We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness

Farías, Ricardo N.

Rapisarda, Viviana A.

Rintoul, María R.

Schurig-Briccio, Lici A.

Journal of Bacteriology

PubMed

We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness. Copyright © 2009, American Society for Microbiology. All Rights Reserved.Fil: Schurig Briccio, Lici Ariane. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; ArgentinaFil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; ArgentinaFil: Rintoul, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; ArgentinaFil: Rapisarda, Viviana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Departamento de Bioquímica de la Nutrición; Argentin

Schurig Briccio, Lici Ariane

Farias, Ricardo Norberto

Rintoul, Maria Regina

Rapisarda, Viviana Andrea

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

JOURNAL OF BACTERIOLOGY, July 2009, p. 4478–4481 Vol. 191, No. 130021-9193/09/$08.000 doi:10.1128/JB.00082-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli IsRelated to Inorganic Polyphosphate LevelLici A. Schurig-Briccio, Ricardo N. Farías, María R. Rintoul, and Viviana A. Rapisarda*Departamento Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional deInvestigaciones Científicas y Técnicas-Universidad Nacional de Tucumán), and Instituto deQuímica Biológica Dr. Bernabé Bloj, Chacabuco 461, San Miguel deTucumán T4000ILI, ArgentinaReceived 22 January 2009/Accepted 9 April 2009We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphatelevel in late stationary phase, which could account for changes in gene expression and enzyme activities thatenhance stationary-phase fitness.Polyphosphate (poly-P) is a long-chain polymer composedof many orthophosphates linked together by high-energy ATP-like bonds. Studied mainly in prokaryotes, poly-P plays animportant role as an energy source, as a regulator of geneexpression, as a store of inorganic phosphate, and as a chelatorof heavy metals (8, 10). The main enzymes associated withpoly-P metabolism in bacteria are the polyphosphate kinase(PPK, encoded by ppk) and the exopolyphosphatase (PPX,encoded by ppx) (1, 2). The ppk ppx double mutant exhibitsgreatly reduced synthesis of poly-P, is deficient in stationary-phase functions, and lacks resistance to different stresses (6,17).Previously, we found that expression of several respiratory(ndh, sdhC, ubiC, nuoAB, and cydA) and defense (katG andahpC) genes was maintained in late stationary phase when themedium’s phosphate concentration was above 37 mM (24, 25).Furthermore, Escherichia coli cells grown in medium contain-ing this critical phosphate concentration had high viability, lowoxidative damage, and elevated resistance to external H2O2stress in late stationary phase (25).We examined the relationship between the medium’s phos-phate concentration and intracellular poly-P levels in the wild-type and ppk ppx mutant strains to see if the previouslyobserved effects on gene expression, enzyme activity, and tol-erance to H2O2 were correlated with elevated poly-P levels.Measurements of poly-P level in whole cells. Intracellularpoly-P was measured in cell suspensions by using a DAPI(4,6-diamidino-2-phenylindole)-based fluorescence approach(3). Cells were washed and resuspended in buffer T (100 mMTris HCl [pH 7.5]). DAPI (Sigma) was added to 10 M incuvettes containing cell suspensions in buffer T at an opticaldensity at 560 nm of 0.02. After 5 min of agitation at 37°C, theDAPI fluorescence spectra (excitation, 415 nm; emission, 445to 650 nm) were recorded using an ISS PCI spectrofluorometer(Champaign, IL). The fluorescence (in arbitrary units) of theDAPI-poly-P complex at 550 nm was used as a measure ofintracellular poly-P because fluorescence emissions from freeDAPI and from DAPI-DNA are minimal at this wavelength (3).We first compared various conditions for preparing the cellsamples, using exponential-phase cells grown aerobically in LBmedium at 37°C (Fig. 1). Untreated cells (buffer T wash only)with or without exposure to heat for 10 min at 60°C gaveessentially identical spectra, similar to those of heat-treatedcompetent cells (3) (Fig. 1). In contrast, competent cells with-out heat treatment did not exhibit a poly-P-DAPI fluorescencesignal (Fig. 1). Thus, it appeared that poly-P levels could bedirectly measured in untreated cells, and we used this methodfor subsequent