Proc. Nati. Acad. Sci. USAVol. 85, pp. 5492-5496, August 1988Biochemistry

Crosstalk between bacterial chemotaxis signal transductionproteins and regulators of transcription of the Ntr regulon:Evidence that nitrogen assimilation and chemotaxis arecontrolled by a common phosphotransfer mechanism

(protein kinase/transcriptional activation/glutamine synthetase)

ALEXANDER J. NINFA*, ELIZABETH GOTTLIN NINFA*, ANDREI N. LUPAS*, ANN STOCK*,BORIS MAGASANIKt, AND JEFF STOCK*t*Department of Molecular Biology, Princeton University, Princeton, NJ 08540; and tDepartment of Biology, Massachusetts Institute of Technology,Cambridge, MA 02149

Contributed by Boris Magasanik, April 13, 1988

ABSTRACT We demonstrate by using purified bacterialcomponents that the protein kinases that regulate chemotaxisand transcription of nitrogen-regulated genes, CheA and NRII,respectively, have cross-specificities: CheA can phosphorylatethe Ntr transcription factor NRI and thereby activate tran-scription from the nitrogen-regulated ginA promoter, and NRIIcan phosphorylate CheY. In addition, we find that a highintracellular concentration of a highly active mutant form ofNRII can suppress the smooth-swimming phenotype of a cheAmutant. These results argue strongly that sensory transductionin the Ntr and Che systems involves a common protein phos-photransfer mechanism.

Bacteria respond to changes in the availability of nutrientssuch as nitrogen, phosphate, and oxygen; changes in mediumosmolarity; and gradients of chemotactic stimuli by means ofa family of homologous signal transduction systems (1-4).These signal transduction systems each contain two inter-acting proteins with conserved domains, a modulator proteinthat processes sensory information and an effector proteinthat is activated by the modulator to produce an appropriateadaptive response. The modulators all contain a homologousC-terminal domain of -200 residues, and the effectors allshare a homologous N-terminal domain of z130 residues.N-terminal portions of the modulators and C-terminal por-tions of the effectors have apparently diverged to provide theappropriate responses to different environmental stimuli.With the exception of the chemotaxis system, all of therelated effectors are transcriptional activators.

In two systems, the modulator and effector proteins havebeen purified and their mechanism of interaction has beenestablished. Enteric bacteria regulate the expression ofnitrogen-regulated (Ntr) genes by responding to changingratios of 2-ketoglutarate and glutamine (5). Information onthis ratio is communicated to the modulator protein, desig-nated NRII or NtrB, which controls the activity of theeffector, designated NRI or NtrC (6). It has been shown thatNRII is a protein kinase that catalyzes an ATP-dependentphosphorylation of NRI (7). In its phosphorylated form, NRIacts as a transcriptional activator at nitrogen-regulated pro-moters, such as that which precedes the glutamine synthetasegene, glnAp2. NRII kinase activity involves the formation ofa high-energy phosphorylated enzyme intermediate, phos-phoryl-NRII, with subsequent phosphotransfer to NRI (V.Weiss and B.M., unpublished data).

In the bacterial chemotaxis system the modulator proteinCheA is a protein kinase that acts to phosphorylate twoeffector proteins: CheY, which interacts with the flagellarmotor to control swimming behavior (8), and CheB, amethylesterase that controls receptor methylation and thussensitivity of the chemotactic sensory system (A.N.L. andJ.S., unpublished data). Just as in the nitrogen regulatorysystem, the chemotaxis phosphorylation reactions proceedvia a high-energy phosphokinase intermediate (8, 9).

Since the homologous regulators NRII and CheA bothapparently exert their effects by means of a mechanisminvolving protein phosphorylation, we examined the possi-bility that these proteins may function by a common mech-anism. In this report, we demonstrate that purified NRII andCheA can each catalyze the phosphorylation of the hetero-logous substrates CheY and NRI. Furthermore, we demon-strate that NRI phosphate formed by the action of CheA isable to activate transcription from the nitrogen-regulatedpromoter glnAp2 in vitro. We also demonstrate with intactcells that a high intracellular concentration of an activatedform of NRII can suppress the smooth-swimming phenotypeof a cheA mutant. Finally, we show that, as was observedpreviously for phosphoryl-CheA (8), the phosphorylatedgroup in the high-energy phosphokinase intermediate phos-phoryl-NR1j is apparently phosphohistidine. On the basis ofthese results, we propose that the homologies betweenconserved modulator and effector proteins reflect conservedkinase and phosphoacceptor functions.

