Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in Escherichia coli

ABSTRACT A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS,relA, and phoB.

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Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

Applied and Environmental Microbiology ◽

10.1128/aem.68.8.4107-4110.2002 ◽

2002 ◽

Vol 68 (8) ◽

pp. 4107-4110 ◽

Cited By ~ 63

Author(s):

Tomohiro Morohoshi ◽

Tatsuya Maruo ◽

Yoko Shirai ◽

Junichi Kato ◽

Tsukasa Ikeda ◽

...

Keyword(s):

Escherichia Coli ◽

Type Strain ◽

Wild Type Strain ◽

Biological Process ◽

Negative Regulator ◽

Wild Type ◽

Inorganic Polyphosphate ◽

Insertional Inactivation ◽

Synechocystis Sp ◽

Polyp Accumulation

ABSTRACT The biological process for phosphate (Pi) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the Pi regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more Pi from the medium than the wild-type strain removed.

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Inorganic Polyphosphate Accumulation inEscherichia coliIs Regulated by DksA but Not by (p)ppGpp

Journal of Bacteriology ◽

10.1128/jb.00664-18 ◽

2019 ◽

Vol 201 (9) ◽

Cited By ~ 13

Author(s):

Michael J. Gray

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Rna Polymerase ◽

Elongation Factor ◽

Stringent Response ◽

Inorganic Polyphosphate ◽

Content Type ◽

Polyphosphate Accumulation ◽

Pathogenic Microbes ◽

Polyp Accumulation

ABSTRACTProduction of inorganic polyphosphate (polyP) by bacteria is triggered by a variety of different stress conditions. polyP is required for stress survival and virulence in diverse pathogenic microbes. Previous studies have hypothesized a model for regulation of polyP synthesis in which production of the stringent-response second messenger (p)ppGpp directly stimulates polyP accumulation. In this work, I have now shown that this model is incorrect, and (p)ppGpp is not required for polyP synthesis inEscherichia coli. However, stringent mutations of RNA polymerase that frequently arise spontaneously in strains defective in (p)ppGpp synthesis and null mutations of the stringent-response-associated transcription factor DksA both strongly inhibit polyP accumulation. The loss of polyP synthesis in a mutant lacking DksA was reversed by deletion of the transcription elongation factor GreA, suggesting that competition between these proteins for binding to the secondary channel of RNA polymerase plays an important role in controlling polyP activation. These results provide new insights into the poorly understood regulation of polyP synthesis in bacteria and indicate that the relationship between polyP and the stringent response is more complex than previously suspected.IMPORTANCEProduction of polyP in bacteria is required for virulence and stress response, but little is known about how bacteria regulate polyP levels in response to changes in their environments. Understanding this regulation is important for understanding how pathogenic microbes resist killing by disinfectants, antibiotics, and the immune system. In this work, I have clarified the connections between polyP regulation and the stringent response to starvation stress inEscherichia coliand demonstrated an important and previously unknown role for the transcription factor DksA in controlling polyP levels.

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Mutations inEscherichia coliPolyphosphate Kinase That Lead to Dramatically IncreasedIn VivoPolyphosphate Levels

Journal of Bacteriology ◽

10.1128/jb.00697-17 ◽

2018 ◽

Vol 200 (6) ◽

Cited By ~ 15

Author(s):

Amanda K. Rudat ◽

Arya Pokhrel ◽

Todd J. Green ◽

Michael J. Gray

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Molecular Mechanisms ◽

Phosphate Removal ◽

Biological Engineering ◽

Inorganic Polyphosphate ◽

Intracellular Magnesium ◽

Content Type ◽

Polyp Accumulation

ABSTRACTBacteria synthesize inorganic polyphosphate (polyP) in response to a wide variety of stresses, and production of polyP is essential for stress response and survival in many important pathogens and bacteria used in biotechnological processes. However, surprisingly little is known about the molecular mechanisms that control polyP synthesis. We have therefore developed a novel genetic screen that specifically links growth ofEscherichia colito polyP synthesis, allowing us to isolate mutations leading to enhanced polyP production. Using this system, we have identified mutations in the polyP-synthesizing enzyme polyP kinase (PPK) that lead to dramatic increases inin vivopolyP synthesis but do not substantially affect the rate of polyP synthesis by PPKin vitro. These mutations are distant from the PPK active site and found in interfaces between monomers of the PPK tetramer. We have also shown that high levels of polyP lead to intracellular magnesium starvation. Our results provide new insights into the control of bacterial polyP accumulation and suggest a simple, novel strategy for engineering bacteria with increased polyP contents.IMPORTANCEPolyP is an ancient, universally conserved biomolecule and is important for stress response, energy metabolism, and virulence in a remarkably broad range of microorganisms. PolyP accumulation by bacteria is also important in biotechnology applications. For example, it is critical to enhanced biological phosphate removal (EBPR) from wastewater. Understanding how bacteria control polyP synthesis is therefore of broad importance in both the fields of bacterial pathogenesis and biological engineering. UsingEscherichia colias a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.

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Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

Journal of Bacteriology ◽

10.1128/jb.180.8.2186-2193.1998 ◽

1998 ◽

Vol 180 (8) ◽

pp. 2186-2193 ◽

Cited By ~ 118

Author(s):

Narayana N. Rao ◽

Shengjiang Liu ◽

Arthur Kornberg

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Response Regulator ◽

Stringent Response ◽

Amino Acid Starvation ◽

Wild Type ◽

Inorganic Polyphosphate ◽

Polyp Accumulation ◽

Phosphate Regulon

ABSTRACT Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (Pi), in media deficient in both Pi and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid,phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during Pi starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.

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