Vol. 166, No. 1JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 205-2110021-9193/86/040205-07$02.00/0Copyright C) 1986, American Society for Microbiology

Nucleotide Pool in pho Regulon Mutants and Alkaline PhosphataseSynthesis in Escherichia coli

N. N. RAO,' E. WANG,' J. YASHPHE,2 AND A. TORRIANI1*Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021391; and Department of

Bacteriology, Hadassah Medical School, The Hebrew University, Jerusalem, Israel2

Received 26 August 1985/Accepted 13 January 1986

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growthin phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvationconditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion ofthe phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is notresponsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expressionof the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excessgrowth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphataseconstitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of beingconstitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulonexpression was supported by the fact that the two nucleotides appearing under phosphate starvation inducedthe synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphateexcess.

Escherichia coli has a complex mechanism for the regula-tion of phosphate (Pi) uptake and metabolism involving thephosphate regulon (Fig. 1). This regulon consists of 20 to 30genes, all of which are induced by the product of the phoBgene and repressed by Pi in the growth medium (A. Torrianiand D. N. Ludke, in M. Schaechter, F. C. Neidhardt, J. L.Ingraham, and N. 0. Kjeldgaard, ed., The Molecular Biol-ogy of Bacterial Growth, in press). One of these genes,phoA, encodes for the enzyme alkaline phosphatase whichcleaves organic phosphomonoesters into Pi.The regulation of the gene phoB depends on the product of

the gene phoR (5, 9, 15). When the cells have an excesssupply of Pi, the phoR gene product acts as a repressor,whereas under Pi limitation the phoR gene product acts as aninducer ofphoB. Thus, the phoR protein has a dual role (11,15).However, alkaline phosphatase can be induced in an

excess phosphate medium when mutants defective in nucle-otide biosynthesis are starved for pyrimidines or for guanine(16). The induction of alkaline phosphatase in this case is notdue to the lowering of the internal Pi pool, but rather toalterations in the cellular nucleotide pool. These results ledWilkins (16) to believe that alkaline phosphatase synthesis isregulated by phosphorylated nucleotides.We have focused our efforts on isolating and identifying

the possible nucleotides involved in the regulation of alkalinephosphatase. The nucleotide pools from mutants of the phoregulon were resolved and analyzed by two-dimensionalpolyethyleneimine-cellulose thin-layer chromatography(TLC) (3) and by autoradiography. In addition, experimentswere performed involving the in vitro synthesis of alkalinephosphatase with permeabilized (2) wild-type repressed cellsto test the inducibility of alkaline phosphatase by isolatednucleotides.

* Corresponding author.

MATERIALS AND METHODS

Materials. Flexible polyethyleneimine-cellulose TLCsheets (nonfluorescent, 20 by 20 cm) were obtained from J. T.Baker Chemical Co. Snake venom phosphodiesterase waspurchased from Boehringer Mannheim Biochemicals. 32p;(carrier free in water) was obtained from ICN Chemical Co.All chemicals used were ofultrapure grade and were obtainedfrom either Schwartz/Mann, Sigma Chemical Co., or AldrichChemical Co.

Bacterial strains. The E. coli K-12 strains used in theseexperiments were mutants of strain W3110 F- trpR rpsL (C.Yanofsky strain) which was constructed by P1 transduction(1). WC4 is a W3110 phoU35 mutant and is alkaline phos-phatase constitutive. WE15 possesses a deletion of the phoAgene and cannot synthesize alkaline phosphatase. The strainK10 Hfr relAl tonA22 was used to prepare the plasmolyzedcells.Growth and 32p labeling of cells. Cultures grown overnight

(18 h) in LB broth (6) were washed with 0.85% salinesolution, diluted 100-fold into MOPS (morpholinopropanesul-fonic acid) minimal medium (7) with limiting glucose (0.25g/liter) and low Pi (0.1 mM), and incubated at 37°C for 16 to18 h in a rotary shaker (250 rpm).For 32p labeling, the cultures grown in limited glucose

were diluted to a concentration of 107 cells per ml withMOPS minimal medium containing 0.1 mM phosphate and 4g of glucose per liter. The growth of the cultures at 37°C wasmonitored by measuring the turbidity at 540 nm. An opticaldensity at 540 nm (OD540) of 1.0 corresponded to a concen-tration of about 5 x 108 cells per ml. When the culturesreached a concentration of 2 x 107 to 3 x 107 cells per ml,500 1xCi of 32P, was added to 5-ml cultures, and the growthwas continued.