MATERIALS AND METHODSMaterials and Radioisotopes. All buffers, salts, electropho-

resis reagents, and nucleotides were standard commerciallyobtained products of reagent or analytical grade and wereused without further purification. Radioisotopes were fromAmersham ([a-32P]UTP), and New England Nuclear/Du-Pont (Uy-32P]ATP). DE52 resin was from Whatman, enzymegrade ammonium sulfate was from Calbiochem, SephadexG-50 and the MONO-Q FPLC column were from Pharmacia,and the GF-200 FPLC column was from Sota (Crompond,NY).

Purified Proteins. Bovine serum albumin fraction V andovalbumin were from Sigma. Salmonella typhimurium CheAand CheY were purified as described (1, 3). Each of thesepurified proteins is at least 95% pure as estimated byinspection of Coomassie blue-stained NaDodSO4/polyacryl-amide protein gels. The preparations of Escherichia coliNRII, NRI, 54, and core RNA polymerase obtained- previ-

*To whom reprint requests should be addressed.

5492

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Proc. Natl. Acad. Sci. USA 85 (1988) 5493

ously (refs. 10 and 11; A.J.N., E. Brodsky, and B.M.,unpublished data) were used. Each of these proteins with theexception of core RNA polymerase is also >90% pure, asestimated from Coomassie blue-stained gels. The core RNApolymerase preparation has been shown to contain no NRI1activity (A.J.N. and B.M., unpublished data). Phosphoryl-CheA and phosphoryl-NRI, were prepared from the purifiedCheA and NRII by autophosphorylation in the presence of[y-32P]ATP, followed by chromatography on a 25-ml Seph-adex G-50 column in 0.1 M sodium phosphate (pH 7.0) toremove free nucleotides.

Transcription Assay. The transcription buffer was 50 mMTris HCl, pH 7.5/50 mM NaCl/10 mM MgCl2/0.1 mMEDTA/1 mM dithiothreitol. Details of the assay are asdescribed (11) except that the [a-32P]UTP was at twice thespecific activity used in previous experiments. The transcrip-tion template was supercoiled pTH8 (12), a derivative ofpTE103 (13) in which the glnAp2 promoter is positioned =300base pairs upstream from a strong rho-independent termina-tor from bacteriophage T7. The assay measures the formationof heparin-resistant transcription complexes formed in theabsence of added UTP, the first nucleotide in the glnAP2transcript. Template, proteins, buffer, and the nucleotidesATP, CTP, and GTP were incubated at 37°C for 30 min,during which time transcription complexes were formed.Heparin and labeled UTP were then added and the sampleswere incubated an additional 10 min to allow the productionof full-length transcripts from transcription complexes; thereactions were then terminated by the addition ofEDTA andthe radioactive transcripts were recovered by ethanol pre-cipitation, subjected to electrophoresis in denaturingurea/polyacrylamide gels, and detected by autoradiography.

Determination of the Chemical Stability of the Phosphoryl-ated Group in Phosphoryl-NRII. This assay was performed asdescribed for phosphoryl-CheA (8). Aliquots of phosphoryl-NRII (4 ,u, 1 pmol) were applied to duplicate 1-cm squares ofImmobilon polyvinylidene difluoride membrane (Millipore),which were then incubated under the following conditions: (i)0.2 M sodium citrate, pH 2.4, 45°C; (ii) 50 mM potassiumphosphate, pH 7.0; (iii) 2 M sodium hydroxide, pH 13.5,45°C; (iv) 0.4 M hydroxylamine hydrochloride, pH 7.6, 25°C;(v) 0.1 M pyridine, 25°C. Membrane squares were removedat 15, 30, 60, 90, and 120 min, rinsed in water, dried, andcounted in Liquiscint (National Diagnostics) fluor in a Beck-man LS-230 liquid scintillation counter. First-order rateconstants were estimated from linear regression analysis ofthe raw data.

