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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

ABSTRACT: Bacteria 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 of Escherichia coli to 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 in in vivo polyP synthesis but do not substantially affect the rate of polyP synthesis by PPK in 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.IMPORTANCE PolyP 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. Using Escherichia coli as a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.

SUBMITTER: Rudat AK

PROVIDER: S-EPMC5826030 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased <i>In Vivo</i> Polyphosphate Levels.

Rudat Amanda K AK Pokhrel Arya A Green Todd J TJ Gray Michael J MJ

Journal of bacteriology 20180223 6

Bacteria 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 of <i>Escherichia coli</i> to polyP synthesis, allowing us to isolate mutatio ...[more]

PMID: 29311274

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Project description:The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased <i>In Vivo</i> Polyphosphate Levels.

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased &lt;i&gt;In Vivo&lt;/i&gt; Polyphosphate Levels.

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Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased <i>In Vivo</i> Polyphosphate Levels.