In Vitro and in Silico Evidence of Phosphatase Diversity in the Biomineralizing Bacterium Ramlibacter tataouinensis

Abstract

Microbial phosphatase activity can trigger the precipitation of metal-phosphate minerals, a process called phosphatogenesis with global geochemical and environmental implications. An increasing diversity of phosphatases expressed by diverse microorganisms has been evidenced in various environments. However, it is challenging to link the functional properties of genomic repertoires of phosphatases with the phosphatogenesis capabilities of microorganisms. Here, we studied the betaproteobacterium Ramlibacter tataouinensis (Rta), known to biomineralize Ca-phosphates in the environment and the laboratory. We investigated the functional repertoire of this biomineralization process at the cell, genome and molecular level. Based on a mineralization assay, Rta is shown to hydrolyse the phosphoester bonds of a wide range of organic P molecules. Accordingly, its genome has an unusually high diversity of phosphatases: five genes belonging to two non-homologous families, phoD and phoX, were detected. These genes showed diverse predicted cis-regulatory elements. Moreover, they encoded proteins with diverse structural properties according to molecular models. Heterologously expressed PhoD and PhoX in Escherichia coli had different profiles of substrate hydrolysis. As evidenced for Rta cells, recombinant E. coli cells induced the precipitation of Ca-phosphate mineral phases, identified as poorly crystalline hydroxyapatite. The phosphatase genomic repertoire of Rta (containing phosphatases of both the PhoD and PhoX families) was previously evidenced as prevalent in marine oligotrophic environments. Interestingly, the Tataouine sand from which Rta was isolated showed similar P-depleted, but Ca-rich conditions. Overall, the diversity of phosphatases in Rta allows the hydrolysis of a broad range of organic P substrates and therefore the release of orthophosphates (inorganic phosphate) under diverse trophic conditions. Since the release of orthophosphates is key to the achievement of high saturation levels with respect to hydroxyapatite and the induction of phosphatogenesis, Rta appears as a particularly efficient driver of this process as shown experimentally.

Keywords: biomineralization; enzymatic activity; genomic repertoire; hydroxyapatite; metal-phosphate mineral phases; microbial phosphatases; organic phosphates; phosphatogenesis.

Figure 1

Calcification assay performed in different media on Rta cells and the extracellular fraction. Three media were used: (A,B) CaGP (10 mM calcium glycerophosphate and 20 mM HEPES at pH 7.5); (C,D) NaGP+Ca (10 mM of sodium glycerophosphate, 0.75 mM of CaCl₂ and 20 mM of HEPES at pH 7.5), and (E,F) NaGP (10 mM sodium glycerophosphate and 20 mM HEPES pH 7.5). The NaGP medium was used as a control assay without Ca. For each assay, time variations of the concentrations of dissolved orthophosphates (A,C,E) and dissolved Ca²⁺ (B,D,F) were measured. Abiotic controls (labeled Medium) consisted in the media without cells. Error bars (smaller than the symbol size) represent the instrumental error: ±0.85 μM and ±50 μM for dissolved orthophosphates and dissolved calcium, respectively.

Figure 2

FT-IR spectra of Rta and recombinant E. coli cells in CaGP medium. The FT-IR spectra of E. coli cells (grown in LB medium) and of a reference hydroxyapatite are shown for comparison. The peaks assigned to hydroxyapatite are numbered as follows. 1: 1,035–1,045 cm⁻¹ corresponding to P-O stretching ν₃; 2: 960–962 cm⁻¹ corresponding to P-O stretching ν₁; 3: 630–633 cm⁻¹ corresponding to librational mode of OH⁻ groups; 4 and 5: 602 and 563 cm⁻¹, respectively, corresponding to P-O-P bending ν₄; 6: 472 cm⁻¹ corresponding to P-OP bending ν₂.

Figure 3

Phosphatase activity of Rta cells toward different substrates. pNPP, p-nitrophenylphosphate (phosphomonoester); bis-pNPP, bis p-nitrophenylphosphate (phosphodiester); TpNPP, Thymidine 5′-monophosphate p-nitrophenylester (phosphodiester, deoxynucleotide analog). Error bars (±0.22 μM) represent the systematic instrumental error.

Figure 4

Structural features of Rta PhoD model. (A) Superimposition of 3D model of Rta PhoD and 3D structure of Bsu PhoD used as template (PDB chain 2yeqA). Bsu PhoD: Ig-like domain and catalytic domain are colored in dark blue and cyan, respectively; C-ter cap in gray. Rta PhoD: Ig-like domain (orange), catalytic domain (yellow). (B) 3D view of the active site of Bsu PhoD. The amino acid residues are labeled according to their PDB numbering. Atom name of co-factors and inorganic phosphate molecule are labeled. Hydrogens are hidden. (C,D) Electrostatic potential maps of Bsu PhoD without C-ter cap and Rta PhoD. The surface potentials are contoured from −5 kT/e (red) to 5kT/e (blue). All structures are shown according to their orientation in the superimposition. The positions of ligand and ions are those from Bsu PhoD; their atoms are represented as spheres of given van der Waals radius (A) or small fixed radius (B–D).

Figure 5

Structural features of Rta PhoX1-4 models. (A) Superimposition of 3D models of Sme PhoX (pink), Rta PhoX1 (green), Rta PhoX2 (orange), Rta PhoX3 (gray), Rta PhoX4 (purple) and 3D structure of Pfl PhoX used as template (PDB chain 4alfA, cyan). (B) 3D view of the active site of Pfl PhoX. The amino acid residues are labeled according to their PDB numbering. Atom name of co-factors and inorganic phosphate molecule are labeled. Hydrogens are hidden. (C–H) Electrostatic potential maps. The surface potentials are contoured from −5 kT/e (red; −15 kT/e for Sme PhoX) to 5kT/e (blue). All structures are shown according to their orientation in the superimposition. The positions of ligand and ions are those from Pfl PhoX; their atoms are represented as spheres of given van der Waals radius (A) or small fixed radius (B–H).

Figure 6

Enzymatic activities of Rta phosphatases. The soluble protein fractions extracted from E. coli clones overexpressing (A) Rta phoD or (B) Rta phoX1 were assayed for phosphatase activity toward monoester (pNPP), diester (bis-pNPP) or nucleotide analog (TpNPP) substrates. (C) Control assay of the soluble protein fraction of E. coli clones over-expressing E. coli phoA. Error bars (±0.22 μM, smaller than the symbol size) represent the systematic instrumental error.

Figure 7

Mineralization of calcium phosphate induced by over-expression of Rta PhoX1 in E. coli clones. (A,B) SEM images in the secondary electron mode. (A) Image of a cluster of cells encrusted by calcium phosphate. (B) Close-up showing the lumen of a cell in the center. The wall is encrusted by calcium phosphate precipitates. (C) Energy dispersive x-ray spectrometry (EDXS) spectrum measured on the mineral phase precipitated on the cells. Emission lines of O, P, and Ca correspond to hydroxyapatite. The emission line of C is due to the cells and the carbon filter on which the sample was deposited.