Characterization of Swimming Behavior. Strains were sub-cultured in L broth medium (14) and grown to midlogarithmicphase at 37°C. Small aliquots were then diluted 1:10 intomotility buffer (50 mM KCl/10 mM KH2PO4, pH 7/0.1 mMEDTA/0.5 uM L-methionine) and incubated for at least 15min at room temperature, after which swimming behaviorwas recorded at x 400 magnification with a Zeiss phase-contrast microscope, Ikegami ITC-510 video camera, and aPanasonic NV8950 video recorder. The video recordingswere analyzed as described (15).

RESULTSCheA Catalyzes the ATP-Dependent Phosphorylation of

NRI. We examined the ability of NR11 and CheA to catalyzethe phosphorylation of NRI (Fig. 1). Both CheA and NRIIwere phosphorylated in the presence of ATP, and no labelwas incorporated into NRI in the absence of other proteins.In Fig. 1, the intensity of the phosphorylated CheA band ismuch greater than that of the autophosphorylated NR1I bandbecause more CheA protein was used. NRI was phosphoryl-ated in reaction mixtures that contained ATP and either NRIIor CheA. Much more NRI-phosphate was produced when

ChANR1NRII

ChaA-NRI -

NRI-

+ 1 + -+ + + +

+ + + +

El

FIG. 1. Phosphorylation ofNRI by CheA and NRII. Proteins wereincubated in transcription buffer (20 ,u) for 3 min at 37°C, 5 ul of[y32P]ATP (final concentration, 0.4 mM; 2 Ci/mmol) was added, andthe incubation was continued for 5 min, after which time 8.3 ,ul of gelsample buffer [124 mM Tris-HCI, pH 6.8/4% NaDodSO4/8%(vol/vol) 2-mercaptoethanol/20% (vol/vol) glycerol] was added toeach reaction mixture. Samples were heated to 60°C for 1 min andapplied directly to a 1o Laemmli-type protein gel (16). Theautoradiograph of the protein gel is shown. Protein concentrations:(where indicated) NRI, 2.7 ,uM; NRII, 80 nM; CheA, 9.3 ,uM.

NRII was present than when CheA was present. Thesefindings suggest that CheA can catalyze the phosphorylationof NR, by ATP, but not as effectively as NRII.

Activation of Transcription from the Nitrogen-RegulatedPromoter glnAp2 by CheA-Generated NR,-Phosphate. Is theNRI-phosphate formed by CheA able to activate transcrip-tion from the nitrogen-regulated promoter gInAp2? Previousresults had indicated that transcription from glnAp2 requiresRNA polymerase containing a54 instead of the usual ou7O (12,17). This transcription is activated by NR,-phosphate but notby unphosphorylated NRI (7). It has also been shown that atlow concentrations of NRI, transcription from glnAp2 isgreatly facilitated by the presence on the template oftwo sitesto which NR, and NRI-phosphate bind (glnA enhancers),located about 100 and 130 base pairs upstream from the siteof transcript initiation (18). When supercoiled templatescontaining the enhancers are used in the transcription assay,very low concentrations of NRI (-1 nM) can readily bedetected (ref. 11; A.J.N. and B.M., unpublished data). Toincrease the sensitivity of the assay even further, we doubledthe specific activity of the labeled UTP used in previousexperiments. We used these most sensitive reaction condi-tions to examine whether CheA could substitute for NRI1 inactivating NRI and, by so doing, activate transcription fromglnAp2. In these reaction conditions a small amount of theglnAp2 transcript was produced by the or" RNA polymerasein the absence of added factors, and the addition of NRI andNRII to the reaction mixture resulted in a huge increase in theamount of glnAp2 transcript produced (Fig. 2), as had beennoted (10, 12). The combination ofCheA and NRI was clearlyable to stimulate transcription from glnAp2 by or54 RNApolymerase, while neither CheA nor NRI alone stimulatedtranscription. This result shows that the small amount ofNRIphosphate formed by CheA and ATP is functionally equiv-alent to that formed by NRII and ATP.

