JOURNAL OF BACTERIOLOGY, Dec. 2000, p. 6687-6693 0021-9193/00/$04.00+0 Vol. 182, No. 23 Copyright © 2000, American Society for Microbiology. All Rights Reserved. Inorganic Polyphosphate in Vibrio cholerae: Genetic, Biochemical, and Physiologic Features NOBUO OGAWA, CHI-MENG TZENG,} CRESSON D. FRALEY, AnD ARTHUR KORNBERG* Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307 Received 12 May 2000/Accepted 8 September 2000 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 mutagen- esis. 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 £. 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,) residues linked by high-en- ergy phosphoanhydride bonds. Among known functions, poly P can serve as a substitute for ATP in kinase reactions, a P; reservoir, and a chelator of divalent metals (9). Poly P is ubiq- uitous 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 Esche- richia coli is poly P kinase (PPK), which catalyzes the polymer- ization of the y phosphate of ATP into a poly P chain (1). Poly P can be hydrolyzed to P,; 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 adap- tive 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, o5, 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 PAOL 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 sur- face attachment (22, 24). Vibrio spp. are among the most common microorganisms in environmental surface waters, such as lakes and rivers. Vibrio * Corresponding author. Mailing address: Department of Biochem- istry, Beckman Center, Stanford University School of Medicine, Stan- ford, CA 94305-5307. Phone: (650) 723-6167. Fax: (650) 723-6783. E-mail: akornber@cmgm.stanford.edu. + Present address: Institute of BioAgricultural Science, Academia Sinica, Nankang, Taiwan. 6687 cholerae O1 is an enteropathogenic gram-negative bacterium that causes severe diarrheal disease. An rpoS mutant of V. cholerae (29) revealed that RpoS is required for V. cholerae persistence in a medium devised to simulate natural aquatic habitats. A gene highly homologous to E. coli ppk was found in the V. cholerae database of The Institute for Genomic Re- search (TIGR) (26). The essential role of poly P for the sta- tionary-phase survival of E. coli and the possibility of a similar role in V. cholerae prompted us to study its PPK and to exam- ine the phenotype of a ppk knockout mutant. V. cholerae O1, biotype El Tor, accumulates much higher levels of poly P than E. coli under normal growth conditions. High accumulations of poly P are stored as granules following a shift from a defined medium lacking P; to one with an excess (20 mM). The ppk null mutant is defective in motility and abiotic surface attachment and fails to adapt to high concen- trations of calcium. MATERIALS AND METHODS Strains and plasmids. E. coli strains MG1655 (A~ F~) and CF5802 (MG1655 Appk Appx::Kan [17]) were the wild-type and mutant strains, respectively. Re- combinant plasmids based on pBluescript H] KS(+) and SK(+) (Stratagene, La Jolla, Calif.) were prepared from DHSqa transformants. Suicide plasmids based on pKNG101 (11) were replicated in £. coli strain S17-1(A pir) (29); pFZY 1 (13) was used as a low-copy-number vector. The 92A1552-Rif* wild-type strain of V. cholerae O1 (El Tor, Inaba [29]}) and the cosmid library of its genomic DNA were provided by F. Yildiz, Department of Microbiology and Immunology, Stanford University. ron Media and growth conditions. A MOPS (morpholinepropanesulfonic acid)- buffered minimal medium (21) was used to impose P,-limiting conditions for the growth of V. cholerae. Media were supplemented with ampicillin (100 pg/ml), kanamycin (150 pg/ml), or streptomycin (100 pg/ml) to select for antibiotic- resistant transformants of V. cholerae. Plasmid construction. The V. cholerae PPK sequence was obtained by a BLAST search of the TIGR genome sequence database using the E. coli se- quence. To obtain the ppk region of V. cholerae, two PCR primers were designed: VCPPKFORI, CCTTCTAGACAACTCTATGACACTAAAGGCAG, and VC PPKREV1, CCTGTCGACTCTGCCGATGAGATAAAGAC. VCPPKFORI1 contains an Xbal site, and VCPPKREV1 contains a SalI site, each located at the 5’ end (underlined). These primers yielded a 1.95-kb PCR product