Guanosine Tetra- and Pentaphosphate Promote Accumulation of Inorganic Polyphosphate in Escherichia coli*

(Received for publication, November 18, 1996, and in revised form, May 30, 1997)

Akio Kuroda Dagger §, Helen Murphy , Michael Cashel and Arthur Kornberg Dagger par

From the Dagger  Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307 and the  Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2785

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

High levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), generated in response to amino acid starvation in Escherichia coli, lead to massive accumulations of inorganic polyphosphate (polyP). Inasmuch as the activities of the principal enzymes that synthesize and degrade polyP fluctuate only slightly, the polyP accumulation can be attributed to a singular and profound inhibition by pppGpp and/or ppGpp of the hydrolytic breakdown of polyP by exopolyphosphatase, thereby blocking the dynamic turnover of polyP. The Ki values of 10 µM for pppGpp and 200 µM for ppGpp are far below the concentrations of these nucleotides in nutritionally stressed cells. In the complex metabolic network of pppGpp and ppGpp, the greater inhibitory effect of pppGpp (compared with ppGpp) leading to the accumulation of polyP, may have some significance in the relative roles played by these regulatory compounds.


INTRODUCTION

Inorganic polyphosphate (polyP),1 a linear polymer of hundreds of phosphate residues linked by high-energy phosphoanhydride bonds, is ubiquitous having been found in all microbes, fungi, plants, and animals examined (1, 2). In Escherichia coli, polyP, which accumulates up to 20 mM (based on Pi residues) in stationary-phase cells,2 is produced from ATP by a membrane-associated enzyme, polyphosphate kinase (PPK) (3).

Mutants lacking PPK are deficient in polyP, fail to adapt to stress, and do not survive in stationary phase (4, 5). A regulatory function is one of the many possible effects of polyP that might account for this essential role. In this regard, the relationship of polyP levels to those of guanosine penta- and tetraphosphate ((p)ppGpp) deserves special attention. Levels of (p) ppGpp rise drastically in response to starvation for amino acids, carbon, or Pi (6, 7). In response to nutritional stress, accompanied by increased levels of (p)ppGpp, large accumulations of polyP have been observed in E. coli,23 Myxococcus xanthus (8), and Pseudomonas aeruginosa3. E. coli mutants that fail to produce (p)ppGpp are also deficient in the accumulation of polyP.3

The present study explores the mechanism of polyP accumulation in nutritionally stressed E. coli and the relationships to (p)ppGpp.


EXPERIMENTAL PROCEDURES

Reagents and Proteins

Sources were as follows: [gamma -32P]ATP was from Amersham Corp.; nonradiolabeled ATP and ADP and bovine serum albumin were from Sigma; polyethyleneimine-cellulose (PEI) TLC plate was from Merck; and creatine phosphate and creatine kinase were from Boehringer Mannheim.

Bacterial Strains

The Delta gppA::kan deletion-insertion allele in strain CF3376 was constructed as follows. A plasmid bearing the wild type gppA region was subcloned from phage lambda 1039 (9). Using synthetic primers, DNA-encoding, amino acid residues 1-452 of the 496 total in GppA were deleted. A kanamycin resistance (Km-r) cassette derived from plasmid pUC4K (Pharmacia Biotech Inc.) was substituted for the deletion. The insertion-deletion allele was recombined into phage lambda 1039 by phage growth on a plasmid-bearing strain and the lysate used to lysogenize a wild type K-12 strain (MG1655) selecting for Km-r. Phage curing and recombinative transfer of the Delta gppA allele from phage lambda  to the chromosome was by heat-pulse curing (10), yielding strain CF3376. Verification of the deletion in genomic DNA was done by: 1) polymerase chain reaction amplification from primers flanking the deletion site and visualization of the shortening of the product due to the presence of the kan cassette, 2) measurement of increased abundance of pppGpp relative to ppGpp during the stringent response induced by serine hydroxamate by a nonuniform labeling procedure (11), i.e. pppGpp/(pppGpp + ppGpp) = 0.072 (± 0.017) for wild type versus 0.60 (± 0.084) for mutant, and 3) observing expected genetic linkage of the Km-r phenotype after P1 phage cotransduction of Km-r with ilv500::Tn10. Strain CF3382 is an example of a Delta gppA::ilv500::Tn10 recombinant.

