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, 74 (17), 5516-23

Cloning and Overexpression of Alkaline Phosphatase PhoK From Sphingomonas Sp. Strain BSAR-1 for Bioprecipitation of Uranium From Alkaline Solutions

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Cloning and Overexpression of Alkaline Phosphatase PhoK From Sphingomonas Sp. Strain BSAR-1 for Bioprecipitation of Uranium From Alkaline Solutions

Kayzad S Nilgiriwala et al. Appl Environ Microbiol.

Abstract

Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn5 random mutagenesis. The corresponding gene, designated phoK, was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 +/- 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in <2 h compared to only 1.5 g U/g dry weight in >7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions.

Figures

FIG. 1.
FIG. 1.
Constitutive expression and pH dependence of the alkaline phosphatase PhoK. (A) Alkaline phosphatase (AP) activity of BSAR-1 cells (equivalent to 100 μl of cells with OD600 of 1) and equivalent spent medium (for extracellular activity) seen at various time points during growth. (B) The pH dependence of the phosphatase activity of BSAR-1. Activity was assayed after 23 h of growth in 100 μl culture aliquots containing BSAR-1 cells with OD600 of 1 and equivalent spent medium. All the assays were conducted with three replicates, and the standard errors for the results are shown in the graph. p-NP, para-nitrophenol.
FIG. 2.
FIG. 2.
Mutagenesis and cloning of phoK from BSAR-1. (A) Schematic representation (not to the scale) of the Tn5 insertion in KN20 showing the clone EK1 and the DNA probes used. (B) The phoK gene in BSAR-1 and clones obtained therefrom. (C to E) Southern blotting and hybridization. (C) SalI-digested genomic DNA of BSAR-1 (lane 1) and of the KN20 mutant (lane 2) hybridized to the nptII probe. (D) Genomic DNA of BSAR-1 hybridized to the SXEK1 probe. The DNA was digested with PstI (lane 1), BglII (lane 2), BamHI (lane 3), PstI-BglII (lane 4), PstI-BamHI (lane 5), or BglII-BamHI (lane 6). (E) Analysis of the clone EK2 with SXEK1 probe. The lanes marked + and − contain the SXEK1 probe and an empty pBluescriptII SK(+) vector, respectively, and are used as the positive and negative controls, respectively. The recombinant plasmid in EK2 was linearized with PstI (lane 1) or digested with SalI-XmnI (lane 2).
FIG. 3.
FIG. 3.
Phenotypic selection of strains for alkaline phosphatase. (A) Bacterial strains were grown on TGY agar (quadrants 1 and 2) or LB agar (quadrants 3 and 4) containing PDP and MG with the appropriate antibiotics at 30°C for 48 h. (B) Zymogram analysis of cell lysates from BSAR-1 and KN20 grown at 30°C for 23 h, E. coli pET29b cells, and EK4 cells induced with 1 mM IPTG at 30°C for 4 h. (C) Phosphatase activities of cells and corresponding spent media in cultures of BSAR-1, KN20, and IPTG-induced EK4 at pHs 5.0 and 9.0. All the assays were conducted with three replicates, and the standard errors for the results are shown in the graph. p-NP, para-nitrophenol.
FIG. 4.
FIG. 4.
Uranium bioprecipitation by various strains. The bioprecipitation assays were conducted with cells (OD600, 1) of BSAR-1 and KN20 and strain EK4 induced with IPTG at 30°C in the presence of 5 mM β-GP (A to C) or 10 mM β-GP (D) as the substrate. Uranium was added to the assays as uranyl carbonate at a concentration of 0.5 mM (A), 2 mM (B), or 5 mM (C and D).
FIG. 5.
FIG. 5.
Identity of bioprecipitated uranium. XRD spectra of BSAR-1 cells before exposure to uranium (A) and after 8 h of exposure to 2 mM uranyl carbonate solution (B) were compared with XRD spectra of EK4 cells before exposure to uranium (C) and after 3 h of exposure to 2 mM uranyl carbonate solution (D).

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