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.

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.Inorganic polyphosphate poly P is a linear polymer of up to hundreds of orthophosphate (P i ) residues linked by high-energy phosphoanhydride bonds. Among known functions, poly P can serve as a substitute for ATP in kinase reactions, a P i reservoir, and a chelator of divalent metals (9). Poly P is ubiquitous in nature, having been found in all organisms examined (15), yet little is known about its physiological roles (14).Several poly P-metabolizing enzymes have been purified, and the genes encoding them have been cloned (14, 28). The enzyme primarily responsible for poly P synthesis in Escherichia coli is poly P kinase (PPK), which catalyzes the polymerization of the ␥ phosphate of ATP into a poly P chain (1). Poly P can be hydrolyzed to P i by an exopolyphosphatase (PPX) (3). In E. coli, the encoding genes, ppk and ppx, respectively, form an operon. The inability to accumulate poly P upon deletion of this operon or upon the overproduction of PPX has produced several striking phenotypes in E. coli (6,20,25): decreased long-term survival in stationary phase; increased sensitivity to oxidative, osmotic, and thermal stresses; and defects in adaptive growth in minimal medium after a shift from rich medium. These phenotypes are likely due to the decreased expression of the rpoS gene, which encodes the principal stationary-phase sigma factor, S , or RpoS (25). These and related results (4) suggest that poly P is an effector signal for responses to acute stringencies and adaptations in the stationary phase.Recently available genome sequences have revealed that PPK is highly conserved in many bacterial species, including some important pathogens (26). This also implies that PPK and/or poly P has fundamental physiological roles in bacteria. The ppk knockout mutant of Pseudomonas aeruginosa PAO1 shows a dramatic deficiency in motility, both flagellar and pilus mediated, an inability to form biofilms, and a loss of virulence (22,23,24). ppk null mutants of several other pathogens and of E. coli also exhibit reduced motility and reduced abiotic surface attachment (22,24).Vibrio spp. are among the most common microorganisms in environmental surface waters, such as lakes and rivers. Vibrio cholerae O1 is an...

) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.

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Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.