The Delta ppkx::kan deletion-insertion allele in strain CF5802 consists of a deletion of the C-terminal portion of ppk fused with a N-terminal deletion of ppx, again with a substitution of a kanamycin resistance cassette. In the ppkx operon this deletion is deduced to remove 98.5% of PPK and 78% of PPX. The source of the residual ppk sequences was plasmid pBC29 (after SacI cleavage), while the source of the residual ppx sequences was the plasmid pBC6 (after PvuII cleavage). After insertion of the pUC4K kan cassette, the plasmid was linearized and used to transform a recBC sbc strain of E. coli selecting for Km-r, yielding strain CF5772. The Delta ppkx deletion-insertion was transferred into strain MG1655 by transduction with phage P1, selecting for Km-r recombinants, which yielded strain CF5802. Verification of the replacement of wild type ppkx locus by the deletion-insertion allele in CF5802 was by: 1) polymerase chain reaction with flanking primers, as above, 2) observation of dramatically decreased levels of PPK and PPX enzymatic activities in extracts, and 3) observing the expected genetic linkage of Km-r after phage P1 transduction to recipients bearing quaBA::Tn10 (86%) or zff-208::Tn10 (10%).

Although the Delta gppA and Delta ppkx alleles are both Km-r, they could be combined in a single strain by transduction of CF5802 with phage P1 grown on a Delta gppA ilv500::Tn10 donor (CF3382), selecting for Rc-r and screening recombinants for the presence of high pppGpp:ppGpp ratios (11), yielding strain CF5986. The ilv500::Tn10 present in CF5986 was removed by transduction with phage P1 grown on a wild type MG1655 parental strain, selecting for prototrophs and screening recombinants for retention of the gpp-phenotype, yielding strain CF6032 Delta ppkx Delta gppA.

Crude Lysate Preparation for Exopolyphosphatase, Polyphosphate Kinase, and Guanosine Pentaphosphate Hydrolase Assay

Cells were grown at 37 °C on LB medium or MOPS medium (12) and harvested by centrifugation. The cell pellet was resuspended in 100 µl (per 1-ml cell culture) of 50 mM Tris-Cl (pH 7.5) and 10% sucrose, frozen in liquid nitrogen, and stored at -80 °C. Lysozyme (250 µg/ml) was added to the thawed cell suspension and incubated at 0 °C for 30 min. The cells were lysed by exposure to 37 °C for 4 min, followed by immediate chilling in ice water. The lysate was further homogenized by sonication (Kontes, ultrasonicator, 50 W, twenty 1-s pulses on ice).

Assay for PPK

The production of [32P]polyP from [gamma -32P]ATP was measured by using glass filters (3). The other method to detect [32P]polyP, using PEI-TLC plates, is reliable only with purified PPK. The reaction mixture contained PPK, PPK buffer (3), 1 mM [gamma -32P]ATP (0.01 µCi/nmol), and 2 mM creatine phosphate and creatine kinase (20 µg/ml) as an ATP-regenerating system. The mixture was incubated at 37 °C for 10 min or the time indicated. 1-µl samples were spotted on a PEI-TLC plate, which was developed with 1.5 M KH2PO4 (pH 3.5). The dried plate was exposed to a screen and visualized in a PhosphorImager scanner (Molecular Dynamics). The ratio between the image intensity of the origin (polyP) and the total was calculated. One unit of activity incorporated 1 pmol of phosphate into [32P]polyP/min (3). The specific activity of the purified PPK was 7 × 107 units/mg of protein.

Assay for PPX

The assay for PPX measured Pi released from [32P]polyP, prepared as described previously (3). The reaction mixture contained PPX, PPX buffer (13), and 0.1 mM [32P]polyP (based on Pi residues). The mixture was incubated at 37 °C for 10 min or the time indicated and then 1-µl samples were spotted on PEI-TLC plates. The development and calculation were described in the assays for PPK. One unit of activity liberated 1 pmol of [32P]Pi/min (13).

Purification of E. coli Exopolyphosphatase (PPX)

Cells were lysed by freezing and thawing, followed by lysozyme treatment, and sonication as described above. The lysate was centrifuged, and the supernatant was precipitated by 60% ammonium sulfate. The precipitate was dissolved in the HEPES buffer (13) and then purified by DE52, S-Sepharose, and Mono Q columns as described (13). The homogeneity of purified PPX was checked by SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue.