using genomic DNA prepared from the wild-type V. cholerae strain 92A1552-Rif as a template, This fragment begins at position — 106 relative to the A in the start codon at +1 and ends at position +1829, After digestion with both Xbal and Sall, this PCR product was cloned into Xbal- and Sall-digested pBiuescript 1 KS(+); the resulting plasmid was designated pVCK1. The cosmid pVCK20 was obtained by colony hybridization from a V. cholerae genome library using the 1.95-kb PCR fragment as a probe; pVCK20 contained an insert of more than 40 kb in which the ppk homologous region was limited to a 2.5-kb Ncol-Bglll fragment (pVCK35 [Fig. 1A]). Plasmids containing the cloned V. cholerae ppk gene were constructed as 6688 OGAWA ET AL. EcoRV2 _EcoRV3 lth Ncol EcoRVi EcoRh | Bell | EcoRl2 Psti j ppx => g I Stem-loop pVCK31/ kan pVCK37 pVCK35 eee B 96 -84 ho box -67 “ACACTAAAGGCA TGCAATAAAAGTGTCAC! TACT AST TT + CTCTTTAATGATGAAAACGTCAACTCAAAACGGCATAAGGTAAAGTATGA ppkORF-start CK AK GQeQET N DN S §S Q ppkORF-stop AAAGCAAAAGGGCAACAAGAAACGAATGACAACAGTAGTCAGTAA ppxORFsat M T T VO OV S N +2062 D +3387 +3413 +3429 TAAGCCAGACTAGTCACTGCAATTCGATGAAAAACGCCCAACTC ppx ORF-stop SEES cd +3432 +3448 AAGTTGGGCGTTTTTCATTTCATCACTCAGTCGA 100 nmol/mg accumulated after 4 h in log phase with 0.1, 0.5, or 2 mM P;. Cultures with 0.5 or 2 mM P, maintained poly P levels of >100 nmol/mg for up to 9h, Le., after entering stationary phase. However, poly P levels in the 0.1 mM P, culture decreased gradually along with the decrease in growth rate (Fig. 2B and C, 5 to 9 h). Given the similarity in the growth curves for the 0.5 and 2 mM P; cultures, the slow growth of the 0.1 mM P, culture is likely due to P, depletion. Thus, poly P levels in V. cholerae depend on the P; concentration in the medium. When cells were shifted from a MOPS medium with no P, added to the same medium with 20 mM P,, dramatic accumu- lations of poly P occurred immediately after the upshift (Fig. 2D). Within 1 h, poly P accumulated to >300 nmol/mg andito near 400 nmol/mg after 2 h without any concomitant growth. With the resumption of growth, the poly P level decreased to 250 nmol/mg and remained there for at Jeast five more hours. Similarly, large poly P accumulations have been reported in Aerobacter aerogenes as the “poly P overplus” phenomenon (9). These massive levels of poly P were observed by electron microscopy as numerous bodies of relatively high electron den- sity (Fig. 3). About 1 in 30 cells contained bodies 20 to 40 nm in diameter (Fig. 3B), while the others had smaller bodies, about 5 nm in diameter (Fig. 3A). Such granules were not found in wild-type cells grown in P,-free medium or in ppk mutant cells (see below) grown in P,-rich medium for 2 h (Fig. 3C and D). Inasmuch as these bodies were correlated with the dmassive accumulations of poly P, they are presumed to be poly P. Similar granules observed in Myxococcus xanthus (9) and Helicobacter pylori are somewhat localized at the flagellar pole and in association with the inner membrane as well as being dispersed in the cytoplasm (5). As determined by thin- section electron microscopy (Fig. 3E), the granules in V. chol- erae are distributed in the cytoplasm but not localized along the inner membrane. The ppk gene. The region in the TIGR V. cholerae genome database surrounding a sequence homologous to that of E. cdli ppk was cloned from a V. cholerae genome library by colétiy hybridization using a PCR fragment of the region as a probe (see Materials and Methods). The E. coli Appk Appx knockout 6690 OGAWA ET AL. J. BACTERIOL. FIG. 3. Electron micrographs of V. cholerae strains. The bacteria were harvested at 0 and 2 h after the addition of P,, as for Fig. 2D. Bars, 0.5 ym. Negatively stained samples were prepared from the wild-type culture after 0 (C) and 2 (A and B) h of incubation and from KCV3 (the ppk mutant) after 2 h of incubation (D). Thin-section samples were prepared from the wild type (E) and KVC3 (F) after 2 h of incubation. Magnifications, X28,000 (A, C, E, and F), <35,000 (D), and <45,000 (B). strain CF5802, transformed with pVCK35 containing the V. cholerae ppk homolog (Fig. 1A), exhibited high levels of PPK activity (38,000 