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Proteins and Regulators of Transcription of the Ntr Regulon

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Proteins and Regulators of Transcription of the Ntr Regulon Proc. Nati. Acad. Sci. USA Vol. 85, pp. 5492-5496, August 1988 Biochemistry Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: Evidence that nitrogen assimilation and chemotaxis are controlled 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 bacterial In the bacterial chemotaxis system the modulator protein components that the protein kinases that regulate chemotaxis CheA is a protein kinase that acts to phosphorylate two and transcription of nitrogen-regulated genes, CheA and NRII, effector proteins: CheY, which interacts with the flagellar respectively, have cross-specificities: CheA can phosphorylate motor to control swimming behavior (8), and CheB, a the Ntr transcription factor NRI and thereby activate tran- methylesterase that controls receptor methylation and thus scription from the nitrogen-regulated ginA promoter, and NRII sensitivity of the chemotactic sensory system (A.N.L. and can phosphorylate CheY. In addition, we find that a high J.S., unpublished data). Just as in the nitrogen regulatory intracellular concentration of a highly active mutant form of system, the chemotaxis phosphorylation reactions proceed NRII can suppress the smooth-swimming phenotype of a cheA via a high-energy phosphokinase intermediate (8, 9). mutant. These results argue strongly that sensory transduction Since the homologous regulators NRII and CheA both in the Ntr and Che systems involves a common protein phos- apparently exert their effects by means of a mechanism photransfer mechanism. involving 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 and Bacteria respond to changes in the availability of nutrients CheA can each catalyze the phosphorylation of the hetero- such as nitrogen, phosphate, and oxygen; changes in medium logous substrates CheY and NRI. Furthermore, we demon- osmolarity; and gradients ofchemotactic stimuli by means of strate that NRI phosphate formed by the action of CheA is a family of homologous signal transduction systems (1-4). able to activate transcription from the nitrogen-regulated These signal transduction systems each contain two inter- promoter glnAp2 in vitro. We also demonstrate with intact acting proteins with conserved domains, a modulator protein cells that a high intracellular concentration of an activated that processes sensory information and an effector protein form of NRII can suppress the smooth-swimming phenotype that is activated by the modulator to produce an appropriate of a cheA mutant. Finally, we show that, as was observed adaptive response. The modulators all contain a homologous previously for phosphoryl-CheA (8), the phosphorylated C-terminal domain of -200 residues, and the effectors all group in the high-energy phosphokinase intermediate phos- share a homologous N-terminal domain of z130 residues. phoryl-NR1j is apparently phosphohistidine. On the basis of N-terminal portions of the modulators and C-terminal por- these results, we propose that the homologies between tions ofthe effectors have apparently diverged to provide the conserved modulator and effector proteins reflect conserved appropriate responses to different environmental stimuli. kinase and phosphoacceptor functions. With the exception of the chemotaxis system, all of the related effectors are transcriptional activators. In two systems, the modulator and effector proteins have MATERIALS AND METHODS been purified and their mechanism of interaction has been Materials and Radioisotopes. All buffers, salts, electropho- established. Enteric bacteria regulate the expression of resis reagents, and nucleotides were standard commercially nitrogen-regulated (Ntr) genes by responding to changing obtained products of reagent or analytical grade and were ratios of 2-ketoglutarate and glutamine (5). Information on used without further purification. Radioisotopes were from this ratio is communicated to the modulator protein, desig- Amersham ([a-32P]UTP), and New England Nuclear/Du- nated NRII or NtrB, which controls the activity of the Pont (Uy-32P]ATP). DE52 resin was from Whatman, enzyme effector, designated NRI or NtrC (6). It has been shown that grade ammonium sulfate was from Calbiochem, Sephadex NRII is a protein kinase that catalyzes an ATP-dependent G-50 and the MONO-Q FPLC column were from Pharmacia, phosphorylation of NRI (7). In its phosphorylated form, NRI and the GF-200 FPLC column was from Sota (Crompond, acts as a transcriptional activator at nitrogen-regulated pro- NY). moters, such as that which precedes the glutamine synthetase Purified Proteins. Bovine serum albumin fraction V and gene, glnAp2. NRII kinase activity involves the formation of ovalbumin were from Sigma. Salmonella