Guanosine Pentaphosphate Hydrolase Assay

(p)ppGpp and 3'-beta [32P]pppGpp were prepared by the procedure of Krohn and Wagner (14). The final concentration was determined at 252 nm (epsilon 252 = 13,100 M-1 cm-1 (15)). The reaction mixture containing PPX buffer (13) or 40 mM Tris acetate (pH 8.0), 1 mM dithiothreitol, 10 mM magnesium acetate, 30 mM ammonium acetate, 0.2 mM EDTA, and the indicated concentrations of [32P]pppGpp were incubated at 37 °C, and 1-µl samples were applied to PEI-TLC plates. The development and calculations were described in the assays for PPK. The ratio between the image intensity of ppGpp and ppGpp plus pppGpp was calculated by the PhosphorImager scanner.


RESULTS

Influence of pppGpp and ppGpp on Activities of PPK and PPX

Accumulation of polyP by E. coli lacking or overproducing PPK has established that this activity is responsible for the synthesis of polyP (5). Similarly, the removal of polyP can be attributed to the presence or absence of PPX, the principal polyphosphatase of E. coli (13). Combination of these two opposing activities can account for a turnover of polyP in growing cells of 12 min or less.4 It was paradoxical then that 100-fold increases in the level of polyP in response to the nutritional stress of amino acid starvation were not accompanied by significant changes in the activities of PPK or PPX as measured in crude cell extracts (Fig. 1). Adding increased amounts of pppGpp and ppGpp had no influence on the activity of PPK: at 100 µM pppGpp and at 400 µM ppGpp, neither the synthesis of polyP from ATP nor the conversion of polyP to ATP were detectably (±5%) affected. However, the effects of these compounds on PPX activity were strikingly inhibitory (Fig. 2). At 100 µM, pppGpp inhibited PPX by 90%; ppGpp also inhibited PPX, but less strongly.


Fig. 1. PolyP accumulation and PPK and PPX activities under stringent conditions. E. coli MG1655 was grown on MOPS medium containing 0.4 mM Pi (10 µCi/ml [32P]orthophosphate) and 40 µg/ml of amino acids. At an A540 near 0.2, serine hydroxamate (SHX) was added (0.5 mg/ml) for induction of amino acid starvation and accumulation of pppGpp and ppGpp.3 At the times indicated after addition of serine hydroxamate, 1-ml cultures were collected and used for the measurement of polyP amount (A) and PPK and PPX activities (B). Symbols are with (square ) and without (diamond ) serine hydroxamate. Units of PPK and PPX in B are 103 units of enzyme activity.
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Fig. 2. Inhibition of PPX by pppGpp and ppGpp. PPX hydrolysis of [32P]polyP was assayed in the presence of pppGpp (A) and ppGpp (B). The reaction mixture (10-µl) in A contained purified PPX (6 ng) and 50 µM [32P]polyP and in B contained purified PPX (3 ng) and 20 µM [32P]polyP. Further details are described under "Experimental Procedures."
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Kinetic Features of Inhibition of PPX by pppGpp and ppGpp

Inhibition of PPX, examined at several levels of pppGpp, was consistent with its behavior as a competitive inhibitor of the polyP substrate with a Ki value of 10 µM (Fig. 3). A similar Lineweaver-Burk plot for inhibition of PPX by ppGpp yielded a Ki value of 200 µM. Binding of pppGpp to PPX was judged to be reversible in that a prior incubation of the nucleotide at 400 µM with the enzyme (200 ng) for 10 or 20 min at 37 °C yielded the expected level of inhibition upon a subsequent 10-fold dilution for assay of enzyme activity.


Fig. 3. Lineweaver-Burk plot for inhibition of PPX by pppGpp. The reaction mixture (10 µl) contained PPX (6 ng), [32P]polyP (10-50 µM), and the indicated concentrations of pppGpp.
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Stimulation of polyP accumulation by pppGpp and ppGpp could be demonstrated in the course of polyP synthesis by PPK and hydrolysis by PPX (Fig. 4). The presence of either of these nucleotides from the outset or an addition later in the reaction led to a large increase in the amount of polyP produced. Further addition of 500 µM ppGpp did not lead to a significant increase in the amount of polyP produced in the presence of 100 µM pppGpp (data not shown).