U/mg), demonstrating that this region did in fact contain the V. cholerae ppk gene; this was confirmed sub- sequently by sequencing. The deduced amino acid sequence of V. cholerae PPK is 701 amino acid residues long, with a calcu- lated molecular mass of 81.6 kDa; it is 64%, identical and 83% similar (in conserved residues) to that of E. coli. Sequencing also revealed a ppx homolog downstream of ppk in V. cholerae, as in E. coli, although with a two-cistron overlap. A transfor- mant of the £. coli Appk Appx mutant strain CF5802 harboring pVCK31, which contains both the ppk and ppx homologs (Fig. 1A), gave low but significant levels of PPX activity (540 U/mg). Its deduced amino acid sequence is 500 amino acid residues in length, with a calculated molecular mass of 56.4 kDa, and is 51% identical and 70% similar (in conserved residues) to E. coli PPX, Upstream of the ppk open reading frame (ORF) (Fig. 1B) lies a putative promoter region which contains a probable pho box sequence with 15 of the 18 consensus base pairs at posi- tions —84 to ~67 from the A in the ppk start codon (Fig. 1B). In E. coli, pho boxes are the binding sites of the two-compo- nent system regulator protein PhoB. Homologs of phoB and PhoR (the cognate sensor) are found in the V. cholerae genome database, suggesting that transcription of the ppk ppx operon may be regulated by this system. Putative pho boxes are also present in the ppk promoter regions of E. coli, K. aerogenes (12), and Acinetobacter sp. strain ADP1 (8). A pair of 17-bp inverted-repeat sequences (from positions 24 to 40 and 43 to 59 from the ppx stop codon) appear to constitute a rho-inde- pendent transcriptional terminator site downstream of the ppx ORF (Fig. 1D). These features imply that ppk and ppx form an operon, as in E. coli and other gram-negative bacteria. The E. coli Appk Appx transformant with a high copy number of the V. cholerae ppk ppx operon [CF5802(pVCK31)] expressed 690,000 U of PPK and 540 U of PPX activity per mg. We have shown previously that the E. coli wild type transformed with a high copy number of the £. coli ppk ppx operon exhibited 630,000 U of PPK and 50,000 U of PPX activity per mg (2, 3). Thus, V. cholerae PPK was expressed in an E. coli host cell at levels similar to those of £. coli PPK from the E. coli operon, but V. cholerae PPX was expressed about 100-fold less than E. VOL. 182, 2000 5 L 1 wt[vector] - 3 - 24 ppk [Vc-ppk] A600 ly 05 ppk [vector} 0.37 ! 0.2 T T T T T {He t 0 2 4 6 8 10 20 22 Time (h) FIG. 4. V. cholerae ppk complements the adaptive growth defect in an E. coli ppk mutant. E. coli strains MG1655(pFYZ1) (squares), CF5802(pFYZ1) (open circles), and CF5802(pVCK37) (solid circles) were grown in 2X YT medium supplemented with 50 yg of ampicillin/ml to an Agog of 0.5. The cells were harvested and washed twice with MOPS medium devoid of nutrients and resus- pended in MOPS medium with 2 mM P, and 0.4% glucose at zero hour. coli PPX. Unlike in E. coli, there is a 20-bp overlap between the ppk and ppx ORFs in the V. cholerae operon (Fig. 1C), which may interfere with translation of the ppy ORF from the ppk-ppx mRNA, accounting for the undetectable level of PPX activity (Table 1). Null mutant of ppk. In a ppk knockout mutant in V. cholerae (KVC3) (see Materials and Methods), the levels of PPK activ- ity and poly P accumulation were undetectable (Table 1 and Fig. 2D), in contrast to the parental strain, 92A1552-Rif, which accumulated more than 300 nmol of poly P per mg when shifted from a P,-free to a 20 mM P, defined medium. We tested KVC3 for the reported phenotypes of the E. coli ppk mutant strain CA10 (5, 18, 20). No phenotypes were observed for long-term survival in synthetic medium (for 30 days at 30°C), sensitivity to heat (at 45 and 55°C) and hydrogen per- oxide (in 10, 3, and 1 mM), adaptive growth following a shift from rich medium to minimal medium, or long-term (10 days at 30°C) survival in artificial seawater (29). With regard to adaptive growth in a nutrient downshift (Fig. 4), the E. coli wild-type strain MG1655 transformed with the plasmid vector (pFZY1) grew in a minimal medium after a 2-h lag following downshift from a rich medium (LB) whereas