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 In addition, we find that a high J.S., unpublished data).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. 11; A.J.N. and B.M., unpublished data). To at 15, 30, 60, 90, and 120 min, rinsed in water, dried, and increase the sensitivity of the assay even further, we doubled counted in Liquiscint (National Diagnostics) fluor in a Beck- the specific activity of the labeled UTP used in previous man LS-230 liquid scintillation counter. First-order rate experiments. We used these most sensitive reaction condi- constants were estimated from linear regression analysis of tions to examine whether CheA could substitute for NRI1 in the raw data. activating NRI and, by so doing, activate transcription from Characterization of Swimming Behavior. Strains were sub- glnAp2. In these reaction conditions a small amount of the cultured in L broth medium (14) and grown to midlogarithmic glnAp2 transcript was produced by the or" RNA polymerase phase at 37°C. Small aliquots were then diluted 1:10 into in the absence of added factors, and the addition of NRI and motility buffer (50 mM KCl/10 mM KH2PO4, pH 7/0.1 mM NRII to the reaction mixture resulted in a huge increase in the EDTA/0.5 uM L-methionine) and incubated for at least 15 amount of glnAp2 transcript produced (Fig. 2), as had been min at room temperature, after which swimming behavior noted (10, 12). The combination ofCheA and NRI was clearly was recorded at x 400 magnification with a Zeiss phase- able to stimulate transcription from glnAp2 by or54 RNA contrast microscope, Ikegami ITC-510 video camera, and a polymerase, while neither CheA nor NRI alone stimulated Panasonic NV8950 video recorder. The video recordings transcription. This result shows that the small amount ofNRI were analyzed as described (15). phosphate formed by CheA and ATP is functionally equiv- alent to that formed by NRII and ATP. Transfer of Phosphate from Phosphoryl-CheA to NRI. We RESULTS next tested the ability of purified phosphoryl-CheA to trans- CheA Catalyzes the ATP-Dependent Phosphorylation of fer its phosphate to NRI in the absence of ATP. We prepared NRI. We examined the ability of NR11 and CheA to catalyze phosphoryl-CheA by gel filtration chromatography after the phosphorylation of NRI (Fig. 1). Both CheA and NRII allowing the phosphorylation reaction to occur in the pres- were phosphorylated in the presence of ATP, and no label ence of [y-32P]ATP and measured the time course of transfer was incorporated into NRI in the absence of other proteins. of labeled phosphate from phosphoryl-CheA preparation to In Fig. 1, the intensity of the phosphorylated CheA band is transfer phosphate to the natural substrate, CheY, and to much greater than that of the autophosphorylated NR1I band ovalbumin. We also tested whether NRI, could serve as a because more CheA protein was used. NRI was phosphoryl- substrate for phosphotransfer. NRI catalyzed the dephos- ated in reaction mixtures that contained ATP and either NRII phorylation of phosphoryl-CheA via a phosphoryl-NRI in- or CheA. Much more NRI-phosphate was produced when termediate (Fig. 3A). After 1 hr of incubation in the presence Downloaded by guest on September 25, 2021 5494 Biochemistry: Ninfa et al. Proc. Natl. Acad. Sci. USA 85 (1988)

Eu54 + + + + + + + + phosphoryl-CheA was nearly complete after 30 sec. We did CheA + + + + + not detect any transfer of phosphate from phosphoryl-CheA NRI + + + + + + to NR,, (Fig. 3B) or to ovalbumin. NR11 Transfer of Phosphate from Phosphoryl-NRI, to CheY. Phosphoryl-NR,,, prepared by gel filtration after phospho- _--__. rylation by labeled ATP, was dephosphorylated by CheY with the formation of a phosphoryl-CheY intermediate (Fig. 4A). Maximal labeling of CheY was observed within 30 sec i.t after the addition of CheY to a reaction mixture containing _ ...... phosphoryl-NR,,, and phosphoryl-NRI was almost com- d*., .gd,.. - ,.,ig. pletely dephosphorylated after 4.5 min of incubation in this