Extraction of nucleotides. When the 32P-labeled culturesreached a concentration of about 108 cells per ml, 0.8 ml wasadded to 0.08 ml of 11 M formic acid, and the mixture was

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206 RAO ET AL.

GENES SENSINGPi LEVEL

GENES CONTROLLINGp.oB EXPRESSION

GENES REQUIRING FUNCTIONALMuOB

jMSA y

IxJ

mR-*PHoR -%I ES PHOSPHATE STARVATIO IN[PHoRWPHOR-Y\ /2 P~~~~~~~~~~ROMC\E,QE OUTER MEMBRANE PORE

eaoM PHOM g e -WiOB 'e$ EHA ALKALINE PHOSPHATASE

e.iR- 4PHOR -'x/ MS PHOSPHATE BINDING PROTEI&UGA8 B GLYCEROL PHOSPHATE

1RANSPORT SYSTEM

DUCEDOTERS

IN

FIG. 1. Scheme of regulation of the phosphate (pho) regulon. The genes belonging to the pho regulon, as represented by phoA (structuralgene for alkaline phosphatase), are derepressed by Pi limitation. Their expression is positively regulated by phoB, phoM, and phoR andnegatively by phoR, phoS, pst, and phoU. The phoS and pst genes are involved in the transport of Pi. They may regulate pho regulon byinfluencing the level of a hypothetical effector(s) through the mediation ofphoU. PhoR (the product ofphoR) presumably binds to a nucleotidecofactor X to form the inducer PhoR-X or to Y to form the repressor PhoR-Y, both of which influence the expression ofphoB. In the absenceof PhoR, the phoM gene product activates the expression of phoB, independent of Pi levels. High levels of PhoB turn on the expression ofgenes like phoE, phoA, and phoS. The product of the phoU gene may change X to Y directly, or it may regulate the gene encoding thisfunction (Torriani and Ludke, in press).

kept at 0°C for 30 min. Another 0.8 ml of the culture wassimilarly treated when the cells reached the stationary phaseof growth (about 3 x 108 cells per ml). The procedure for theextraction of nucleotides with formic acid and the precipita-tion of unincorporated 32p was similar to the one describedby Bochner and Ames (3), except 1 mM NaF was presentduring the extraction procedure to prevent the possibledegradation of nucleotides by acid phosphatase (10).

Two-dimensional TLC. The separation of nucleotides fromformic acid extracts was carried out by two-dimensionalTLC on polyethyleneimine-cellulose plates (3). Before spot-ting the extracts (10 ,ul per plate), the sheets were prerunovernight in the first-dimension solvent. This eliminated theinterfering impurities. The prerun plates were washed inmethanol for 20 min and allowed to dry at 25°C before use.The solvents used for the first dimension were 0.9 M

guanidine hydrochloride or a mixture of 0.75 M Tris, 0.45 MHCl, and 0.5 M LiCl (used in the experiments described inFig. 5). The guanidine hydrochloride solvent required arunning time of 90 to 120 min, whereas the Tris-HCl-LiClsolvent required about 3 h for completion of the run in thefirst dimension. After the first-dimensional run, the plateswere washed in methanol for 20 min and air dried as before.The chromatograms were then run in the second dimension.The solvent used was a solution containing 43.5 g of(NH4)2SO4, 0.4 g of (NH4)HSO4, and 4 g of disodium EDTAin 100 ml of distilled water. The time required for thecompletion of the run in the second dimension was about 6 h.The chromatograms after the second-dimensional run werewashed in methanol and dried as described above.

Autoradiography. The chromatograms were exposed onKodak XAR-5 X-ray films at -70°C. After exposure for 72 to76 h, the X-ray films were developed by standard proce-dures.