the E. coli Appk Appx mutant CF5802 harboring pFZY1 was un- able to grow even 22 h after the downshift. However, CF5802 bearing the V. cholerae ppk plasmid pVCK37 grew in minimal medium after the downshift with only a 4-h lag, after which the growth rate and final OD were similar to that of the wild type. Thus, the V. cholerae ppk complements the E. coli Appk mu- tation in response to this stress condition. A P. aeruginosa PAO] ppk mutant shows no defects in adap- tive responses but is severely impaired in motility and surface attachment (22, 23, 24), and the V. cholerae ppk mutant was found to be deficient in these features as well (23). The swim area of the ppk mutant was 57% that of the wild type on 0.3% agar plates (Table 2), The ppk mutant also exhibited a signif- icantly lower ability for surface attachment (Table 2). Deficien- cies in motility and surface attachment have also been observed in ppk mutants of E. coli, K. pneumoniae, and Salmonella spp. (22). POLYPHOSPHATE IN V. CHOLERAE 6691 TABLE 2. Swimming and surface attachment Strain Genotype Swim area Surface attachment (cm?)* (Asos)” 92A1552-Rif™ wt 1.52 + 0.07 2.14 + 0.70 KVC3 ppk::Kan 0.87 + 0.07 1.46 + 0.30 * Data are from Rashid et al. (23). ’ Average of 16 measurements; two-tail P value by ¢ test with a 0.05 threshold is 0.0012. ta ° WT, wild type. In view of the capacity of poly P to function as a chelator of divalent metals (9), the calcium sensitivity of the V. cholerae ppk mutant was tested (Fig. 5). The growth lag time of the wild-type strain (92A1552-Rif") in LB containing 200 mM CaCl, was 5 h, more than 4 h longer than in the absence of CaCl, (Fig. 2A). The lag time of the ppk mutant KVC3 was significantly longer, at 7h. When complemented with the high- copy-number plasmid pVCK31 harboring the ppk gene, the lag time was shortened to 3 h. As both the wild type and the ppk mutant of V. cholerae had the same lag time when grown in LB in the presence of 400 mM NaCl (data not shown), these results imply that the ppk gene is involved in an adaptation to excess levels of calcium but not chloride or osmolality. Another phenotype of the ppk mutant was observed with regard to P, uptake (Fig. 6). After growth in a P,-free medium for 2 h, wild-type cells displayed a linear (nonsaturable) P, uptake for up to 25 min when incubated in 0.1 mM P, (the P,-limited condition) (Fig. 2B and C). The Appk mutant had unique saturable profiles for P; uptake. It showed a rate’ of uptake similar to that of the wild type from 0 to 3 min, but the uptake after 5 min was minimal and the rate was decreased significantly. These data suggest that ppk is required for the continual high-rate uptake of P,; under low-P, conditions. Characterization of PPK. PPK was purified from a transfor- mant of the E. coli CF5802 strain (Appk Appx::Kan) bearing the V. cholerae ppk ppx operon on a high-copy-number vector, pVCK31. Homogeneity of the purified protein was verified as 10 IL 7 iW 5 | wt[vector] A600 3 2 q 1-4 ppk [Ve-ppk]_ 0.5 4 ppk {vector] 0.2 0.14 0.05 4 a —l} T TTT To 6 8 10 12 14 22 24 Time (h) FIG. 5, Growth adaptation to an excess amount of calcium. V. cholerae strains 92A1552-Rif*(pBluescript H) (squares), KVC3(pBluescript II) (open cir- cles), and KVC3(pVCK31) (solid circles) were grown in LB medium supple- mented with 10 mM KH,PQ, and 50 yg of ampicillin/ml overnight. The cells were inoculated in LB supplemented with 200 mM CaCl, and shaken at 37°C. 6692 OGAWA ET AL. 1500 wt S sO S ] | 1000 5 E | Qo & 4 7 ppk & 500-4 - 4 0 ' T qT t t 0 5 10 15 2 25 30 Time (min) FIG. 6. P; uptake of V. cholerae. P; uptake activities of 92A1552-Rif (squares) and KVC3 (circles) were tested as described in Materials and Methods. a single band on a Coomassie-stained sodium dodecyl sulfate- polyacrylamide gel (data not shown) with a molecular mass estimated at 87 kDa, compared to the calculated mass of 81.6 kDa. Comparison of the PPK retention time to those of ref- erence proteins in a high-performance liquid chromatography gel filtration column showed a molecular mass of 310 kDa (data not shown), indicating that V. cholerae PPK is a homotet- ramer like E. coli PPK (1). The final fraction has a specific activity of 40 x 10° U/mg, slightly higher than that of E. coli PPK (29 x 10° U/mg) (1). The optimal reaction conditions for V. cholerae PPK are almost the same as those for E. coli PPK, e.g., a pH optimum of 7.2 in HEPES buffer and 4 mM Mg?