experiments.The poly-P level was determined for cells of wild-type andppk ppx mutant strains (Table 1) grown in LB medium (6 mMphosphate) and in minimal medium M9 (64 mM phosphate)(23) or MT (2 mM phosphate) (26) (Fig. 2). All media weresupplemented with 0.5% glycerol, and minimal medium wasalso supplemented with 0.1% tryptone. The poly-P level in LBmedium-grown wild-type cells was always low, whereas it washigh in M9 medium-grown cells even at 72 h (Fig. 2a and b).However, the poly-P level in MT medium-grown cells was highup to 24 h but decreased at 48 and 72 h (Fig. 2c). The ppk ppxstrain exhibited low poly-P levels regardless of the culturemedium or growth stage, confirming that the fluorescence sig-* Corresponding author. Mailing address: INSIBIO, DepartamentoBioquímica de la Nutrición, Charabuco 461, San Miguel de TucumánT4000ILI, Argentina. Phone and fax: 54-381-4248921. E-mail: vrapisarda@fbqf.unt.edu.ar. Published ahead of print on 17 April 2009.FIG. 1. DAPI-poly-P complex fluorescence in live E. coli cells. Flu-orescence emission spectra of DAPI-poly-P were measured in un-treated (solid black line), untreated and heated (solid gray line), com-petent (dotted black line), and heated competent (dotted gray line)exponential MC4100 cells. Data are representative of results of at leastthree separate experiments. AU, arbitrary units.4478 by on June 15, 2009 jb.asm.orgDownloaded from nals were specific for poly-P (Fig. 2d to f). The basal level ofDAPI-poly-P fluorescence in the mutant strain could be pro-duced by short-chain poly-P synthesized through an alternativepathway (5) or by some lack of specificity of the DAPI stain.A high phosphate concentration maintains a high poly-Plevel in stationary phase. To determine if the extracellularphosphate concentration in the growth medium influenced thepoly-P level in stationary phase, we measured poly-P levels at48 h in the wild-type and mutant strains grown in MT mediumwith the addition of different phosphate concentrations. In thewild type, the poly-P level was maintained only when the phos-phate concentration was 37 mM (Fig. 3). This effect could bea consequence of PPX inhibition by high phosphate concen-trations in the medium, as described for Saccharomyces cerevi-siae (9). It is important to note that even in MTP medium(defined as MT medium with added 40 mM buffer phosphate [pH7]) (24), the poly-P level was low in the ppk ppx mutant (Fig. 3).Unusual Pi-dependent stationary-phase events are corre-lated with poly-P level. Using the ppk ppx mutant strain, westudied some of the stationary-phase physiological events thatare triggered by growth at high phosphate concentrations (25).By using chromosomal lacZ transcriptional fusions (Table 1),the expression of sdhC (encoding succinate dehydrogenase)and ubiC (chorismate pyruvate-lyase) was assayed as -galac-tosidase activity (15) in MTP medium-grown cells (Fig. 4aand b). Expression of rpoS (encoding sigma S factor), which isknown to be positively regulated by poly-P levels (22), wasmeasured similarly to a control (Fig. 4c). The sdhC, ubiC, andrpoS gene expression remained high in the wild type, but in theppk ppx mutant strain, expression decreased as the cells pro-gressed into stationary phase. We cannot yet say whetherpoly-P directly regulates expression of these respiratory genes,but Kusano and Ishihama (12) have reported that poly-P phys-ically interacts with RNA polymerase and under certain con-ditions activates rpoS transcription, although the precise mech-anism is still unclear.In MTP medium-grown cells, membrane NDH-2 and suc-cinate dehydrogenase activities (18, 21, 24) decreased throughstationary phase in the mutant strain, whereas they remainedhigh in the wild-type strain (Table 2). In addition, catalaseTABLE 1. Strains used in this workStrain Relevant genotype Reference or sourceMC4100 araD lac rpsL flbB deoC ptsF rbsR relA1 4TSDH00 MC4100 	(sdhC-lacZ) 16MC4100(MO2) MC4100 	(ubiC-lacZ) 13NR629 MC4100 	(rpoS477-lacZ) 22CA10 (lac-proAB) supE thi-1/F traD36 proAB lacIq lacZM15(ppk ppx)::Km6LSB022 MC4100 (ppk ppx)::Km P1(CA10)  MC4100LSB023 MC4100 	(sdhC-lacZ) (ppk ppx)::Km P1(CA10)  TSDH00LSB024 MC4100 	(ubiC-lacZ) (ppk ppx)::Km P1(CA10)  MC4100(MO2)LSB025 MC4100 	(rpoS477-lacZ) (ppk ppx)::Km P1(CA10)  NR629FIG. 2. DAPI-poly-P fluorescence in a wild-type and a ppk ppx mutant strain. Fluorescence