Identification of 32P-labeled spots on autoradiograms. Thecorrespondence of a given spot on the autoradiogram with aknown nucleotide was confirmed by the following tests aspreviously described (3). (i) The spots were cochromato-graphed with chemical standards located by their UV fluo-rescence with the aid of a UV light source (Mineralight lamp,model UVGL-25; UVP, Inc.); (ii) the extracts were treatedwith charcoal, (iii) oxidized by periodate, or (iv) treated with

snake venom phosphodiesterase. For phosphodiesterasetreatment the selected nucleotide spots on TLC plates werecut out, and the polyethyleneimine-cellulose was scrapedinto microfuge tubes containing 50 p1l of 0.1 M Tris hydro-chloride (pH 8.9) containing 0.1 M NaCl, 0.015 M MgCl2,and phosphodiesterase (0.0014 U/ml; Boehringer Mannheim)and incubated overnight at 37°C. The tubes were thencentrifuged for 5 min at 4°C in a microfuge; the supernatantfluids were collected and analyzed by TLC.

Preparation of permeabilized cells. E. coli K10 cells wererendered permeable to nucleotides by a method developedby Ben-Hamida and Gros (2).

In vitro system for alkaline phosphatase synthesis. As asource of DNA and ribosomes, premeabilized cells (corre-sponding to 5 x 108/ml) of E. coli K10 were used with an invitro mix (17). Phenethyl alcohol was omitted because it isknown to arrest the conversion of the inactive monomersubuntis of alkaline phosphatase into the active dimers (14).To promote the dimerization and activation of the enyzme,0.1 mM ZnCl2 was added (13).To test the possible induction of alkaline phosphatase by

any of the isolated nucleotides, the polyethyleneimine-cellulose containing the individual nucleotides was scrapedand placed in a corresponding well in a Cell Well (ComingGlass Works). A 10-,ul sample of 1 M Tris hydrochloride (pH8.2) containing 2 mM MgSO4 and 0.2 ml of in vitro mix withpermeabilized cells was added to each well. The reactionmixture was incubated at 37°C for 24 h. Then each samplewas dialyzed against 0.1 M Tris hydrochloride (pH 8.2) with2 mM MgSO4 and 0.1 mM ZnCl2 to eliminate the free Pi,which is a competitive inhibitor of alkaline phosphatase. Totest the efficiency of this in vitro system, the synthesis ofP-galactosidase was also measured by using o-nitrophenylP-D-galactoside as a substrate (6). The presence of alkalinephosphatase was detected in two ways. In the first proce-dure, a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (50 ,ug/ml) in N,N-dimethylformamide was addedto each well, and the appearance of the blue-colored productwas registered. For the second method, the contents of eachwell were transfered to small test tubes to which 0.5 ml ofalkaline phosphatase assay mix (equal volumes of 1.2 M Trishydrochloride [pH 8.2] with 2 mM MgSO4 and 2 mM

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E. COLI NUCLEOTIDE POOL AND ALKALINE PHOSPHATASE

p-nitrophenyl phosphate) was added, and the mix incubatedovernight at 27°C. Each sample was centrifuged at 7,500 x gfor 5 min, and the absorption of the supernatant fluid at 410nm was measured.

RESULTSSynthesis of alkaline phosphatase in E. coli W3110. During

exponential growth, Pi in the medium is in excess; thus thepho regulon is repressed in the wild-type strain W3110, andno alkaline phosphatase is synthesized. When Pi in themedium is depleted the cells enter the stationary phase ofgrowth, and the pho regulon is derepressed; hence alkalinephosphatase is synthesized (12). In a constitutive strain likeWC4 phoU35 both at the exponential (Pi excess) and thestationary (Pi limited) phase of growth, the pho regulon isexpressed; hence alkaline phosphatase is synthesized all thetime (Table 1). In this paper, the terms "exponential" and"stationary" are retained to indicate Pi-excess and Pi-limitedconditions in both the wild-type and mutant strains of thepho regulon.