_: .; l, reaction mixture. In the absence of CheY, phosphoryl-NRI, _._.. w.; i..: g was almost entirely stable for this period of time. Transfer of ,.. phosphate from phosphoryl-NRI, to NRI was more efficient; 1 2 3 4 5 6 7 8 in this case, dephosphorylation of phosphoryl-NR11 was essentially complete within 30 sec (Fig. 4B). In control FIG. 2. Activation of transcription from glnAp2 by CheA- experiments, we did not detect any transfer of phosphate generated NR, phosphate. The autoradiograph of a transcription gel is shown. All reaction mixtures contained template at 5 nM, o54 at 400 from phosphoryl-NR1, to bovine serum albumin or to CheA. nM, and core RNA polymerase at 100 nM. Other protein concen- Effect of Overproducing NRII on the Swimming Behavior of trations are as follows: lane 1, NRI at 185 nM and NR1I at 20 nM; lane a cheA Mutant. It has been proposed that phosphoryl-CheY 2, NRI at 370 nM; lane 3, CheA at 4.6 ,uM; lane 4, NRI at 185 nM and interacts with the flagellar motor to cause tumbly behavior CheA at 4.6 ,uM; lane 5, NRI at 185 nM and CheA at 9.3 ,uM; lane (8). Mutants in cheA are unable to tumble, presumably 6, NR, at 370 nM and CheA at 9.3 uM; lane 7, NRI at 185 nM, NRII because they are deficient in phosphoryl-CheY. We exam- at 20 nM, and CheA at 4.6 ,4M; lane 8, no NRI, NRII, or CheA ined whether or not an increased intracellular concentration present. of NRII could suppress the smooth-swimming phenotype of a cheA mutant. For these experiments, we used a plasmid, of NR,, most of the phosphate from phosphoryl-CheA had pTH814, that causes the overproduction (to -1% of cell been released in a low molecular weight form that runs at the protein) of a mutant form of NR,,, NR112302 (10). Previous dye front of the acrylamide gel, but in the absence ofNRI the results had indicated that in intact cells NR112302 causes the phosphoryl-CheA was almost entirely stable for this period of activation of glnA transcription in the presence of ammonia time. The transfer of phosphate from phosphoryl-CheA to (19); analysis of the activity of purified NR112302 had indi- CheY was very rapid; in that case dephosphorylation of cated that this protein, unlike wild-type NRII, will catalyze the phosphorylation of NR, in the presence of the Ntr signal A transduction protein that acts as the intracellular signal of ChM-P + NRI ChA-P nitrogen excess (7). We introduced pTH814 and the parent min at 370C 0 7.5 15 30 60 15 30 60 vector pBR322 into S. typhimurium strains containing cheA A NRII-P + CheY NRII-P ChA - min at 300C 0 0.5 1.5 4.5 13.5 0.5 1.5 4.5 13.5 NR- V: NRI-

CheY 0 ChA-P + CheY NRI NRII B min at 370C 0 0.5 0.5 7.5 0.5 7.5 NRII-P + ...... i NRI CheY Ch.A min at 300C 0 0.5 0.5 5 0.5 5 CheA - Ch.A - NRI NRI - NRii - NRII - CheY CheY FIG. 3. Transfer ofphosphate from phosphoryl-CheA to NRI and FIG. 4. Transfer of phosphate from phosphoryl-NR,, to CheY CheY. (A) Time course of phosphotransfer to NRI. Purified phos- and NR,. (A) Time course of phosphotransfer to CheY. Purified phoryl-CheA (final concentration, 85 nM) was incubated in a buffer phosphoryl-NR,, (final concentration, 300 nM) was incubated in 0.1 containing 82 mM Tris-HCI, 82 mM NaCl, 12 mM potassium M potassium phosphate buffer, pH 7.0/5 mM MgCl2 at 30°C for 30 phosphate, 10 mM MgCl2, 1.6mM dithiothreitol, and 0.16mM EDTA sec, after which either CheY (final concentration, 71 MM) or buffer (pH.7.5) at 37°C with either NRI (final concentration, 12 ,M) or was added to a final vol of50 Ml. At the indicated times, 10-MLI samples buffer in a final vol of 105 ,ul. At the indicated times, 25-Mul samples were removed, added to sample buffer, and subjected to electro- were removed, added to 8.3 ul of sample buffer, and subjected to phoresis and autoradiography (see legend to Fig. 1). (B) Comparison electrophoresis and autoradiography (see legend to Fig. 1). (B) of phosphotransfer to NR,, CheY, and CheA. The experiment is Comparison of phosphotransfer to CheY, NRII, and NRI. The similar to that shown in A except that phospho-NR,, was present