*, and 40 mM ammo- nium sulfate increases activity twofold. Autophosphorylated PPK was observed in a reaction with [y-32P]ATP. Like the E. coli enzyme (16), the V. cholerae enzyme can catalyze the synthesis of ATP from ADP and poly P and can catalyze the synthesis of GTP and ppppG (linear guanosine 5’-tetraphos- phate) from GDP and poly P. Significant differences in the kinetic parameters for the PPKs of V. cholerae and E. coli are shown by the 40-fold decrease in the V. cholerae K,, value for ATP for the forward (poly P synthesis) reaction (Table 3). The Kea/K,, ratio for the ATP synthesis reaction of V. cholerae PPK is more than 10 times higher than that for E. coli. On the other hand, the k,,,/K,,S ratios for the GTP and ppppG synthesis reactions of V. cholerae PPK are 5 and 20 times lower, respec- tively, than those for £. coli. These data suggest that the V. cholerae PPK is more specific for the generation of ATP than GTP or ppppG. DISCUSSION Accumulations of poly P in V. cholerae are remarkable for being so great and sustained compared to those in E. coli. The levels in excess of 50 nmol/mg of protein during exponential growth in a rich medium (Fig. 2A) and 150 nmol/mg in sta- tionary phase in a defined medium (Fig. 2C) are roughly 100 times those in E. coli. Yet the PPK activities in extracts are nearly the same (Table 1). Some of the reason may lie in the undetectable levels of PPX activity in extracts of V. cholerae (Table 1). In that organism, as in E. coli (3), an operon con- tains the ppk as well as the ppx gene (Fig. 1). Unlike in E. coli, J. BACTERIOL. where the ppx gene is separated by 7 bp from the upstream ppk gene, the amino terminus-encoding region of ppx in V. cholerae overlaps the carboxy terminus-encoding region of ppk by 20.bp (Fig. 1C). The E. coli transformant with the V. cholerae ppx gene on a high-copy-number plasmid did express PPX activity. How these genes and possibly others are regulated pre- and posttranscriptionaily remains to be determined. When cells are switched from a P; starvation medium to one with adequate P,, there is a massive accumulation of poly P (Fig. 2D), as observed in A. aerogenes (9) and designated the poly P overplus phenomenon. The accumulation of poly P is evident as electron-dense granules up to 40 nm in diameter with an ordered matrix structure as in crystal complexes (Fig. 3B). The granules appear to be largely cytoplasmic (Fig. 3E), unlike some of the granules in H. pylori, which have polar and membrane-oriented locations (5). The dynamic accumulation and removal of poly P and its mobilization at a molecular level, as well as the nature of the granules and their cellular loca- tions, need to be clarified. V. cholerae PPK resembles that of E. coli in size and in its multiple activities: processive poly P synthesis from ATP, nu- cleoside diphosphate kinase action on ADP and GDP by donor poly P, pyrophosphoral transfer to GDP to form ppppG, and autophosphorylation by ATP. The most notable differences are in the kinetic parameters (Table 3), e.g., a K,, for ATP of 0.2 mM for V. cholerae PPK compared to 2.0 mM for E. coli PPK. Also, kinetic parameters for ATP, GTP, and ppppG synthesis reactions indicate that V. cholerae PPK is more specific for the generation of ATP than for GTP or ppppG compared to E. coli PPK. These enzyme characteristics are similar to those of H. pylori PPK purified as a recombinant protein (27; C.