Fig. 4. Stimulation of polyP accumulation by pppGpp and ppGpp in the course of polyP synthesis by PPK and hydrolysis by PPX. The reaction mixture (20 µl) contained 8000 units of PPK, 3600 units of PPX, 1 mM ATP ([gamma -32P]ATP, 0.01 µCi/nmol), 2 mM creatine phosphate, and 20 µg/ml of creatine kinase. 1-µl samples were used for estimation of polyP by TLC as described under "Experimental Procedures." To a portion of the sample without (p)ppGpp, 100 µM pppGpp was added after 10 min as indicated by the arrow.
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PPX Possesses pppGpp Hydrolase Activity

An awareness of the profound inhibitory effect of pppGpp on PPX activity and the far lesser effect of ppGpp makes it important to examine the enzymatic routes whereby pppGpp is hydrolyzed to ppGpp. The principal known route is the action of GppA (16), which also proved to have exopolyPase activity (17). In view of the considerable sequence homology between GppA and PPX (18), the latter was examined and also proved to have pppGpp hydrolase activity (Fig. 5). The Km value of 7 µM for pppGpp as substrate (Table I) as expected was virtually identical to its Ki value of 10 µM as an inhibitor of polyPase activity and even lower than the Km of 110-130 µM pppGpp determined for GppA (16, 17). Yet, as indicated by the kcat value, PPX still qualifies more as a polyPase than as a pppGppase (Table I).


Fig. 5. Hydrolysis of pppGpp to ppGpp by PPX. Reaction mixtures (10 µl) containing up to 30 ng of PPX and 10 µM [32P]pppGpp were incubated at 37 °C. A, 1-µl samples were used to estimate ppGpp by TLC as described under "Experimental Procedures." B, amounts of [32P]ppGpp formed.
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Table I. Enzyme kinetics of PPX as an exopolyphosphatase and a guanosine pentaphosphate hydrolase


Km kcat kcat/Km

µM s-1 M-1 s-1 × 10-6
PolyPase 0.033a 8.3 251
pppGppase 6.7 0.33 0.049

a Calculated on basis of polymer size of 750 residues. Ki for polyPase activity of pppGpp is 10 µM. Ki for polyPase activity of ppGpp is 200 µM.

Relative pppGppase Contributions by GppA and by PPX

To evaluate the relative activities of GppA and PPX in the hydrolysis of pppGpp, extracts of several strains with various levels of these activities were compared. The strain lacking both ppkx and GppA was strikingly deficient in the hydrolytic activity compared with the strain that lacked only ppkx (Fig. 6). However, gppA and ppx mutants complemented with multicopy plasmids bearing ppx did recover pppGppase activity. Thus, GppA appears to be the major source of pppGppase activity, but PPX may function in an auxiliary role.


Fig. 6. Guanosine pentaphosphate hydrolase activities in wild type strains and mutants. pppGppase activities were measured in the lysate (1.5 µg of protein) of E. coli MG1655 (wild type): Delta ppkx, Delta ppkx Delta gppA, and Delta ppkx Delta gppA harboring pBC10 (PPX+++(10)). The reaction mixtures containing 15 µM [32P]pppGpp were incubated at 37 °C; amounts of [32P]ppGpp were measured by TLC as described under "Experimental Procedures."
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DISCUSSION

Whereas the polyP levels in E. coli may increase 1000-fold in response to nutritional stress, the levels of the enzyme activities responsible for polyP synthesis (PPK) and polyP degradation (PPX) hardly change at all.2 This was shown to be true when the stringent response was provoked using an amino acid analog serine hydroxamate (Fig. 1). In this study, we show that this phenomenon can be explained by the profound and singular inhibition of PPX by the stress-responsive nucleotides, ppGpp and pppGpp, without any effect on PPK (Figs. 2 and 3). Thus, the continued synthesis of polyP without degradation results in its extensive accumulation.

In keeping with these in vitro findings are several in vivo observations. (i) Accumulation of polyP follows the buildup of ppGpp and pppGpp in response to amino acid starvation.2, 3 (ii) In amino acid-starved cells, the levels of ppGpp increases from 20 µM to 1 mM and that of pppGpp from 0.5 µM to 200 µM; these elevated levels far exceed those required to inhibit PPX in vitro (i.e. a Ki value of 200 µM for ppGpp and 10 µM for pppGpp (Figs. 2, 3)). The fact that the activity of PPX was unchanged in extracts of stressed cells can be attributed to the large dilution of ppGpp and pppGpp suffered in the preparation of the cell extracts. (iii) Turnover of polyP (resulting from its cyclic synthesis by PPK and hydrolysis by PPX) was found to be 12 min or less,4 a result simulated by the accumulation of polyP in a mixture containing purified PPK and PPX responding to the presence of ppGpp and pppGpp (Fig. 4). (iv) Finally, mutants that fail to produce ppGpp and pppGpp (e.g. relA and spoT) also fail to accumulate any polyP2, 3 when treated with serine hydroxamate.