Transfer of Phosphate from Phosphoryl-CheA to NRI. Wenext tested the ability of purified phosphoryl-CheA to trans-fer its phosphate to NRI in the absence of ATP. We preparedphosphoryl-CheA by gel filtration chromatography afterallowing the phosphorylation reaction to occur in the pres-ence of [y-32P]ATP and measured the time course of transferof labeled phosphate from phosphoryl-CheA preparation totransfer phosphate to the natural substrate, CheY, and toovalbumin. We also tested whether NRI, could serve as asubstrate for phosphotransfer. NRI catalyzed the dephos-phorylation of phosphoryl-CheA via a phosphoryl-NRI in-termediate (Fig. 3A). After 1 hr of incubation in the presence

Biochemistry: Ninfa et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

Eu54CheANRINR11

+ + + + + + + +

+ + + + ++ +_ - - __.i.t_ ......._. .. w.; i.

d*., .gd,....- ,.,ig.

_: .; l,_ .: g

,1 2 3

+ + + +

4 5 6 7 8

FIG. 2. Activation of transcription from glnAp2 by CheA-generated NR, phosphate. The autoradiograph of a transcription gelis shown. All reaction mixtures contained template at 5 nM, o54 at 400nM, and core RNA polymerase at 100 nM. Other protein concen-trations are as follows: lane 1, NRI at 185 nM and NR1I at 20 nM; lane2, NRI at 370 nM; lane 3, CheA at 4.6 ,uM; lane 4, NRI at 185 nM andCheA at 4.6 ,uM; lane 5, NRI at 185 nM and CheA at 9.3 ,uM; lane6, NR, at 370 nM and CheA at 9.3 uM; lane 7, NRI at 185 nM, NRIIat 20 nM, and CheA at 4.6 ,4M; lane 8, no NRI, NRII, or CheApresent.

of NR,, most of the phosphate from phosphoryl-CheA hadbeen released in a low molecular weight form that runs at thedye front of the acrylamide gel, but in the absence ofNRI thephosphoryl-CheA was almost entirely stable for this period oftime. The transfer of phosphate from phosphoryl-CheA toCheY was very rapid; in that case dephosphorylation of

AChM-P + NRI ChA-P

min at 370C 0 7.5 15 30 60 15 30 60

ChA -

NR-

phosphoryl-CheA was nearly complete after 30 sec. We didnot detect any transfer of phosphate from phosphoryl-CheAto NR,, (Fig. 3B) or to ovalbumin.

Transfer of Phosphate from Phosphoryl-NRI, to CheY.Phosphoryl-NR,,, prepared by gel filtration after phospho-rylation by labeled ATP, was dephosphorylated by CheYwith the formation of a phosphoryl-CheY intermediate (Fig.4A). Maximal labeling of CheY was observed within 30 secafter the addition of CheY to a reaction mixture containingphosphoryl-NR,,, and phosphoryl-NRI was almost com-pletely dephosphorylated after 4.5 min of incubation in thisreaction mixture. In the absence of CheY, phosphoryl-NRI,was almost entirely stable for this period of time. Transfer ofphosphate from phosphoryl-NRI, to NRI was more efficient;in this case, dephosphorylation of phosphoryl-NR11 wasessentially complete within 30 sec (Fig. 4B). In controlexperiments, we did not detect any transfer of phosphatefrom phosphoryl-NR1, to bovine serum albumin or to CheA.