at experiment is similar to that shown in A except that the phosphoac- 168 nM and the phosphoaccepting species were varied as indicated. cepting species was varied as indicated. Protein concentrations were Protein concentrations were as follows: NRI, 1.5 MM; CheY, 34,uM; as follows: CheY, 85 nM; NRI, 12 MM; NRII, 2.6 ,uM. CheA, 17 MM. Downloaded by guest on September 25, 2021 Biochemistry: Ninfa et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5495 and che Y mutations, and we determined the swimming Table 2. Chemical stability of phosphorylated group in phos- behavior of these strains and their parents as well as of the phoryl-NRII, phosphoryl-CheA, and phosphoryl-enzyme I wild-type strain. We found that pTH814, but not pBR322, Rate of hydrolysis, min-1 suppressed the smooth-swimming phenotype of the cheA kl mutant (Table 1). The cheA mutant containing pTH814 Condition NRII* CheAt Enzyme It tumbled even more than wild type; swimming behavior was pH 2.4 0.017 0.021 0.025 uncoordinated, with many extended runs and tumbles lasting pH 7.0§ 0.001 0.000 0.008 up to 10 sec. The effect ofpTH814 was entirely dependent on pH 13.5 0.003 0.000 0.008 the presence of CheY. These findings suggest that the NH20H 0.022 0.014 0.041 CheY-phosphate formed from phosphoryl-NRI, is function- Pyridine 0.020 0.009 0.031 ally equivalent to that formed from phosphoryl-CheA. *This study. The Phosphorylated Group in Phosphoryl-CheA and Phos- tData from ref. 8. phoryl-NRI, Have Similar Chemical Stability and Are Proba- tData from ref. 20. bly Phosphohistidine. We examined the stability of the phos- §Enzyme I was examined at pH 6.5 (20); CheA and NR,, were phorylated group in phosphoryl-NRI, in the presence of examined at pH 7.0 (ref. 8; this study). hydroxlyamine and pyridine, and at pH 2.4 (citrate buffer), pH 7.0 (phosphate buffer), and pH 13.5 (sodium hydroxide). Crosstalk between heterologous modulator/effector pairs We found that the phosphorylated group was stable at neutral provides an explanation for the complex phenotypes often or alkaline pH but was relatively unstable in acid (Table 2). associated with modulator mutations. These include phoM- Pyridine and hydroxylamine both catalyzed the dephos- dependent expression ofphoA in phoR mutants (26), residual phorylation reaction. These results are very similar to those regulation of gInA in mutants lacking NRII (27), and the obtained previously with phosphoryl-CheA and phosphoryl- tumbly swimming behavior of cheA mutants in which CheY enzyme I of the phosphotransferase system (8, 20). The is overproduced from a multicopy plasmid with a strong phosphoryl group in phosphoryl-enzyme I has been shown to promoter (28). Whether or not crosstalk between heterolo- be a 3-phosphohistidine (20). gous modulator/effector pairs actually occurs in wild-type cells under normal physiological conditions is at this time not known. Our results raise the possibility that the family of DISCUSSION related modulator/effector pairs may constitute a network of The results presented in this report indicate that the homol- sensory transducers that process information to coordinate ogous modulator proteins NR,, and CheA utilize a common cellular responses to environmental stimuli. phosphotransfer mechanism to regulate the activity of their We thank Austin Newton for his advice and support, and David corresponding effectors, NR, and CheY. This conclusion is Wylie and Thomas Chen for their technical assistance. This work based on our ability to observe crosstalk between heterolo- was supported by grants from the Public Health Service (AI-20980) gous modulator/effector pairs with purified bacterial com- and American Cancer Society (NP-515). A.S. was supported by a ponents and on the effect that overproducing NRII has on the grant from the Damon Runyon-Walter Winchell Cancer Research swimming behavior ofa cheA mutant. The apparent chemical Fund (DRG-933). identity of the phosphate group in the high-energy phospho- kinase intermediates tends to confirm this conclusion. 