-M. Tzeng and A. Kornberg, unpublished data). The strong PPK sequence homologies of 20 or more bacte- tial species inchide a number of the major pathogens (26), V. cholerae among them. In view of the striking dependence of E. coli on PPK for a variety of adaptive responses in the stationary phase and the expression of virulence factors in the stationary phases of some pathogens (7), the phenotypes of null mutants of ppk in these pathogens have been sought. Furthermore, there is the attractive possibility that PPK might prove a novel target for an antimicrobial drug with a broad spectrum and minimal side effects, inasmuch as PPK has not been found in animal species. Among the pathogenic features of the PPK null mutants, a decrease in motility (commonly associated with a loss of viru- lence [19]) and weakened attachment to abiotic surfaces (a TABLE 3. Kinetic parameters of PPK re Reaction and Kin max Kot Keat/Kn source (mM) (pmol mg? min7?) (s7?) (s7? mM~?) Poly P synthesis V. cholerae 0.05 1.6 X 10° 8.2 164 E. coli* 2 51 x 10° 59 29.5 ATP synthesis V. cholerae 0.12" 95 x 10° 65.3 544 E. coli€ 0.25 3.7 x 10° 10.5 42 GTP synthesis V. cholerae 2.5° 1.5 x 10* 0.076 0.03 E. coli€ 0.63 5.7 x 104 0.088 0.14 ppppG synthesis V. cholerae 2.3" 1.1 x 103 0.006 0.002 E. coli 0.46 9.0 x 108 0.027 0.04 * E. coli data for poly P synthesis are from Ahn and Kornberg (1). * Values for ADP or GDP. ° E. coli data for ATP and GTP synthesis are from Kuroda and Kornberg (16). VOL. 182, 2000 frequent correlate of poor biofilm formation) have been ob- served in several pathogens. Particularly striking are mutants of P. aeruginosa (22, 23, 24) which are defective in quorum sensing and have also lost their virulence in mouse models. The V. cholerae ppk mutant also had diminished attachment to an abiotic surface (Table 2), with the activity decreased by 68% compared to that of the wild type. Although this was not a dramatic reduction as with P. aeruginosa (22, 24), it could be emphasized by using the rugose colony variant of V. cholerae O1 (30), which prefers to form biofilm, compared to the smooth colony variant which was used in this study. The failure to adapt to stress and the lack of survival in stationary phase observed in the E. coli ppk mutant (6) have not been apparent in the V. cholerae mutant. In addition to decreased motility and decreased attachment to abiotic sur- faces (Table 2), other defects have been observed. One is a delayed adaptation to high calcium levels in the medium (Fig. 5); complementation of the mutant with the ppk gene more than corrects for the extended lag in growth. It is plausible that a large amount of poly P can trap excess calcium in the cell and maintain the calcium level at a low enough level to permit growth. Another defect of the ppk mutant is an abnormal rate of uptake of P; from the medium (Fig. 6). Whereas the initial rate resembled that of the wild type, the subsequent rate was considerably reduced. Not enough is known about P; uptake in V. cholerae to identify which system might be affected by the lack of PPK function. Interestingly, budding-yeast mutants de- ficient in poly P accumulation also showed a similar saturable curve for P; uptake (18a). The phenotypic tests of the V. cholerae ppk mutant were patterned on those performed on bacterial species which differ sharply from V. cholerae in their physiologic features, commen- sal interactions, and host invasiveness (10). In view of the unique aspects of the aquatic ecology of V. cholerae, much needs to be studied to evaluate what effect the lack of PPK and poly P might have on its survival and pathogenesis. ACKNOWLEDGMENTS We thank F. H. Yildiz and G. Schoolnik in the Department of Microbiology and Immunology for providing strains, plasmids, the gene library, and technical advice, N. Ghori in the same department for electron microscopy, S. Handy in the Department of Chemistry for high-performance liquid chromatography analyses, N. Rao in our lab- oratory for performing surface attachment assays, and L. Bertsch for help with the manuscript. This work was supported by a grant from the National Institute of General Medical Sciences. REFERENCES . Ahn, K,, and A. Kornberg. 1990. Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J. Biol. Chem. 265:11734--11739. 2. Akiyama, M., E. Crooke, and A. Kornberg. 1992. The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and mem- brane location of the protein. J. Biol. Chem. 267:22556-22561. 3. Akiyama, M., E. Crooke, and A. Kornberg. 1993. An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J. Biol. Chem. 268:633-639, 4, Ault-Riché, D., C. D. Fraley, C.-M. Tzeng, and A. Kornberg. 1998. 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