Uranyl Precipitation byPseudomonas aeruginosavia Controlled Polyphosphate Metabolism

Abstract

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.Biological methods for the removal of heavy metals and actinides have recently been investigated due to their cost effectiveness at moderate metal concentrations. Many strains have been isolated that sorb (14,23,51,59,61), reduce (10, 18, 52, 56), and precipitate (4, 33, 41, 44, 45) metals, usually on the outer membrane of the cell. Sorption is generally achieved in a reversible process and is dependent on the composition of the water being treated, as other chelating agents compete with the complexing moieties on the cell. Thus, the applicability of this method in more complex waste streams or environments may be somewhat suspect due to the relatively low binding affinities of cellular components for metals compared to chelators, such as humic or organic acids. Reduction is generally limited to anaerobic processes and is ineffective against single-oxidationstate metals. Precipitation has the advantage of producing chemically stable forms of metal, and its use is not limited to reducible metals.With respect to the biological precipitation of metals, both metal sulfides (12,57,58,60) and metal phosphates (4, 33, 45) have been investigated due to their low solubilities. While some work has been done to produce sulfide aerobically (57), its production is generally an anaerobic process, limiting its applicability. The release of phosphate via the hydrolysis of an organic phosphate has been shown to be an effective method for the precipitation of metals on cell membranes. Macaskie and Dean (30) isolated a cadmium-resistant strain of Citrobacter that precipitated numerous metals, such as uranyl ion, as metal phosphates through the use of a membranebound acid phosphatase (26,31,32). Using glycerol-2-phosphate as a phosphate source, the organism cleaved phosphate from the source in the periplasmic space, leaving it to bind with metals that had been complexe...

Horizontal Gene Transfer of P IB -Type ATPases among Bacteria Isolated from Radionuclide- and Metal-Contaminated Subsurface Soils

“…The tolerance to U exhibited by gram-positive and gramnegative microorganisms may be explained by several different cellular mechanisms, as previously reported by other investigators (30,49,55). One mechanism, bioadsorption, has recently been hypothesized to occur in a well-characterized Arthrobacter type strain, the A. nicotianae type strain (59).…”

Phosphate-Mediated Remediation of Metals and Radionuclides

“…Electron microscopy analyses of cells exposed to these elements demonstrated intracellular localization with phosphate-rich granules, suggesting that contaminant sequestration may be achieved by polyphosphates and may protect sensitive cytosolic molecules from oxidative damage. In addition to polyphosphate chelation of metals and radionuclides, an engineered Pseudomonas aeruginosa strain overexpressing the ppk gene was shown to enhance intracellular phosphate concentrations when compared to the wildtype strain [133]. Upon nutrient starvation, polyphosphate depolymerization and efflux of phosphate into the media containing U(VI) promoted the removal of 80% of the soluble U(VI) as a uranyl phosphate precipitate.…”

“…In response to the local geochemistry of contaminated environments, production of intracellular polyphosphate provides microorganisms with a means to sequester toxic ions within the cell cytosol as well as the regulation of gene(s) expressed in response to cellular stress (e.g., DNA repair, RNA polymerase sigma factor, and pH extremes) [55,[127][128][129][130][131]. The reactivity of cytosolic polyphosphates has been shown to facilitate intracellular sequestration of Cd, Cu, Hg, Pb, U, and Zn in genetically engineered bacterial strains as well as naturally occurring archaeal and bacterial strains [52,55,[132][133][134][135][136]. Electron microscopy analyses of cells exposed to these elements demonstrated intracellular localization with phosphate-rich granules, suggesting that contaminant sequestration may be achieved by polyphosphates and may protect sensitive cytosolic molecules from oxidative damage.…”

Unusual Versatility of the Filamentous, Diazotrophic Cyanobacterium Anabaena torulosa Revealed for Its Survival during Prolonged Uranium Exposure

“…Such intracellular sequestration within phosphate-rich granules or polyphosphates decreases the intracellular U concentration thereby protecting sensitive cytosolic molecules from U toxicity (6,20,21). On the other hand, the hydrolysis or degradation of polyphosphates in response to heavy metals or nutrient stress has been proposed to precipitate heavy metals extracellularly, enabling metal detoxification (23,24). The overexpression of the polyphosphate kinase (ppk) gene in Pseudomonas aeruginosa results in the significant accumulation of polyphosphates which degrade under carbon-starved conditions, and the phosphates released therefrom precipitate uranyl out of the solutions (24).…”

“…On the other hand, the hydrolysis or degradation of polyphosphates in response to heavy metals or nutrient stress has been proposed to precipitate heavy metals extracellularly, enabling metal detoxification (23,24). The overexpression of the polyphosphate kinase (ppk) gene in Pseudomonas aeruginosa results in the significant accumulation of polyphosphates which degrade under carbon-starved conditions, and the phosphates released therefrom precipitate uranyl out of the solutions (24). Several environmental strains, such as Cellulomonas, Arthrobacter, Rahnella, and Bacillus, isolated from contaminated subsurface soils of the Department of Energy's Field Research Centre (ORFRC) were shown to immobilize uranium as a biogenic phosphate mineral resulting from polyphosphate metabolism or organophosphate hydrolase activity (25)(26)(27).…”