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MICROBIOLOGICAL REVIEWS, Dec. 1989, p. 450-490 Vol. 53, No. 40146-0749/89/040450-41$02.00/0Copyright © 1989, American Society for Microbiology

Protein Phosphorylation and Regulation of AdaptiveResponses in Bacteria

JEFFRY B. STOCK,* ALEXANDER J. NINFA,t AND ANN M. STOCK

Departments of Biology and Chemistry, Princeton University, Princeton, New Jersey 08544

INTRODUCTION .......................................... 450HOMOLOGOUS REGULATORY PROTEINS.......................................... 451

Adaptive Responses and Homologous Regulators .......................................... 451Histidine Protein Kinases .......................................... 451Response Regulators .......................................... 451

PROTEIN PHOSPHOTRANSFERASE CHEMISTRY .......................................... 455Protein Phosphorylation at Imidazole Nitrogens .......................................... 455Phosphorylation at Aspartate Residues .......................................... 458Phosphatase Reactions.......................................... 459

CHEMOTAXIS .......................................... 460Physiology and Genetics .......................................... 460Sensory Inputs.......................................... 460Phosphorylation and Signal Transduction .......................................... 460

NITROGEN REGULATION .......................................... 463Adaptive Response to Nitrogen Starvation .......................................... 463Signal Transduction.......................................... 465Mechanism of Transcriptional Activation .......................................... 466

PHOSPHATE REGULATION .......................................... 467Phosphate Uptake .......................................... 467Pho Regulon .......................................... 467Signal Transduction Pathways That Regulate PhoB .......................................... 467

OSMOREGULATION .......................................... 469Solute Relationships across the Envelope .......................................... 469Regulation of Porin Expression .......................................... 469Roles of EnvZ and OmpR .......................................... 470

OTHER SYSTEMS .......................................... 472Sporulation .......................................... 472Competence .......................................... 473Secretion of Degradative Enzymes .......................................... 473Alginate Production .......................................... 473Exoprotein Synthesis.......................................... 473Symbiotic Nitrogen Fixation .......................................... 474Dicarboxylate Transport .......................................... 474Tricarboxylate Transport.......................................... 474Oxygen Regulation .......................................... 474Nitrate Reductase.................................... 475Hydrogenase .................................... 475Phosphoglycerate Transport .................................... 475Uptake of Hexose Phosphate.................................... 475UvrC-ORF2 .................................... 476Agrobacterium Virulence .................................... 476Salmonella Virulence.................................... 476Frizzy.................................... 477Flagellar Biogenesis .................................... 477

CROSS-TALK AND GLOBAL REGULATION .................................... 477ACKNOWLEDGMENTS .................................... 478LITERATURE CITED.................................... 478

INTRODUCTION

Adaptive responses in bacteria range from rapid transientchanges in motility to long-term global reorganizations of

* Corresponding author. gene expression and cell morphology. Signals from withint Current address: Department of Biochemistry, Wayne State the cytoplasm and from the environment are used to control

University School of Medicine, Detroit, MI 48201. the cellular activities that mediate a particular type of

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PROTEIN PHOSPHORYLATION AND REGULATORY SYSTEMS 451

response. The molecular mechanisms responsible for stimu-lus-response coupling often involve two types of enzymaticcomponents: histidine protein kinases (HPK), and theirassociated response regulators (RR). Signal transductionoccurs through the transfer of phosphoryl groups from adeno-sine triphosphate (ATP) to histidine residues in the histidinekinases, from the HPK-phosphohistidine side chains toaspartic acid residues in the RR, and, finally, from theresponse regulator-phosphoaspartate side chains to water:

ATP + HPK-His ADP + HPK-His - PHPK-His - P + RR-Asp HPK-His + RR-Asp - P

H20 + RR-Asp - P RR-Asp + Piwhere ADP is adenosine diphosphate and Pi is inorganicphosphate. The level of phosphorylation of a responseregulator controls its activity. In principle, therefore, theregulation of any of the reactions shown above can contrib-ute to the control of an adaptive response.

