Modulation of the phosphate-starvation response in Escherichia coli by genetic manipulation of the polyphosphate pathways

Sharfstein, Susan T.; Dien, Stephen J. Van; Keasling, Jay D.

doi:10.1002/(sici)1097-0290(19960820)51:4<434::aid-bit6>3.0.co;2-j

Cited by 10 publications

(2 citation statements)

References 19 publications

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“…In the presence of uranyl, this assay does not work, due to the interference of uranyl with the binding of polyphosphate to Glassmilk. In this case, polyphosphates were precipitated selectively from cell lysates (48), hydrolyzed with 0.1 M HCl at 95°C for 1 h, and analyzed for phosphate.…”

Section: Methodsmentioning

confidence: 99%

Uranyl Precipitation byPseudomonas aeruginosavia Controlled Polyphosphate Metabolism

Renninger¹,

Knopp²,

Nitsche

et al. 2004

Appl Environ Microbiol

108

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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...

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Section: Methodsmentioning

confidence: 99%

Uranyl Precipitation byPseudomonas aeruginosavia Controlled Polyphosphate Metabolism

Renninger¹,

Knopp²,

Nitsche

et al. 2004

Appl Environ Microbiol

108

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“…The ability of polyphosphate to alter the cell's response to starvation for inorganic phosphate (P i ) may also be exploited to improve expression from phosphate‐starvation promoters (Sharfstein et al, 1996). These promoters form part of the Pho regulon, a group of genes controlled by the external P i concentration.…”

Section: Introductionmentioning

confidence: 99%

Effect of Polyphosphate Metabolism on the Escherichia coli Phosphate-Starvation Response

Dien

Keasling

1999

Biotechnol. Prog.

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A previously developed dynamic model of the Escherichia coli Pho regulon was extended to investigate the effect of polyphosphate synthesis and degradation on this control system. Differential equations for ATP and polyphosphate were formulated, and the model was applied to the growth of cells containing the ppk and ppx genes under control of separate, inducible promoters. In agreement with recent experimental observations, the degradation of polyphosphate by PPX during a period of phosphate limitation could repress the phosphate-starvation response. This is attributed to the release of phosphate from the cell into the periplasm, where it can be detected by the external phosphate sensor. A segregated model was then developed to account for differences in K(I), the dissociation constant for the repression complex, among cells of the population. Since K(I) is the key parameter in determining whether the Pho response is induced or repressed at a particular surface phosphate concentration, this permitted the induction of some cells while others remained repressed. The induction profiles resulting from the population-averaged values more closely matched experimental results than did those with the nonsegregated model.

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Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Dien

Keasling

1998

Biotechnol. Bioeng.

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Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium‐ and high‐copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate‐starved cells; excess phosphate was secreted into the medium, leading to a down‐regulation of the phosphate‐starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:754–761, 1998.

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Modulation of the phosphate-starvation response in Escherichia coli by genetic manipulation of the polyphosphate pathways

Cited by 10 publications

References 19 publications

Uranyl Precipitation byPseudomonas aeruginosavia Controlled Polyphosphate Metabolism

Uranyl Precipitation byPseudomonas aeruginosavia Controlled Polyphosphate Metabolism

Effect of Polyphosphate Metabolism on the Escherichia coli Phosphate-Starvation Response

Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

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