typhimurium CheA a high-energy phosphorylated enzyme intermediate, phos- and CheY were purified as described (1, 3). Each of these phoryl-NRII, with subsequent phosphotransfer to NRI (V. purified proteins is at least 95% pure as estimated by Weiss and B.M., unpublished data). inspection of Coomassie blue-stained NaDodSO4/polyacryl- amide protein gels. The preparations of Escherichia coli 54, and core RNA polymerase obtained- previ- The publication costs of this article were defrayed in part by page charge NRII, NRI, payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5492 Downloaded by guest on September 25, 2021 Biochemistry: Ninfa et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5493 ously (refs. 10 and 11; A.J.N., E. Brodsky, and B.M., ChA + 1 + - unpublished data) were used. Each of these proteins with the NR1 + + + + exception of core RNA polymerase is also >90% pure, as NRII + + + + estimated from Coomassie blue-stained gels. The core RNA polymerase preparation has been shown to contain no NRI1 El activity (A.J.N. and B.M., unpublished data). Phosphoryl- ChaA- CheA and phosphoryl-NRI, were prepared from the purified NRI - CheA and NRII by autophosphorylation in the presence of [y-32P]ATP, followed by chromatography on a 25-ml Seph- NRI- adex G-50 column in 0.1 M sodium phosphate (pH 7.0) to remove free nucleotides. Transcription Assay. The transcription buffer was 50 mM Tris HCl, pH 7.5/50 mM NaCl/10 mM MgCl2/0.1 mM EDTA/1 mM dithiothreitol. Details of the assay are as FIG. 1. Phosphorylation ofNRI by CheA and NRII. Proteins were incubated in transcription buffer (20 ,u) for 3 min at 37°C, 5 ul of described (11) except that the [a-32P]UTP was at twice the [y32P]ATP (final concentration, 0.4 mM; 2 Ci/mmol) was added, and specific activity used in previous experiments. The transcrip- the incubation was continued for 5 min, after which time 8.3 ,ul ofgel tion template was supercoiled pTH8 (12), a derivative of sample buffer [124 mM Tris-HCI, pH 6.8/4% NaDodSO4/8% pTE103 (13) in which the glnAp2 promoter is positioned =300 (vol/vol) 2-mercaptoethanol/20% (vol/vol) glycerol] was added to base pairs upstream from a strong rho-independent termina- each reaction mixture. Samples were heated to 60°C for 1 min and tor from bacteriophage T7. The assay measures the formation applied directly to a 1o Laemmli-type protein gel (16). The of heparin-resistant transcription complexes formed in the autoradiograph of the protein gel is shown. Protein concentrations: absence of added UTP, the first nucleotide in the glnAP2 (where indicated) NRI, 2.7 ,uM; NRII, 80 nM; CheA, 9.3 ,uM. transcript. Template, proteins, buffer, and the nucleotides ATP, CTP, and GTP were incubated at 37°C for 30 min, NRII was present than when CheA was present. These during which time transcription complexes were formed. findings suggest that CheA can catalyze the phosphorylation Heparin and labeled UTP were then added and the samples of NR, by ATP, but not as effectively as NRII. were incubated an additional 10 min to allow the production Activation of Transcription from the Nitrogen-Regulated of full-length transcripts from transcription complexes; the Promoter glnAp2 by CheA-Generated NR,-Phosphate. Is the reactions were then terminated by the addition of EDTA and NRI-phosphate formed by CheA able to activate transcrip- the radioactive transcripts were recovered by ethanol pre- tion from the nitrogen-regulated promoter gInAp2? Previous cipitation, subjected to electrophoresis in denaturing results had indicated that transcription from glnAp2 requires urea/polyacrylamide gels, and detected by autoradiography. RNA polymerase containing a54 instead of the usual ou7O (12, Determination of the Chemical Stability of the Phosphoryl- 17). This transcription is activated by NR,-phosphate but not ated Group in Phosphoryl-NRII. This assay was performed as by unphosphorylated NRI (7). It has also been shown that at described for phosphoryl-CheA (8). Aliquots of phosphoryl- low concentrations of NRI, transcription from glnAp2 is NRII (4 ,u, 1 pmol) were applied to duplicate 1-cm squares of greatly facilitated by the presence on the template oftwo sites Immobilon polyvinylidene difluoride membrane (Millipore), to which NR, and NRI-phosphate bind (glnA enhancers), which were then incubated under the following conditions: (i) located about 100 and 130 base pairs upstream from the site 0.2 M sodium citrate, pH 2.4, 45°C; (ii) 50 mM potassium of transcript initiation (18). When supercoiled templates phosphate, pH 7.0; (iii) 2 M sodium hydroxide, pH 13.5, containing the enhancers are used in the transcription assay, 45°C; (iv) 0.4 M hydroxylamine hydrochloride, pH 7.6, 25°C; very low concentrations of NRI (-1 nM) can readily be (v) 0.1 M pyridine, 25°C. Membrane squares were removed detected (ref.
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