This protein phosphotransfer chemistry has only recentlybeen discovered. Molecular genetic studies had indicatedthat many bacterial signal transduction systems containmembers of two families of homologous proteins, one ofwhich controls the activity of the other (270, 372). Studies ofthe nitrogen regulation, chemotaxis, osmoregulation, andphosphorous regulation systems in Escherichia coli andSalmonella typhimurium have demonstrated that phosphor-ylation of the response regulators is an essential feature ofthe signal transduction mechanism and that a commonphosphotransfer enzymology is involved (133, 145, 262, 264,443; K. Makino, H. Shinagawa, M. Amemura, T.Kawamoto, M. Yamada, and A. Nakata, J. Mol. Biol., inpress). Finally, solution of the three-dimensional X-raycrystal structure of one of the response regulators hasallowed the interpretation of sequence homologies in termsof a conserved phosphoaccepting active site (375; J. B.Stock, A. M. Stock, and J. M. Mottonen, Nature [London],in press).

Studies with purified proteins from the Che, Ntr, and Ompsignal transduction systems have indicated a high degree ofcross-reactivity between histidine kinases and response reg-ulators (145, 264). These results underscore the possibility,originally hypothesized from genetic studies (421), of cross-talk between parallel signal transduction pathways. Phos-photransfer elements within the cytoplasm may functiontogether as an information-processing network that inte-grates signals from a wide range of sensory inputs tocoordinate the activities of an equally diverse array of outputresponse strategies.

This review begins by summarizing current knowledge ofthe structure and enzymology of the homologous families ofhistidine protein kinases and phosphorylated response regu-lators. The functions of these signal transduction compo-nents are then examined within the context of systems thatcouple specific types of environmental signals to specificadaptive responses. Finally, an attempt is made to placethese systems within the context of global regulation of cellfunction. For previous reviews of this family of related signaltransduction systems, see references 46, 240, 315, and 378and Stock et al., in press. For reviews of specific adaptivesystems within this family, see Table 1.

HOMOLOGOUS REGULATORY PROTEINS

Adaptive Responses and Homologous RegulatorsAs the sequences of signal transduction proteins have

begun to be determined, it has become clear that a large

number of bacterial adaptive responses are controlled bymembers of two homologous families of proteins (Table 1).Because of the central importance of these two components,these signal transduction systems have been designated"two-component" systems (270). This is a misleading over-simplification since these systems generally contain addi-tional signal transduction components and in some casesmore than one kinase or response regulator or both. Thekinases have been referred to as sensors or modulators (270).Although in some cases kinases appear to function asclassical membrane receptor proteins, in others it is quiteclear that the kinase is not the sensor. In this review, we usethe nomenclature kinase (or kinase-phosphatase when appli-cable) and response regulator since these terms seem to bestrepresent the essential activities of these proteins.

Histidine kinases and response regulators are involved inadaptive responses ranging from chemotaxis and control ofgene expression in enteric bacteria to the control of complexdevelopmental pathways in sporulating, symbiotic, andpathogenic bacteria. The homologs are found in both gram-negative and gram-positive bacteria, including the generaAgrobacterium, Bradyrhizobium, Bacillus, Bordetella, En-terobacter, Escherichia, Klebsiella, Myxococcus, Pseudo-monas, Rhizobium, Staphylococcus, and Salmonella. In E.coli alone, 10 kinases and 11 response regulators have beenidentified to date. Since approximately 20% of the E. coligenome has been sequenced (182), there may be as many as50 of these types of signal transduction systems operatingsimultaneously within a single E. coli cell.

Histidine Protein KinasesThe histidine protein kinase family is defined by a region

of conserved sequence generally located near the C terminus(Fig. 1). Within this region of conservation, there are sixresidues that are especially conserved: N..(15 to 45residues)..DXGXG..(20 to 50 residues)..GXG (regions IIand III in Fig. 2). In addition, all of the kinases except CheAand FrzE have a conserved histidine that precedes theconserved asparagine by approximately 100 residues (regionI in Fig. 2). This histidine is presumably the site of autophos-phorylation in these proteins. In CheA, this portion of theconserved region is missing, and the protein is phosphory-lated at His-48 within a nonconserved N-terminal domainthat precedes the conserved histidine kinase region (132,374). FrzE contains a histidine residue at position 49, and thesurrounding sequence is very similar to that found aroundHis-48 in CheA.The remaining portions of each of the kinases tend to be

quite variable. In a few proteins, such as CheA, FrzE, VirA,and the Bordetella Vir protein, there are additional se-quences beyond the C-terminal end of the conserved region.Many of the histidine kinases are membrane associated, withtwo hydrophobic transmembrane sequences bordering adomain that is presumably localized to the outer surface ofthe cytoplasmic membrane. It has been postulated that theseproteins function as membrane receptors, their N-terminalextracytoplasmic domains interacting with stimulatory li-gands in the periplasm and transmembrane signals acting tocontrol the kinase or phosphatase activities, or both, of theircytoplasmic C-terminal domains (106). NR,, and CheA arecytoplasmic proteins that receive signals within the cyto-plasm (267, 374).