Effect of Overproducing NRII on the Swimming Behavior ofa cheA Mutant. It has been proposed that phosphoryl-CheYinteracts with the flagellar motor to cause tumbly behavior(8). Mutants in cheA are unable to tumble, presumablybecause they are deficient in phosphoryl-CheY. We exam-ined whether or not an increased intracellular concentrationof NRII could suppress the smooth-swimming phenotype ofa cheA mutant. For these experiments, we used a plasmid,pTH814, that causes the overproduction (to -1% of cellprotein) of a mutant form of NR,,, NR112302 (10). Previousresults had indicated that in intact cells NR112302 causes theactivation of glnA transcription in the presence of ammonia(19); analysis of the activity of purified NR112302 had indi-cated that this protein, unlike wild-type NRII, will catalyzethe phosphorylation of NR, in the presence of the Ntr signaltransduction protein that acts as the intracellular signal ofnitrogen excess (7). We introduced pTH814 and the parentvector pBR322 into S. typhimurium strains containing cheA

ANRII-P + CheY NRII-P

min at 300C 0 0.5 1.5 4.5 13.5 0.5 1.5 4.5 13.5

NRI-

CheY0 ChA-P +

CheY NRI NRIImin at 370C 0 0.5 0.5 7.5 0.5 7.5

CheA -

NRINRii -

CheY

..........iB

min at 300CCh.A -

NRI -NRII -

NRII-P +

NRI CheY Ch.A0 0.5 0.5 5 0.5 5

CheY

FIG. 3. Transfer of phosphate from phosphoryl-CheA to NRI andCheY. (A) Time course of phosphotransfer to NRI. Purified phos-phoryl-CheA (final concentration, 85 nM) was incubated in a buffercontaining 82 mM Tris-HCI, 82 mM NaCl, 12 mM potassiumphosphate, 10mM MgCl2, 1.6mM dithiothreitol, and 0.16mM EDTA(pH.7.5) at 37°C with either NRI (final concentration, 12 ,M) orbuffer in a final vol of 105 ,ul. At the indicated times, 25-Mul sampleswere removed, added to 8.3 ul of sample buffer, and subjected toelectrophoresis and autoradiography (see legend to Fig. 1). (B)Comparison of phosphotransfer to CheY, NRII, and NRI. Theexperiment is similar to that shown in A except that the phosphoac-cepting species was varied as indicated. Protein concentrations wereas follows: CheY, 85 nM; NRI, 12 MM; NRII, 2.6 ,uM.

FIG. 4. Transfer of phosphate from phosphoryl-NR,, to CheYand NR,. (A) Time course of phosphotransfer to CheY. Purifiedphosphoryl-NR,, (final concentration, 300 nM) was incubated in 0.1M potassium phosphate buffer, pH 7.0/5 mM MgCl2 at 30°C for 30sec, after which either CheY (final concentration, 71 MM) or bufferwas added to a final vol of50 Ml. At the indicated times, 10-MLI sampleswere removed, added to sample buffer, and subjected to electro-phoresis and autoradiography (see legend to Fig. 1). (B) Comparisonof phosphotransfer to NR,, CheY, and CheA. The experiment issimilar to that shown in A except that phospho-NR,, was present at168 nM and the phosphoaccepting species were varied as indicated.Protein concentrations were as follows: NRI, 1.5 MM; CheY, 34,uM;CheA, 17 MM.

V:

5494 Biochemistry: Ninfa et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 5495

and che Y mutations, and we determined the swimmingbehavior of these strains and their parents as well as of thewild-type strain. We found that pTH814, but not pBR322,suppressed the smooth-swimming phenotype of the cheAmutant (Table 1). The cheA mutant containing pTH814tumbled even more than wild type; swimming behavior wasuncoordinated, with many extended runs and tumbles lastingup to 10 sec. The effect ofpTH814 was entirely dependent onthe presence of CheY. These findings suggest that theCheY-phosphate formed from phosphoryl-NRI, is function-ally equivalent to that formed from phosphoryl-CheA.The Phosphorylated Group in Phosphoryl-CheA and Phos-

phoryl-NRI, Have Similar Chemical Stability and Are Proba-bly Phosphohistidine. We examined the stability of the phos-phorylated group in phosphoryl-NRI, in the presence ofhydroxlyamine and pyridine, and at pH 2.4 (citrate buffer),pH 7.0 (phosphate buffer), and pH 13.5 (sodium hydroxide).We found that the phosphorylated group was stable at neutralor alkaline pH but was relatively unstable in acid (Table 2).Pyridine and hydroxylamine both catalyzed the dephos-phorylation reaction. These results are very similar to thoseobtained previously with phosphoryl-CheA and phosphoryl-enzyme I of the phosphotransferase system (8, 20). Thephosphoryl group in phosphoryl-enzyme I has been shown tobe a 3-phosphohistidine (20).