In a standard experiment in which 0.1 mM Pi was depletedwhen the culture reached an OD540 0.7 (ca. 3 x 108 cells perml), alkaline phosphatase was fully synthesized by the cells.The addition of P1 to the medium allowed further exponentialgrowth but repressed alkaline phosphatase synthesis. Theconstitutive mutants continued the synthesis at the samelevel, however.These conditions (0.1 mM K2HPO4) ofgrowth and alkaline

phosphatase synthesis were adopted for the experiments inwhich we analyzed the pool of nucleotides.

Nucleotide pools of W3110. The nucleotide pools werestudied by TLC (3) of extracts of strain W3110 and itsAphoA, phoU, and phoR mutants. In our experiments, 32P,was added during exponential growth at an OD540 of 0.05(Fig. 2). Samples were withdrawn during exponential growth(OD540 of 0.2) and after Pi depletion measured by the onset ofalkaline phosphatase synthesis in the wild type (OD540 of0.65) (Fig. 2).The two-dimensional TLC employing 0.9 M guanidine

hydrochloride in the first dimension and ammonium sulfatesolvent in the second dimension separated the nucleotidesby the charge or phosphate content in the first dimension andby the base in the second dimension with a good resolution,as shown in the TLC plates of Fig. 3 and 4. The standardnucleotides were located by their fluorescence under UVlight. These standards were superimposed on the autoradio-grams of all samples. To each 32P-labeled sample, ATP andGTP were added as unlabeled standards in the chromato-gram. Of 25 to 30 32P-labeled spots, about 15 were recog-

TABLE 1. Alkaline phosphatase activities in cultures of E. coliAlkaline phosphatase activitya (U/4 x 108

Strain cells)Pi excess Pi depletion

W3110 (wild type) 0.8 43.2W3110 phoU35 (C4) 182.9 241.8W3110 phoR68 (C2) 31.4 12.2W3110 AphoA8

(E15) <0.03 <0.03a The cultures were grown in MOPS minimal medium containing 0.1 mM

K2HPO4, and 4 g of glucose per liter. Samples were withdrawn during theexponential phase of growth (Pi excess) or during Pi depletion (time points Eand S in Fig. 2). The alkaline phosphatase activity was expressed asnanomoles of p-nitrophenol formed per minute at 27'C from 1 mM p-nitrophenol phosphate in 0.6 M Tris hydrochloride buffer (pH 8.2).

160

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to 0 Q0 0.130.32p0 32~~~~~~~~0

0 - 2~~~00 0..

32p E

0.012 4 6 8 10 12

TIME (hours)FIG. 2. Growth and alkaline phosphatase synthesis by E. ccli

W3110. The culture was labeled with 32P (100 pCi/ml) during growthin MOPS medium with 0.1 mM K2HP04 and 4 g of glucose per liter.E and S represent the sampling points at the exponential andstationary phases of growth, respectively. The arrow indicates thetime of addition of 32P1. Symbols: 0, growth; 0, alkaline phospha-tase activity as units per 4 x 108 cells per ml.

nized as identical to the cochromatographed standards. Theother spots were recognized as nucleotides because theywere eliminated when the extract was treated with activatedcharcoal. The autoradiograms from extracts of wild-typecells growing exponentially (conditions of alkaline phospha-tase repression) (Fig. 3A and B) were compared with thosefrom wild-type cells in the stationary phase (alkaline phos-phatase-derepressed conditions) (Fig. 3C and D). Threespots, S2, S14, and S15, present in stationary-phase cellswere absent in the exponential-phase cells (Table 2). On theother hand, the exponentially growing wild-type cells hadfour spots, S3, S4, S8, and S12, which were missing inwild-type stationary-phase cells (Table 2).Three questions may arise from these results. (i) Is the

modified nucleotide pattern due to the presence of activealkaline phosphatase? (ii) Is it due to P1 starvation? (iii) Is thechanging pattern related to genetic properties of the phoregulon?These questions were approached by using various mu-

tants and comparing their nucleotide patterns with those ofthe wild type.