Response RegulatorsThe response regulator family is defined by a conserved

domain of about 100 amino acids that generally extends from

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TABLE 1. Adaptive responses controlled by homologous signal transduction proteins

System Histidine Response Adaptive response Organism" Reference(s)protein kinase regulator(s)

Che CheA CheB/CheY Chemotaxis Ea/Ec/St 209, 366Ntr NRI NRI Nitrogen regulation Bp/Ec/Kp/Ka/St 216, 217Pho PhoR PhoB Phosphate regulation Ec/Bsh 254, 419Omp EnvZ OmpR Porin gene expression Ec/St 71, 106Spo SpoIIJ' SpoOA/SpoOF Sporulation Bs 199, 351Dct DctB DctD Dicarboxylate transport RI 312, 313Hyd HydHd HydG Hydrogenase Ec 386Pgt PgtB PgtA Phosphoglycerate transport St 160, 322Arc CpxA' ArcA Oxygen regulation Ec 154, 428PhoM PhoM ORF2 ND' Ec 11, 426Vir VirAtf VirA/VirG Agrobacterium virulence At 257, 361Vir PhoQ PhoP Salmonella virulence St 241Tct TctD Tricarboxylate transport St 435Uhp UhpB UhpA Uptake hexose phosphates Ec 433Fix FixL FixJ Nitrogen fixation Rm 73Nar NarX NarL Nitrate reductase Ec 368Deg DegS DegU Secretion of enzymes Bs 23, 131UvrC ORF2 ND Ec 248Com ComA Competence Bs 6, 124Alg AlgR Alginate production Pa 80, 82Agr ORF2 AgrA Exoprotein synthesis Sa 292Frz FrzE1 FrzE Motility/development Mx 231, 318Vir Vir"d Vir Bordetella virulence Bp 240, 371

" At, Agrobacterium tuimefaciens; Bp, Bradyrhizobioin paraspoticae; Bs, Bacilluis siubtilis; Bt, Bordetella petutissis; Ea, Enterobacter aerogenes; Ec,Escherichia coli; Ka, Klebsiella aerogenes; Kp, Klebsiella pneunoniae; Mx, Myxococcus xanthus; Pa, Pseudototmonas aeruiginosa-, RI, Rliizobiium legu,nminosarumn;Rm, Rhizobiutm mneliloti; Sa, Staphylococcus auirelus; St, Salmtioniella tvphimuiriuim. This list is limited to organisms in which genes encoding histidine proteinkinase or response regulator components or both have been sequenced.

b In B. suibtilis, a gene involved in Pho regulation with a sequence that is very similar to phoB in E. (oli has been termed phoP (335), while another gene involvedin Pho regulation, with a sequence that is homologous but somewhat less similar to the E. coli plioR gene, has been designated plioR (336).

' In these systems, there is little genetic evidence for a specific interaction between the indicated kinase and response regulators.d In these instances, only fragmentary sequence information is available.e ND, Not defined.f These proteins contain regions homologous to both histidine protein kinase and response regulator families.

the N terminus of each of these proteins (Fig. 3). Sequencealignments between any two response regulator domainsshow 20 to 30% identical amino acids at correspondingpositions. The residues that correspond to Asp-13, Asp-57,and Lys-109 in the CheY sequence tend to be conservedamong all sequences (Fig. 4). The three-dimensional struc-ture of the S. typhimurium CheY protein has been solved to0.27-nm resolution (375; Stock et al., in press). The proteinis composed of a central core of five parallel ,B-strandssurrounded by five u.-helices (Fig. 5). Comparison of theCheY sequence with that of other response regulators indi-cates that structural features such as the hydrophobic core

are conserved, suggesting that all of the response regulatorshave an N-terminal domain of similar structure (Stock et al.,in press). Within these structures, the highly conservedresidues corresponding to Asp-13, Asp-57, and Lys-109would be clustered at the carboxyl end of the r-sheet just as

they are in CheY. Domains of oi/r structure similar to CheYare found in numerous enzymes, including dehydrogenasesand reductases, kinases, proteases, and the periplasmicbinding proteins (309). In every case the active site of theprotein is located at the carboxyl end of the r-sheet (49). Itis therefore highly likely that this portion of the response

