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Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1

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Abstract

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.010.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s−1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

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References

  1. Abe K, Yoshida S, Suzuki Y, Mori J, Doi Y, Takahashi S, Kera Y (2014) Haloalkylphosphorus hydrolases purified from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1. Appl Environ Microbiol 80(18):5866–5873. https://doi.org/10.1128/AEM.01845-14

  2. Abe K, Mukai N, Morooka Y, Makino T, Oshima K, Takahashi S, Kera Y (2017) An atypical phosphodiesterase capable of degrading haloalkyl phosphate diesters from Sphingobium sp. strain TCM1. Sci Rep 7(1):2842. https://doi.org/10.1038/s41598-017-03142-9

  3. Alves MP, Salgado RL, Eller MR, Vidigal PMP, Fernandes de Carvalho A (2016) Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity. J Dairy Sci 99(10):7842–7851. https://doi.org/10.3168/jds.2016-11236

  4. Ansai T, Awano S, Chen X, Fuchi T, Arimoto T, Akifusa S, Takehara T (1998) Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia. FEBS Lett 428(3):157–160. https://doi.org/10.1016/s0014-5793(98)00514-6

  5. Bartolommei G, Moncelli MR, Tadini-Buoninsegni F (2013) A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases). PLoS One 8(3):e58615. https://doi.org/10.1371/journal.pone.0058615

  6. Bihani SC, Das A, Nilgiriwala KS, Prashar V, Pirocchi M, Apte SK, Ferrer JL, Hosur MV (2011) X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases. PLoS One 6(7):e22767. https://doi.org/10.1371/journal.pone.0022767

  7. Chaudhuri G, Chatterjee S, Venu-Babu P, Ramasamy K, Thilagaraj WR (2013) Kinetic behaviour of calf intestinal alkaline phosphatase with pNPP. Indian J Biochem Biophys 50(1):64–71

  8. Choi JJ, Park JW, Shim H, Lee S, Kwon M, Yang JS, Hwang H, Kwon ST (2006) Cloning, expression, and characterization of a hyperalkaline phosphatase from the thermophilic bacterium Thermus sp T351. J Microbiol Biotechnol 16(2):272–279

  9. Forsberg CW, Cheng KJ (1980) The constitutive nature of alkaline phosphatase in rumen bacteria. Can J Microbiol 26(2):268–272. https://doi.org/10.1139/m80-043

  10. Green MR, Sambrook J (2014) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New York

  11. Hauksson JB, Andrésson ÓS, Ásgeirsson B (2000) Heat-labile bacterial alkaline phosphatase from a marine Vibrio sp. Enzym Microb Technol 27(1–2):66–73. https://doi.org/10.1016/s0141-0229(00)00152-6

  12. Hou CI, Gronlund AF, Campbell JJ (1966) Influence of phosphate starvation on cultures of Pseudomonas aeruginosa. J Bacteriol 92(4):851–855

  13. Hsieh YJ, Wanner BL (2010) Global regulation by the seven-component Pi signaling system. Curr Opin Microbiol 13(2):198–203. https://doi.org/10.1016/j.mib.2010.01.014

  14. Iglesias Neves H, Pereira TF, Yagil E, Spira B (2015) Ugp and PitA participate in the selection of PHO-constitutive mutants. J Bacteriol 197(8):1378–1385. https://doi.org/10.1128/JB.02566-14

  15. Kadurugamuwa JL, Beveridge TJ (1997) Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother 40(5):615–621. https://doi.org/10.1093/jac/40.5.615

  16. Lee DH, Choi SL, Rha E, Kim SJ, Yeom SJ, Moon JH, Lee SG (2015) A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments. BMC Biotechnol 15(1):1. https://doi.org/10.1186/s12896-015-0115-2

  17. Lin HY, Shih CY, Liu HC, Chang J, Chen YL, Chen YR, Lin HT, Chang YY, Hsu CH, Lin HJ (2013) Identification and characterization of an extracellular alkaline phosphatase in the marine diatom Phaeodactylum tricornutum. Mar Biotechnol (NY) 15(4):425–436. https://doi.org/10.1007/s10126-013-9494-3

  18. McComb RB, Bowers GN, Posen S (1979) Alkaline phosphatase. Plenum Press, New York

  19. Nilgiriwala KS, Alahari A, Rao AS, Apte SK (2008) Cloning and overexpression of alkaline phosphatase PhoK from Sphingomonas sp. strain BSAR-1 for bioprecipitation of uranium from alkaline solutions. Appl Environ Microbiol 74(17):5516–5523. https://doi.org/10.1128/AEM.00107-08

