNPP was 113.5 U/mg and K0.5 = 65 μM (Figure 5). Kinetic data revealed that the hydrolysis of PNPP exhibited cooperativity with n = 1.3.
The ability of this enzyme to dephosphorylate phosphoesteres and the observed magnitudes of the kinetic values are consistent with those obtained for acid phosphatase from other sources [33, 31]. In addition, the enzymatic properties of acid phosphatase were virtually identical to the acid phosphatase of E. histolytica, which catalyzes p-nitrophenylphosphate hydrolysis under acid pH conditions.
The effects of several compounds on p-nitrophenylphosphatase activity of the enzyme are shown in Table 1. EDTA and tartrate reagents that were present in concentrations up to 10 mM showed only minor effects on enzymatic activity. The lack of inhibition by EDTA and sodium tartrate on PNPPase activity and the lack of dependence on magnesium, calcium, cobalt and zinc was also observed, suggesting that metal ions are not involved in maintaining enzyme activity [35–37, 26, 38]. The significant inhibition observed for vanadate (94%), does not imply, unequivocally, that this membrane-bound enzyme is a specific P-type ATPase. This interpretation is supported by the lack of inhibition by ouabain, which is an inhibitor of the Na+/K+-ATPase. Bafilomycin is also a highly specific inhibitor of vacuolar H+-ATPases (V-ATPase) type in animal cells, plant cells and microorganisms  and does not inhibit the enzyme studied here. In addition, vanadate ions form a trigonal bipyramidal transition state at the active site of phosphatase, which bears some resemblance to the metastable intermediate occurring during the hydrolysis of phosphate esters . Phosphate and arsenate are well known as inhibitors of acid phosphatase activity; the inhibition of PNPPase activity by phosphate and the more efficient inhibition by sodium arsenate, which is its structural analog, suggest a common mode of binding to the active site . Similar to other phosphohydrolases, membrane-bound acid phosphatase is significantly inhibited by sodium orthovanadate [41, 42] and p-hydroxymercuribenzoate [43, 44], which suggests that the sulfhydryl residue of cysteine is essential for its activity.
The main mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil . Considering that acid phosphatase is bound to the external membrane surface and therefore exposed to extracellular medium, our results bring an important insight on the mechanism of mineral phosphate solubilization by this bacterium and on plant nutrition through the increase in P uptake by the plant, mainly in soils with low levels of phosphate that are found in many regions of the world.
Although Burkholderia gladioli is known as a pathogen in some plant species  and causes opportunistic infection in severely immunocompromised humans , Bae  reported that a strain of this species have the ability to suppress pathologies caused by Pythium ultimum. In addition, B. gladioli has been described as a possible biofertilizer because of its capacity to fix nitrogen, mobilize phosphorus and stimulate plant growth [48–50]. It should be emphasized that B. gladioli also promotes beneficial effects as plant growth and nitrogen fixation in sugarcane crops . Therefore, it may be possible to use this bacterium as a biofertilizer for specific crops, besides biochemical studies can contribute to elucidate its mechanisms.
Through analysis of the 16S rDNA our strain was classified as Burkholderia gladioli (GenBank BankIt accession nº JN 700991), therefore, phylogenetically distant from the Burkholderia cepacia complex (Bcc species). The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element for specific crops.
Coenye T, Vandamme P: Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ Microbiol. 2003, 5 (9): 19-29.
Compant S, Nowak J, Coenye T, Clément C, Ait Barka E: Diversity and occurrence of Burkholderia spp. in the natural environment. FEMS Microbiol Rev. 2008, 32 (4): 607-626. 10.1111/j.1574-6976.2008.00113.x.
Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, Hashimoto Y, Ezaki T, Arakawa M: Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia Palleroni and Holmes 1981. Microbiol Immunol. 1992, 36: 1251-1275. 10.1111/j.1348-0421.1992.tb02129.x.
Caballero-Mellado J, Onofre-Lemus J, Estrada de LosSantos P, Martínez-Aguilar L: The tomato rhizosphere, an environment rich in nitrogen-fixing Burkholderia species with capabilities of interest for agriculture and bioremediation. Appl Environ Microbiol. 2007, 73 (16): 5308-5319. 10.1128/AEM.00324-07.
Barea JM, Pozo MJ, Azcón R, Azcón-Aguilar C: Microbial co-operation in the rhizosphere. J Exp Bot. 2005, 56 (417): 1761-1778. 10.1093/jxb/eri197.
Lin TF, Huang HI, Shen FT, Young CC: The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-Al74. Bioresour Technol. 2006, 97 (7): 957-960. 10.1016/j.biortech.2005.02.017.