emission spectra of DAPI-poly-P were measuredin untreated MC4100 (a to c) or LSB022 (d to f) cells grown in LB, M9, or MT medium for different periods of time as indicated. Data arerepresentative of results of at least three separate experiments. AU, arbitrary units.VOL. 191, 2009 NOTES 4479 by on June 15, 2009 jb.asm.orgDownloaded from activity (7, 25) increased in the wild-type strain only duringstationary phase (Table 2). These unusual enzyme activities instationary phase require a persistent protein synthesis. In high-phosphate media, an elevated poly-P level may prolong theamino acid supply required for de novo synthesis, because itpromotes ribosomal protein degradation by the Lon protease(11).The ability to tolerate the exogenous oxidative stress gener-ated by H2O2 was also studied. After 1 h of incubation with 10mM H2O2, the numbers of CFU on LB agar plates incubatedfor 24 h at 37°C were determined. The wild-type cells werehighly tolerant to exogenous peroxide, as evidenced by the factthat the viability was maintained, whereas the viability of theppk ppx mutant strain decreased around 2 orders of magnitude.These results for MTP could be explained by the high poly-Plevel and the induction of several antioxidant enzymes (e.g.,catalase).A phosphate concentration of around 30 mM (similar to thecritical concentration used here) could be present in the hu-man intestinal lumen in healthy adults consuming an averageWestern diet (14, 27). The PPK sequence was found in severalhuman pathogens in which the role of poly-P is related togrowth advantages, motility, quorum sensing, biofilm forma-tion, and virulence (19, 20). Therefore, our observation of therelationship between exogenous phosphate concentration andpoly-P level may help to elucidate the importance of highphosphate as a physiological signal.We thank Y. J. Seok, R. Meganathan, T. J. Silhavy, and E. Crookefor providing strains TSDH00, MC4100(MO2), NR629, and CA10,respectively.This research was supported by the Consejo de Investigaciones de laUniversidad Nacional de Tucumán (CIUNT) and by the Consejo Na-cional de Investigaciones Científicas y Técnicas (CONICET) of Ar-gentina. L.A.S.-B. is a predoctoral fellow of CONICET (Argentina).REFERENCES1. 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 kinasegene of Escherichia coli. Isolation and sequence of the ppk gene and mem-brane location of the protein. J. Biol. Chem. 267:22556–22561.3. Aschar-Sobbi, R., A. Y. Abramov, C. Diao, M. E. Kargacin, G. J. Kargacin,R. J. French, and E. Pavlov. 2008. High sensitivity, quantitative measure-ments of polyphosphate using a new DAPI-based approach. J. Fluoresc.18:859–866.4. Casadaban, M. J., and S. N. 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Gene expression. sdhC (a), ubiC (b), and rpoS (c) expres-sion was measured as -galactosidase activity in the wild type (filledsymbols) and the ppk ppx mutant (open symbols), with correspondingfusions, at different times of growth in MTP. Data are representativeof results of at least four separate experiments performed in duplicate.MU, Miller units.TABLE 2. Enzymatic activities in a wild-type and a ppk ppx mutantE. coli straincLength ofculture(h)aRate of MTTb reduction(nmol min1 mg protein1)Rate of H2O2decomposition(mol min1 mgprotein1)NADHdehydrogenase-2SuccinatedehydrogenaseMC4100 LSB022 MC4100 LSB022 MC4100 LSB0227 1,284  65 1,230  68 602  22 611  44 16  3 15  148 1,257  54 257  48 624  28 188  21 26  1 14  2a Cells were grown in MTP.b MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.c Values are averages  standard deviations.4480 NOTES J. 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Phosphate-enhanced stationary-phase fitness of Escherichia coli is related to inorganic polyphosphate level

CONICET Digital

Lici A. Schurig-Briccio

Ricardo N. Farías

María R. Rintoul

Viviana A. Rapisarda

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Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level

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Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level▿

Phosphate-Enhanced Stationary-Phase Fitness of Escherichia coli Is Related to Inorganic Polyphosphate Level▿

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LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

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