  20. Ordonez-Robles M, Santos-Beneit F, Rodriguez-Garcia A, Martin JF (2017) Analysis of the Pho regulon in Streptomyces tsukubaensis. Microbiol Res 205:80–87. https://doi.org/10.1016/j.micres.2017.08.010

  21. Pop O, Martin U, Abel C, Muller JP (2002) The twin-arginine signal peptide of PhoD and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system. J Biol Chem 277(5):3268–3273. https://doi.org/10.1074/jbc.M110829200

  22. Post MA (2006) Alkaline phosphatase from psychrophile TAB5 and cold-adapted, northern shrimp (Pandalus borealis) are structurally similar yet functionally distinct. FASEB J 20(4):A479

  23. Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57(1):50–108

  24. Roy NK, Ghosh RK, Das J (1982) Monomeric alkaline phosphatase of Vibrio cholerae. J Bacteriol 150(3):1033–1039

  25. Takahashi S, Kawashima K, Kawasaki M, Kamito J, Endo Y, Akatsu K, Horino S, Yamada RH, Kera Y (2008) Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures. J Biosci Bioeng 106(1):27–32. https://doi.org/10.1263/jbb.106.27

  26. Takahashi S, Satake I, Konuma I, Kawashima K, Kawasaki M, Mori S, Morino J, Mori J, Xu H, Abe K, Yamada RH, Kera Y (2010) Isolation and identification of persistent chlorinated organophosphorus flame retardant-degrading bacteria. Appl Environ Microbiol 76(15):5292–5296. https://doi.org/10.1128/AEM.00506-10

  27. Takahashi S, Abe K, Kera Y (2013) Microbial degradation of persistent organophosphorus flame retardants. In: Petre M (ed) Environmental biotechnology – new approaches and prospective applications. IntechOpen, London, pp 91–122. https://doi.org/10.5772/53749

  28. Takahashi S, Katanuma H, Abe K, Kera Y (2017) Identification of alkaline phosphatase genes for utilizing a flame retardant, tris(2-chloroethyl) phosphate, in Sphingobium sp. strain TCM1. Appl Microbiol Biotechnol 101(5):2153–2162. https://doi.org/10.1007/s00253-016-7991-9

  29. Wu JR, Shien JH, Shieh HK, Hu CC, Gong SR, Chen LY, Chang PC (2007) Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) from Pasteurella multocida strain X-73. FEMS Microbiol Lett 267(1):113–120. https://doi.org/10.1111/j.1574-6968.2006.00542.x

  30. Yu Plisova E, Balabanova LA, Ivanova EP, Kozhemyako VB, Mikhailov VV, Agafonova EV, Rasskazov VA (2005) A highly active alkaline phosphatase from the marine bacterium cobetia. Mar Biotechnol (NY) 7(3):173–178. https://doi.org/10.1007/s10126-004-3022-4

  31. Yurchenko JV, Budilov AV, Deyev SM, Khromov IS, Sobolev AY (2003) Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium Meiothermus ruber and characterization of the recombinant enzyme. Mol Gen Genomics 270(1):87–93. https://doi.org/10.1007/s00438-003-0899-y

  32. Zaheer R, Morton R, Proudfoot M, Yakunin A, Finan TM (2009) Genetic and biochemical properties of an alkaline phosphatase PhoX family protein found in many bacteria. Environ Microbiol 11(6):1572–1587. https://doi.org/10.1111/j.1462-2920.2009.01885.x

  33. Zappa S, Rolland JL, Flament D, Gueguen Y, Boudrant J, Dietrich J (2001) Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi. Appl Environ Microbiol 67(10):4504–4511. https://doi.org/10.1128/aem.67.10.4504-4511.2001

  34. Zhang Y, Ji C, Zhang X, Yang Z, Peng J, Qiu R, Xie Y, Mao Y (2008) A moderately thermostable alkaline phosphatase from Geobacillus thermodenitrificans T2: cloning, expression and biochemical characterization. Appl Biochem Biotechnol 151(1):81–92. https://doi.org/10.1007/s12010-008-8166-7

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Correspondence to Shouji Takahashi.

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Takahashi, S., Morooka, Y., Kumakura, T. et al. Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1. Appl Microbiol Biotechnol 104, 1125–1134 (2020). https://doi.org/10.1007/s00253-019-10291-6

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Keywords

  • Sphingobium sp. strain TCM1
  • PhoK alkaline phosphatase
  • Enzymatic characteristics
  • Gene expression