Nucleotide pools in various mutants with mutations of thepho regulon. (i) To investigate whether the modified patternobserved during Pi depletion was due to the activity of theinduced alkaline phosphatase, we used WE15, a AphoAstrain which is devoid of alkaline phosphatase activity. Thenucleotide pool of WE15 (AphoA8) (results not shown) wasvery similar to that of the wild-type W3110 strain, except forspots S4 and S12, which were present in WE15 at both theexponential and stationary phases of growth, and S14, whichwas observed in wild-type stationary cells but was absentfrom WE15 cells (Table 2). (ii) The nucleotide pattern of thepho constitutive strain WC4 phoU35 during exponentialgrowth was identical (except for S16) to that during station-ary growth (Fig. 4 and Table 2). However, when comparison

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208 RAO ET AL.

A

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FIG. 3. Two-dimensional TLC separation of E. coli W3110 cellular nucleotides. Guanidine hydrochloride (0.9 M) was used for the firstdimension, and ammonium sulfate solvent [43.5 g (NH4)2SO4, 0.4 g of (NH4)HSO4, and 4 g of disodium EDTA in 100 ml of distilled water]was used for the second dimension. A and C are photographic reproductions of the autoradiograms; B and D are tracings of theautoradiograms and give the identities of metabolites. A and B are autoradiograms from an exponentially growing (repressed) culture; C andD are from a stationary-phase (derepressed) culture.

was made between the nucleotide patterns of WC4 phoU35(either at the exponential or stationary phase of growth) withthat of stationary-phase wild-type cells, spots Si, S7, S10,and S14 were absent in WC4 phoU35 cells, but they showedthree additional spots S4, S12, and S16 (Table 2). The resultsobtained with another constitutive mutant, phoR (WC2phoR68), on the other hand, resembled those of the strainsW3110 wild type and WE15 phoA8 (results not shown); i.e.,the pattern was modified from the exponential to the station-ary phase of growth (Table 2), although alkaline phosphatasewas synthesized at all times (Table 1). This suggested thatthe nucleotide pool modifications were under the control ofgenes of the pho regulon. The results presented in Table 2point out that only two spots, S2 and S15, were present inthe stationary phase (P1 starvation) of all strains and presentas well in the exponential phase (P1 excess) in the WC4phoU35 constitutive mutant. Thus, these two nucleotides

may be the positive cofactors required for the synthesis ofalkaline phosphatase. On the other hand, two spots, S3 andS8, are present only in the exponential phase of growth butabsent in stationary phase cultures and in all growth condi-tions of phoU35 constitutive mutant, suggesting a negativeeffect on alkaline phosphatase synthesis.

Effect of isolated nucleotides on the in vitro synthesis ofalkaline phosphatase. If the nucleotides present in the pool ofphoU35 mutant when alkaline phosphatase was synthesizedin excess phosphate were those necessary to overcome Pirepression, it should be possible to test them in vitro. Thus,we used wild-type K10 cells grown in rich medium with ahigh concentration of Pi and repressed for alkaline phospha-tase. These cells were permeabilized. Experiments of incor-poration of [3H]UTP and the requirement for GTP in thesynthesis of f-galactosidase proved that the permeabilizedcells were indeed permeable to nucleotides (Table 3).

B

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GYP2

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E. COLI NUCLEOTIDE POOL AND ALKALINE PHOSPHATASE

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FIG. 4. Two-dimensional TLC separation of E. coli (phoU35) cellular nucleotides. Solvents and legend as in Fig. 3; E and F are

autoradiograms and identities of metabolites from exponentially growing cells, and G and H are from stationary-phase cells.

The permeabilized cells were added to an in vitro mixwhich also contained a high level of Pi (17). 32P-labelednucleotide spots were cut out from the TLC plates corre-

sponding to WC4 phoU35 (pho constitutive cells). Thepolyethyleneimine-cellulose was carefully scraped into cellwalls containing the permeabilized cells and the in vitro mixand incubated for 24 h, and alkaline phosphatase activitywas then measured as described in Materials and Methods.Activity of viable cells was 43.2 U per 4 x 108 cells. Of the30 spots tested (which included 10 unidentified nucleotides),only S2 and S15 consistently (alkaline phosphatase activitiesof 2.79 + 0.03 and 1.75 ± 0.07 U per 4 x 108 cells,respectively, in three repeat experiments) provoked thehydrolysis of p-nitrophenylphosphate above background.The background value (0.35 ± 0.01 U per 4 x 108 cells)corresponded to the alkaline phosphatase activity of thepermeabilized cells used in the in vitro mix (0.35 U). Noincreased hydrolysis was obtained by separate addition of 50

known nucleotide standards including pyrophosphate,ppGpp, pppGpp, XMP, and atnino imidazole carboxamideriboside 5'-monophosphate to the in vitro mix.

