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J Bacteriol, April 1998, p. 1841-1847, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Novel Assay Reveals Multiple Pathways Regulating Stress-Induced
Accumulations of Inorganic Polyphosphate in Escherichia
coli
Dana
Ault-Riché,
Cresson D.
Fraley,
Chi-Meng
Tzeng, and
Arthur
Kornberg*
Department of Biochemistry, Stanford
University School of Medicine, Stanford, California 94305-5307
Received 24 November 1997/Accepted 27 January 1998
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.
*
Corresponding author. Mailing address: Department of
Biochemistry, Stanford University School of Medicine, Stanford, CA
94305-5307. Phone: (650) 723-6161. Fax: (650) 723-6783. E-mail:
akornber{at}cmgm.stanford.edu.
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