DISCUSSIONThe results presented in this report indicate that the homol-ogous modulator proteins NR,, and CheA utilize a commonphosphotransfer mechanism to regulate the activity of theircorresponding effectors, NR, and CheY. This conclusion isbased on our ability to observe crosstalk between heterolo-gous modulator/effector pairs with purified bacterial com-ponents and on the effect that overproducing NRII has on theswimming behavior of a cheA mutant. The apparent chemicalidentity of the phosphate group in the high-energy phospho-kinase intermediates tends to confirm this conclusion.

In light of our findings, it seems likely that the homologiesbetween modulator proteins reflect conserved protein kinasefunction and that the homologies between effector proteinsreflect conserved phosphoacceptor activities. Thus, for in-stance, in phosphate regulation (21), PhoR is probably aphosphate-regulated kinase and PhoB is a phosphorylatedtranscription factor; in osmoregulation of porin expression(22), EnvZ is probably a kinase that phosphorylates OmpR;and in regulation of the Dct regulon (23), DctB probablyphosphorylates DctD. Moreover, we can now predict thatone or more kinases functions to phosphorylate SpoOA andSpoOF to control sporulation of Bacillus subtilis (24); simi-larly, the Arc repressor that controls the expression oftricarboxylic acid cycle enzymes in E. coli (25) is probablyregulated by a kinase that processes information concerningthe availability of environmental oxygen.

Table 1. A high intracellular concentration of NR112302 sup-presses the smooth-swimming phenotype of a cheA mutant

Average

No. % smooth duration, secStrain Plasmid examined swimming Run Tumble

PSi (wt) 20 89 2.1 0.26PS34 (cheA) 20 >99 >10.0 NDPS34 pBR322 20 >99 >10.0 NDPS34 pTH814 105 69 2.3 1.10PS257 (cheY) 20 >99 >10.0 NDPS257 pBR322 20 >99 >10.0 NDPS257 pTH814 20 >99 >10.0 ND

ND, no detectable tumbly behavior.

Table 2. Chemical stability of phosphorylated group in phos-phoryl-NRII, phosphoryl-CheA, and phosphoryl-enzyme I

Rate of hydrolysis, kl min-1Condition NRII* CheAt Enzyme ItpH 2.4 0.017 0.021 0.025pH 7.0§ 0.001 0.000 0.008pH 13.5 0.003 0.000 0.008NH20H 0.022 0.014 0.041Pyridine 0.020 0.009 0.031

*This study.tData from ref. 8.tData from ref. 20.§Enzyme I was examined at pH 6.5 (20);examined at pH 7.0 (ref. 8; this study).

CheA and NR,, were

Crosstalk between heterologous modulator/effector pairsprovides an explanation for the complex phenotypes oftenassociated with modulator mutations. These include phoM-dependent expression ofphoA in phoR mutants (26), residualregulation of gInA in mutants lacking NRII (27), and thetumbly swimming behavior of cheA mutants in which CheYis overproduced from a multicopy plasmid with a strongpromoter (28). Whether or not crosstalk between heterolo-gous modulator/effector pairs actually occurs in wild-typecells under normal physiological conditions is at this time notknown. Our results raise the possibility that the family ofrelated modulator/effector pairs may constitute a network ofsensory transducers that process information to coordinatecellular responses to environmental stimuli.

We thank Austin Newton for his advice and support, and DavidWylie and Thomas Chen for their technical assistance. This workwas supported by grants from the Public Health Service (AI-20980)and American Cancer Society (NP-515). A.S. was supported by agrant from the Damon Runyon-Walter Winchell Cancer ResearchFund (DRG-933).

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