1. Stock, A., Koshland, D. E., Jr., & Stock, J. B. (1985) Proc. In light of our findings, it seems likely that the homologies Natl. Acad. Sci. USA 82, 7989-7993. 2. Ronson, C. W., Nixon, D. T. & Ausubel, F. M. (1987) Cell 49, between modulator proteins reflect conserved protein kinase 579-581. function and that the homologies between effector proteins 3. Stock, A., Chen, T., Welsh, D. & Stock, J. B. (1988) Proc. reflect conserved phosphoacceptor activities. Thus, for in- Natl. Acad. Sci. USA 85, 1403-1407. stance, in phosphate regulation (21), PhoR is probably a 4. Stock, J. B. (1987) BioEssays 6, 199-203. phosphate-regulated kinase and PhoB is a phosphorylated 5. Magasanik, B. (1982) Annu. Rev. Genet. 6, 135-168. transcription factor; in osmoregulation of porin expression 6. Bueno, R., Pahel, G. & Magasanik, B. (1985) J. Bacteriol. 164, (22), EnvZ is probably a kinase that phosphorylates OmpR; 816-822. and in regulation of the Dct regulon (23), DctB probably 7. Ninfa, A. J. & Magasanik, B. (1986) Proc. Natl. Acad. Sci. phosphorylates DctD. Moreover, we can now predict that USA 83, 5909-5913. one or more to and 8. Wylie, D., Stock, A. M., Wong, C.-Y. & Stock, J. B. (1988) kinases functions phosphorylate SpoOA Biochem. Biophys. Res. Commun. 151, 891-8%. SpoOF to control sporulation of Bacillus subtilis (24); simi- 9. Hess, J. F., Oosawa, K., Matsumura, P. & Simon, M. I. (1987) larly, the Arc repressor that controls the expression of Proc. Natl. Acad. Sci. USA 84, 7609-7613. tricarboxylic acid cycle enzymes in E. coli (25) is probably 10. Ninfa, A. J., Ueno-Nishio, S., Hunt, T. P., Robustell, B. & regulated by a kinase that processes information concerning Magasanik, B. (1986) J. Bacteriol. 168, 1002-1004. the availability of environmental oxygen. 11. Ninfa, A. J., Reitzer, L. J. & Magasanik, B. (1987) Cell 50, 1039-1046. Table 1. A high intracellular concentration of NR112302 sup- 12. Hunt, T. P. & Magasanik, B. (1985) Proc. Natl. Acad. Sci. presses the smooth-swimming phenotype of a cheA mutant USA 82, 8453-8457. 13. Elliot, T. & Geiduschek, E. P. (1984) Cell 36, 211-219. Average 14. Miller, J. H., ed. (1972) Experiments in Molecular Genetics No. % smooth duration, sec (Cold Spring Harbor Lab., Cold Spring Harbor, NY), p. 433. Strain Plasmid examined swimming Run Tumble 15. Stock, J., Borczuk, A., Chiou, F. & Burchenal, J. E. B. (1985) Proc. NatI. Acad. Sci. USA 82, 8364-8368. PSi (wt) 20 89 2.1 0.26 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. PS34 (cheA) 20 >99 >10.0 ND 17. Hirshman, J., Wong, P. K., Sei, K., Keener, J. & Kustu, S. PS34 pBR322 20 >99 >10.0 ND (1985) Proc. Natl. Acad. Sci. USA 82, 7525-7529. PS34 pTH814 105 69 2.3 1.10 18. Reitzer, L. J. & Magasanik, B. (1986) Cell 45, 785-792. PS257 (cheY) 20 >99 >10.0 ND 19. Chen, Y. M., Backman, K. & Magasanik, B. (1982) J. Bacte- PS257 pBR322 20 >99 >10.0 ND riol. 150, 214-229. 20. Weigel, N., Kukuruzinska, M. A., Nakazawa, A., Waygood, PS257 pTH814 20 >99 >10.0 ND E. B. & Roseman, S. (1982) J. Biol. Chem. 257, 14477-14491. ND, no detectable tumbly behavior. 21. Wanner, B. L. (1987) in Escherichia coli and Salmonella Downloaded by guest on September 25, 2021 5496 Biochemistry: Ninfa et al. Proc. Nadl. Acad. Sci. USA 85 (1988)

typhimurium, ed. Neidhardt, F. C. (Am. Soc. Microbiol., 25. luchi, S. & Lin, E. C. C. (1988) Proc. Nati. Acad. Sci. USA 85, Washington, DC), Vol. 2, pp. 1326-1333. 1888-1892. 22. Slauch, J. M., Garrett, S., Jackson, D. E. & Silhavy, T. J. 26. Wanner, B. L., Wilmes, M. R. & Young, D. C. (1988) J. (1988) J. Bacteriol. 170, 439-441. Bacteriol. 170, 1092-1102. 23. Ronson, C. W. Astwood, P. M., Nixon, B. T. & Ausubel, 27. Backman, K. C., Chen, Y. M., Ueno-Nishio, S. & Magasanik, F. M. (1987) Ni cleic Acids Res. 15, 7921-7950. B. (1983) J. Bacteriol. 154, 516-519. 24. Losick, R., Youngman, P. & Piggot, P. J. (1986) Annu. Rev. 28. Clegg, D. 0. & Koshland, D. E., Jr. (1984) Proc. Nati. Acad. Genet. 20, 625-670. Sci. USA 81, 5056-5060. Downloaded by guest on September 25, 2021