Preliminary characterization of the nucleotides of interest inalkaline phosphatase synthesis. Formic acid extracts contain-ing cellular nucleotides were treated with sodium periodate:all nucleotides, the coenzyme A derivatives, and S1 were

found to be resistant to oxidation, whereas the other nucle-otides, including ppGpp and pppGpp, were sensitive.For a further characterization, two of the isolated spots

(S3 and S8) present in pho repressed wild type and S2,observed in pho derepressed cells, were incubated withsnake venom phosphodiesterase, and the enzyme-treatedsamples were chromatogrammed individually on TLCplates. The locations of the products of enzyme digestion as

seen by autoradiography were marked on a composite

diagram (Fig. 5). S8 resulted in two products, both of whichmigrated close to S2. S2 was not modified by phosphodies-

F

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210 RAO ET AL.

TABLE 2. Pattern of some specific 32P-labeled nucleotide spots of E. coli cell extracts resolved on TLC autoradiogramsa

Presence of spots:Strain Phase

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Sll S12 S13 S14 S15 S16

W3110 wild type Exponentialb + - + + + + + + + + + + + - - -Stationaryc + + - - + + + - + + + - + + +

WE15AphoA8 Exponential + - + + + + + + + + + + + - -

Stationary + + - + + + + - + + + + + - -

WC4 phoU35 Exponential - + - + + + - - + - + + + - + +Stationary - + - + + + - - + - + + + - +

WC2 phoR68 Exponential + - + + + + + + + + + + + -

Stationary + + - + + + + - + + + + + - +

a The guanidine HCI-ammonium sulfate solvent system was used for TLC separation of nucleotides extracted form 32P-labeled cells.b Exponential cells growing in excess Pi (Fig. 1).c Stationary cells stopped growing due to Pi limitation (12).

terase. Spot S3, on the other hand, produced two nucleo-tides; one resolved close to S8, and the other resolved in theregiop between ATP and GTP.

DISCUSSIONThe comparative analysis of the pool of nucleotides be-

tween the wild type and various mutants of the regulatorygenes of the pho regulon revealed three main features. (i) Atleast two nucleotides (S3 and S8) present during the phoregulon-repressed condition in the exponential phase of thewild type were absent in stationary phase during Pi starva-tion. These modifications, however, were independent fromthe synthesis of alkaline phosphatase, since a AphoA mutantbehaved like the wild type. It was not solely dependent uponthe level of Pi since a constitutive mutant (phoU35) exhibitsthe same pattern of nucleotides during exponential andstationary phase of growth. (ii) It appears that the nucleotidepattern depends on the regulatory genes of the pho regulon.(iii) The pattern of the phoR68 alkaline phosphatase-constitutive mutant strain was similar to that of wild type.This is as expected on the basis of our working hypothesis(Fig. 1). The genes of the pho regulon are coinduced by theproduct of phoB gene, the expression of which is in turnregulated by the bifunctional product of phoR. Hypotheti-cally, the phoR product has a negative function when it bindswith a cofactor Y, synthesized under the control ofphoU inthe presence of Pi. IfY is not present (as in phoU mutants or