While ppGpp is more abundant than pppGpp, the relative effects of these two regulatory nucleotides have not been thoroughly examined. In the case of polyP accumulation, pppGpp appears to be more effective than ppGpp (Fig. 2). The levels of these nucleotides depend on the synthesis of pppGpp by RelA and SpoT, the hydrolysis of pppGpp to ppGpp, and the conversion of ppGpp to GDP by SpoT. With regard to the generation of ppGpp from pppGpp, the action of the GppA enzyme is likely the principal route (Fig. 6). However, the facts that GppA is an exopolyphosphatase and also bears strong amino acid homologies to PPX promoted our discovery that PPX, like GppA, can also hydrolyze pppGpp to ppGpp (Fig. 5).

Based on studies with extracts of various mutants, the PPX generation of ppGpp from pppGpp may be auxiliary to the action of GppA (Fig. 6). Other possibilities for minor contributions toward the formation of ppGpp from pppGpp include the translation elongation factors, EF-G and EF-Tu (6).

The kinetic parameters of PPX actions (Table I) show the same Km value for pppGpp as a substrate as the Ki value as an inhibitor of exopolyphosphatase, indicating that the same active center is employed for both activities.

A scheme of polyP metabolism (Fig. 7), based on information mentioned in this report, is surely incomplete. For example, the role of RpoS, the sigma factor induced by (p)ppGpp and responsible for the expression of some 50 genes important in the response to starvation, needs to be included. Complementation of ppk mutants by a multicopy rpoS plasmid restored hydrogen peroxidase II activity, indicating an interaction relevant to polyP metabolism (4). Another example is the behavior of phoB, the regulatory gene of the phosphate regulon (19). Mutants of phoB, which produce (p)ppGpp in a stringent response, nevertheless fail to accumulate polyP.2 Attempts to reconstitute transcriptional systems with an RNA polymerase holoenzyme containing RpoS acting on an RpoS-activated gene (e.g. katE, the hydroperoxidase II gene) have resulted in a profound inhibition by polyP rather than the anticipated activation.5 One must conclude that factors, such as a polyP-binding protein, may be operating in vivo and are lacking in the reconstituted systems. Clues might also be supplied from studies of other microbial systems, such as Myxococcus xanthus (8) and Pseudomonas aeruginosa,3 in which starvation responses, heralded by increased levels of (p)ppGpp are followed, as in E. coli, by a large accumulation of polyP.


Fig. 7. Provisional scheme for polyphosphate metabolism in E. coli. Metabolism of pppGpp and ppGpp as described in Ref. 6 and the text. Ndk is nucleoside diphosphate kinase.
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FOOTNOTES

*   This work was supported by grants from the General Medical Sciences Institute of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a fellowship from Human Frontier Science Program. Present address: Dept. of Molecular Biotechnology, Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739, Japan.
par    To whom correspondence should be addressed. Tel.: 415-723-6167; Fax: 415-723-6783.
1   The abbreviation used are: polyP, inorganic polyphosphate; polyPase, inorganic polyphosphatase; ppGpp, guanosine 5'-diphosphate 3'-diphosphate; pppGpp, guanosine 5'-triphosphate 3'-diphosphate; (p)ppGpp, ppGpp and pppGpp; PEI, polyethylenimine, PPK, polyphosphate kinase; PPX, exopolyphosphatase, PPX+++, overexpressed PPX; GppA, guanosine pentaphosphate phosphohydrolase; Km-r, kanamycin resistance; MOPS, 4-morpholinepropanesulfonic acid.
2   N. N. Rao and A. Kornberg, unpublished results.
3   H.-Y. Kim and A. Kornberg, unpublished results.
4   S.-J. Park and A. Kornberg, unpublished results. Cells growing in the MOPS medium with [32P]Pi were transferred to the same medium without [32P]Pi. Cells removed from the culture every 3 min after the chase were used to determine [32P]polyP. The time required for the radioactivity of [32P]polyP to be reduced to one-half its initial value was regarded as the turnover of polyP.
5   H. Wurst and A. Kornberg, unpublished results.

ACKNOWLEDGEMENT

We thank Dr. H.-Y. Kim for the use of unpublished data, Dr. N. N. Rao for technical help and discussion, and L. Bertsch for critical reading of this manuscript.


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