regulators corresponds to the active site of these proteins.This hypothesis is supported by the finding that Asp-57 is thesite of phosphorylation in the CheY protein (D. A. Sanders,B. L. Gillece-Castro, A. Stock, A. L. Burlingame, andD. E. Koshland, Jr., J. Biol. Chem., in press). Thus, thesequence similarities that define the response regulator N-terminal domain can be understood in terms of a common

structure and a conserved phosphotransfer enzymology(377, 431).The response regulators may be placed into subfamilies

based on sequence similarities between their C-terminaldomains (Fig. 3). CheY and SpoOF consist of only theconserved response regulator N-terminal domain. Conceiv-ably, in these proteins the response regulator domain itselfdirectly controls the adaptive response. In the case of CheY,a number of lines of evidence suggest that P-CheY interactswith the flagellar motor to effect the behavioral response;thus, in this case a direct role for the phosphorylatedresponse regulator domain is indicated (264, 285, 377).NR,, DctD, HydG, and PgtA share a homologous C-

terminal domain that is also found in proteins such as F1bD,NifA, and TyrR that lack the conserved N-terminal responseregulator region (57, 69, 88). NRI, DctD, and NifA have beenshown to activate transcription from promoters that arerecognized by EB54 ribonucleic acid (RNA) polymeraseholoenzyme (76, 142, 238, 314); thus, it seems likely that thisconserved domain is involved in the interaction with EU54.At their extreme C terminus, NRI and NifA contain a regionthought to be involved in deoxyribonucleic acid (DNA)binding (88, 238), and each of these proteins binds to sitesupstream from the regulated promoters (13, 54, 306). Phos-phorylation of NR, within the N-terminal response regulatordomain stimulates its ability to bind DNA and is requiredfor activation of transcription (262, 266); thus, activitiesassociated with the C-terminal domains are controlled byphosphorylation of the N-terminal response regulator re-gion.

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PROTEIN PHOSPHORYLATION AND REGULATORY SYSTEMS

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by solid boxed regions near the N termini. The sequences corresponding to regions I, II, and III are shown in Fig. 2. Region I, containingthe conserved histidine residue, is indicated by a filled X; region II, containing the conserved asparagine, is indicated by a stippled box; andthe glycine-rich region III is indicated by a hatched box. Boxed regions at the extreme C termini of VirA and FrzE are homologous to theresponse regulator domains shown in Fig. 3 and 4. CheA, NR,,, SpoIIJ, DegS, and FrzE lack any discernible membrane-spanning sequences.FrzE has no clearly defined region II, and NarX and Agr-ORF2 appear to lack portions of the glycine-rich region III (Fig. 2). Numbers to theright designate the total number of amino acid residues in each protein as predicted from the sequences of the corresponding genes. Sequencesused are indicated in the legend to Fig. 2.

PhoB, OmpR, ArcA, PhoM-ORF2, VirG, PhoP, and TctDhave another type of C-terminal domain. This conserved Cterminus is found in at least one protein, ToxR, that lacks anN-terminal response regulator domain. Each member of theOmpR subfamily, including ToxR, functions to regulate the

expression of a specific set of target genes at promoters thatare thought to be recognized by the major form of RNApolymerase, corresponding to Ea70 in E. coli. It has beenshown that a proteolytically generated C-terminal fragmentof OmpR binds to specific DNA sequences upstream from

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the regulated promoters (396). In the intact protein, phos-phorylation of the N-terminal domain serves to stimulate thisDNA-binding activity (4) and can also stimulate the activa-tion of transcription by OmpR (144, 145). Thus, as with theNR, family, phosphorylation of the N-terminal domain ofOmpR controls the activity of the associated C-terminaldomain.UhpA, FixJ, ComA, NarL, DegU, and UvrC-ORF2 share

a third type of C-terminal domain. These proteins are alsothought to function as transcriptional regulators, but theirmechanisms of action have not been characterized.The response regulators CheB, AlgR, AgrA, and SpoOA,

have C termini that are not significantly similar to otherresponse regulators. Among these, CheB is the best charac-terized. The C-terminal portion of CheB is a protein methy-lesterase enzyme that can function in the complete absenceof the N-terminal regulatory domain (348, 367). Phosphory-lation of CheB within its response regulator domain has beenshown to cause a dramatic activation of esterase activity(205, 377). A similar activation is obtained when the N-terminal domain is removed by genetic deletion or proteo-lytic cleavage (348). Thus, in the CheB protein the regulatorydomain, and phosphorylation removes this inhibitory effect.