Song OR, Lee SJ, Lee YS, Lee SC, Kim KK, Choi YL: Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil. Braz J Microbiol. 2008, 39 (1): 151-156. 10.1590/S1517-83822008000100030.
Li L, Qiu X, Li X, Wang S, Lian X: The expression profile of genes in rice roots under low phosphorus stress. Sci China Life Sci. 2009, 52 (11): 1055-1064. 10.1007/s11427-009-0137-x.
Torriani-Gorini A: Regulation of phosphate metabolism and transport. Phosphate in microorganisms: cellular and molecular biology. Edited by: Torriani-Gorini A, Yagil E, Silver S. 1994, Washington D.C: American Society for Microbiology, 1-4.
Caddick MX, Brownlee AG, Arst JRHN: Phosphatase regulation in Aspergillus nidulans: Responses to nutritional starvation. Genet Res. 1986, 47: 93-102. 10.1017/S0016672300022916.
Nozawa SR, Maccheroni JW, Stábeli RG, Thedei JG, Rossi A: Purification and properties of pi-repressible acid phosphatases from Aspergillus nidulans. Phytochemistry. 1998, 49 (6): 1517-1523. 10.1016/S0031-9422(98)00205-2.
Pedrinho EAN, Galdiano-Júnior RF, Campanharo JC, Carareto-Alves LM, Lemos EGM: Identificação e avaliação de rizobactérias isoladas de raízes de milho. Bragantia. 2010, 69 (4): 905-911. 10.1590/S0006-87052010000400017.
Weisburg WG, Barns SM, Pelletier DA, Lane DJ: 16S ribossomal DNA amplification for phylogenetic study. J Bacteriol. 1991, 173 (2): 697-703.
Astschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.
Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987, 4: 406-425.
Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985, 39: 783-791. 10.2307/2408678.
Nei M, Kumar S: Molecular Evolution and Phylogenetics. 2000, New York: Oxford University Press
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.
Hartree EF: Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972, 48: 422-427. 10.1016/0003-2697(72)90094-2.
Pizauro JM, Demenis MA, Ciancaglini P, Leone FA: Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochondral ossification. Biochemica et Biophysics Acta- Biomembranes. 1998, 1368 (1): 108-114. 10.1016/S0005-2736(97)00174-0.
Ullah AH, Cummins BJ: Aspergillus ficuum extracellular pH 6.0 optimum acid phosphatase: purification, N-terminal amino acid sequence, and biochemical characterization. Prep Biochem. 1988, 18 (1): 37-65.
Olczak M, Morawiecka B, Watorek W: Plant purple acid phosphatases-genes, structures and biological function. Acta Biochemica Polonica. 2003, 50: 1245-1256.
Lung SC, Leung A, Kuang R, Wang Y, Leung P, Lim BL: Phytase activity in tobacco Nicotiana tabacum root exudates is exhibited by a purple acid phosphatase. Phytochemistry. 2008, 69 (2): 365-373. 10.1016/j.phytochem.2007.06.036.
Yeung SL, Cheng C, Lui TKO, Tsang JSH, Chan W, Lim BL: Purple acid phosphatase-like sequences in prokaryotic genomes and the characterization of an atypical purple alkaline phosphatase from Burkholderia cenocepacia J2315. Gene. 2009, 440: 1-8. 10.1016/j.gene.2009.04.002.
Barton PL, Futerman AH, Silman I: Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C. Biochem J. 1985, 231: 237-240.
Curti C, Pizauro JM, Ciancaglini P, Leone FA: Kinetic characteristic of some inhibitors of matrix-induced alkaline phosphatase. Cell Mol Biol. 1987, 33: 625-635.
Pizauro JM, Ciancaglini P, Leone L: Osseous Plate alkaline phosphatase is anchored by GPI. Braz J Med Biol Res. 1994, 27: 453-456.
Pizauro JM, Ciancaglini P, Leone FA: Characterization of the hosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. Mol Cell Biochem. 1995, 152 (2): 121-129. 10.1007/BF01076074.
Boyce A, Walsh G: Purification and characterization of an acid phosphatase with phytase activity from Mucor hiemalis Wehmer. J Biotechnol. 2007, 132 (1): 82-87. 10.1016/j.jbiotec.2007.08.028.
Kondo E, Kurata T, Naigowit P, Kanai K: Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei. Southeast Asian J Trop Med Public Health. 1996, 27: 592-599.
Rossolini GM, Schippa S, Riccio ML, Berlutti F, Macaskie LE, Thaller MC: Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology. Cell Mol Life Sci. 1998, 54 (8): 833-850. 10.1007/s000180050212.