TABLE 3. In vitro synthesis by permeabilized cells of E. coliK10

[3HIUTPb GaatsdePrepna incorporation" 3Gaatsde(cpm) activityc

Viable cells 361 11.2IVM, IPTG 7,672.0

Permeabilized cells (no GTP) 361Permeabilized cells with GTP 2,700IVM (no GTP) 10.0IVM, IPTG 506.0a IVM, In vitro mix added. IPTG, Isopropyl-3-D-thiogalactopyranoside (1

mM) added as an inducer of ,3-galactosidase."Total [3H]UTP incorporated (in 120 min) in the presence of the complete

in vitro mix or without GTP (2).c ,-Galactosidase activity as micromoles of o-nitrophenol produced per

minute per 4 x 108 permeabilized cells.

in Pi starvation), the phoR product cannot represF phoB, andthe regulon is induced by PhoR-X, an alternative form ofphoR product (as in phoR69) or by the product ofphoM (asin phoR68) (4, 11, 15; Torriani and Ludke, in press).The specific nucleotide effectors of the pho regulon were

classified (Table 2) as positive (with the patterns of wild-typestationary phase) or negative (with the patterns of wild-typeexponential phase). The two nucleotides S2 and S15, whentested in vitro for their effect in bypassing the Pi repressionof phQA in wild type K10-permeabilized rqpressed cells,showed a small (2.44 ± 0.03 and 1.4 ± 0.07 units, respec-

FIG. 5. Composite diagram of two-dimensional TLC location of32Pi-labeled nucleotide isolates and their product(s) of hydrolysiswith snake venom phosphodiesterase (see Materials and Methods).The solvent systems were a mixture of 0.75 M Tris, 0.45 M HCI, and0.5 M LiCl for the frist dimension and ammopium sulfate solvent (asin Fig. 3) for the second dimension. Open spots represent theuntreated nucleotides, and lined spots represent the products aftertreating with phosphodiesterase. Symbols: 0, S2 and its product; A,S3 and its products; a, S8 and its products.

A G I c U

Mono -

Di -

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S3

- b 2nd

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E. COLI NUCLEOTIDE POOL AND ALKALINE PHOSPHATASE

tively) but reproducible effect on the synthesis of alkalinephosphatase.The negative factors (S3 and S8) could not be tested on

alkaline phosphatase synthesis, since the in vitro systemgenerates excess free Pi, which keeps phoA repressed. Atotal in vitro reconstruction tnay eventually give an answer.

It was interesting to find that the phoR68 mutant behavedlike the wild type as predicted by the position ofphoR in thescheme (Fig. 1) past the X=Y reaction.The activity of alkaline phosphatase observed in the

presence of specific nucleotides in vitro was small, but wascomparatively higher (7- to 10-fold) than the one reported byWilkins as escape synthesis in vivo by the mutants of thepyrimidine pathway (16). In Wilkins' experiments (which wehave repeated with identical results), the escape synthesiswas 1% or less than the expected activity by wild-typederepressed cells. The efficiency previously observed for invitro experiments in the Zubay system (8) was also about 1to 2% of the expected in vivo synthesis. (The units ofalkaline phosphatase in reference 8 are nanomoles [notmicromoles as printed] of p-nitrophenol produced perminute.) In the present experitnents the in vitro synthesis isabout 7 to 10%, comparatively 10 times higher than the oneobserved in Pi excape synthesis both in vivo (16) and in vitro(8).The identification of the spots is preliminary and incom-

plete, but we can conclude that they are all nucleotides andexclude some obvious hypotheses. For instance, spot S2,which was not digested by phosphodiesterase, may be PP1(3), but its position in the chromatogram was different fromthat of PP, (detected as 32pp, generated from 32P ATP byphosphodiesterase digestion). S2 responds to the pattern of apositive factor and has a positive effect on alkaline phospha-tase synthesis, whereas PP, was present in all conditions andhad no effect on alkaline phosphatase synthesis when addedin vitro. Although the chemical analysis is insufficient todetermine the nature of the negative factors S3 and S8, thephosphodiesterase hydrolysis suggests that these are proba-bly complex, highly phosphorylated nucleotides.

ACKNOWLEDGMENTSWe thank John Chen and Jane Ko for their technical assistance.E.W., John Chen, and Jane Ko were supported by the Under-

graduate Research Opportunity program funds of MassachusettsInstitute of Technology. This work was supported by Public HealthService Grant GM24009 from the National Institutes of Health.

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