Ferreira AS, Leitão JH, Sousa SA, Cosme AM, Sá-Correia I, Moreira LM: Functional Analysis of Burkholderia cepacia Genes BCED and bceF, Encoding a Phosphotyrosine Phosphatase and a Tyrosine Autokinase, Respectively: Role in Exopolysaccharide Biosynthesis and Biofilm Formation. ApplEnviron Microbiol. 2007, 73 (2): 524-534.
Toh-E A, Ueda Y, Kakimoto SI, Oshima Y: Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiae. J Bacteriol. 1973, 113 (2): 727-738.
Anaya-Ruiz M, Pérez-Santos JL, Talamás-Rohana P: An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells. Int J Parasitol. 2003, 33 (7): 663-670. 10.1016/S0020-7519(03)00029-8.
Anderson RA, Bosron WF, Kennedy FS, Vallee BL: Role of magnesium in Escherichia coli alkaline phosphatase metal content/metalloenzyme regulation/spectral properties/tritium exchange. Proc Natl Acad Sci USA. 1975, 72: 2989-2999. 10.1073/pnas.72.8.2989.
Mccomb RB, Bowers GN, Posen S: Alkaline phosphatase. 1979, New York: Plenum Press
Fortuna R, Anderson HC, Carty R, Sajdera SW: Enzymatic characterization of the matrix vesicle alkaline phosphatase isolated from bovine fetal epiphyseal cartilage. Calcif Tissue Int. 1980, 30 (1): 217-225. 10.1007/BF02408631.
Ciancaglini P, Pizauro JM, Grecchi MJ, Curti C, Leone FA: Effect of ZnII and MgII on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase. Mol Cell Biol. 1989, 35: 503-510.
Lopez V, Stevens T, Lindquist RN: Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis. Arch Biochem Biophys. 1976, 175 (1): 31-38. 10.1016/0003-9861(76)90482-3.
Durmus A, Eicken C, Sift BH, Kratel A, Kappl R, Hüttermann J, Krebs B: The active site of purple acid phosphatase from sweet potatoes Ipomoea batatas Metal content and spectroscopic characterization. Eur J Biochem. 1999, 260 (3): 709-716. 10.1046/j.1432-1327.1999.00230.x.
Fukami Y, Lipmann F: Purification of a specific reversible tyrosine-O-phosphate phosphatase. Proc Natl Acad Sci USA. 1982, 79: 4275-4279. 10.1073/pnas.79.14.4275.
Lau KHW, Farley JR, Baylink DJ: Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase. J Biol Chem. 1985, 260: 4653-4660.
Granjeiro JM, Ferreira CV, Jucá MB, Taga EM, Aoyama H: Bovine kidney low molecular weight acid phosphatase: FMN-dependent kinetics. Biochem Mol Biol Int. 1997, 41: 1201-1206.
Buzalaf MAR, Granjeiro JM, Ferreira CV, Lourenção VA, Ortega MM, Poletto DW: Kinetic characterization of bovine lung lowmolecular-weight protein tyrosine phosphatase. Exp Lung Res. 1998, 24 (3): 269-272. 10.3109/01902149809041534.
Rodríguez H, Fraga R: Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv. 1999, 17 (4–5): 319-339.
Graves M, Robin T, Chipman AM: Four additional cases of Burkholderia gladioli infection with microbiological correlates and review. Clin Infect Dis. 1997, 25 (4): 838-842. 10.1086/515551.
Bae YS, Park K, Choi OH: Laboratory culture media-dependent biocontrol ability of Burkholderia gladioli strain B543. Plant Pathol J. 2007, 23 (3): 161-165. 10.5423/PPJ.2007.23.3.161.
Babalola OO, Akindolire AM: Identification of native rhizobacteria peculiar to selected food crops in Mmabatho municipality of South Africa. Biol Agric Hortic An International Journal for Sustainable Production Systems. 2011, 27 (3–4): 294-309.
Mamta P, Rahi R, Pathania V, Gulati A, Singh B, Bhanwra RK, Tewari R: Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside and rebaudioside-A contents of Stevia rebaudiana Bertoni. Appl Soil Ecol. 2010, 46: 222-10.1016/j.apsoil.2010.08.008.
Pereira APA, Silva MCB, Oliveira JRS, Ramos APS, Freire MBGS, Freire FJ, Kuklinsky-Sobral J: Salinity influence on the growth and production of indole acetic acid by endophytic Burkholderia spp. from sugarcane. Bioscience J. 2012, 28 (1): 112-121.
This research was supported by FAPESP and CNPq. We would like to thank Dra. Silvana Pompéia Val-Moraes for the reading and scientific advice.
The authors declare that they have no competing interests.
THR executed the enzymatic assays and enzyme extraction and analyzed the data, along with AMG and LFJS. EANP and EGML performed the genetic studies and analysis. JMPJ conceived of the study and wrote the manuscript. All authors read and approved the final manuscript.