PROTEIN PHOSPHORYLATION CHEMISTRY

Phosphorylation at Histidine Imidazole Nitrogens

Four members of the histidine protein kinase family havebeen purified and characterized, CheA (374), NR1I (267),PhoR (Makino et al., in press), and EnvZ (144). Each ofthese proteins is labeled with 32p when it is incubated iny-32P-labeled ATP (134, 146, 164, 264, 431, 443; Makino etal., in press). The phosphorylated residues in CheA andNR,, have been identified as histidines (377, 431). From theirrates of hydrolysis under a variety of different conditions,the N-3 position of the imidazole ring appears to be the siteof phosphorylation (264, 443), and this supposition has beenconfirmed in the case of CheA by 31P-nuclear magneticresonance (G. Lucat and J. Stock, unpublished observation).Phosphoramidates such as the phosphohistidine groups inCheA and NRI1 exhibit a characteristic instability in acidcompared with alkali. This relative acid lability distinguishesthem from phosphorylated tyrosine, serine, threonine, oraspartate residues, which tend to be more stable at acidicthan alkaline pH. The reported disappearance of 32P-labeledCheA and EnvZ after treatment with strong base (134, 146)was more likely due to peptide bond cleavage than tohydrolysis of the phosphoramidate linkage. In fact, thephosphohistidine in CheA has been obtained as the freephosphorylated amino acid in alkaline hydrolysates of 32p_labeled CheA protein (377).The site of phosphorylation in CheA, His-48 (132), is

located in a domain of CheA that is distinct from theconserved histidine kinase domain indicated in Fig. 1. Pro-teolytic cleavage of CheA releases a phosphorylated N-terminal polypeptide of approximately 18,000 molecularweight that can serve as a phosphodonor to CheY and CheBjust as well as the intact P-CheA protein (132). The peptideappears to be totally incapable of autophosphorylation,however. Thus, the conserved histidine kinase domain neednot function as a site of interaction with response regulatorproteins. Instead, the sequence similarities that define thehistidine kinase domain reflect a conserved catalytic mech-anism that involves ATP binding and transfer of a y-phos-phoryl moiety to an imidazole nitrogen.

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Aspl3 Asp57 LyslO9

FIG. 5. Schematic diagram showing the topology of the second-ary structural elements of CheY. A central parallel a-sheet with,-strands 1 through 5 depicted as arrows is flanked by five a-helicalsegments, A through E, represented as cylinders (375). The loca-tions of the highly conserved residues, Asp-13, Asp-57, and Lys-109, are indicated at the C-terminal ends of ,-strands 1, 3, and 5,respectively. Asp-57 has been shown to be phosphorylated by CheA(Sanders et al., in press).

There are numerous other examples of proteins that are

phosphorylated at the N-1 or N-3 imidazole nitrogens ofhistidine residues (Table 2). In most cases, the phosphohis-tidine may be regarded as a high-energy phosphoenzymeintermediate mediating passage of a phosphoryl moiety froma donor to an acceptor species. This contrasts with thegeneral role of phosphoesters such as phosphoserine, phos-phothreonine, and phosphotyrosine residues, in which phos-phorylation generally serves to modulate rates of catalysiswithout any direct involvement in the enzymatic mecha-nism. The reason for this difference may relate to the factthat phosphoramidate bonds in proteins tend to have a

higher standard free energy of hydrolysis than ATP by as

much as -7 kcal (-29 kJ)/mol (430; Stock et al., in press).The standard free energy of hydrolysis of P-CheA is about-12 kcal (-50 kJ)/mol compared with a value for ATP undercomparable conditions of approximately -8 kcal (-33 kJ)/

TABLE 2. Phosphohistidinyl proteins

o

0o~0

4 co ._

0

C: 4a

a

C00

3 . .a

o Q X Cu

On 64

IL)0

0.

o_C U

O

0

>

,0 8 ,

r L ->

*-~-,=Q.;

E co0=04- *

oo

o o

>-c)_

4..

0

" " U0

Protein Source Refer-ence

Histones H1/H4 Rat liver 62ATP-citrate lyase Rat liver 436Fructose-2,6-bisphosphatase Rat liver 294Glucose-6-phosphatase Rat liver 95Phosphoglycerate mutase Rat liver 294Diphosphoglycerate kinase Rabbit muscle 317

Saccharomyces cere- 317visiae

Nucleoside diphosphate kinase Bovine heart 67Saccharomyces cere- 91

visiaeBisphosphoglycerate synthase Horse erythrocytes 317Succinyl thiokinase Mitochondria 179Succinyl coenzyme A synthe- Escherichia coli 418

taseIsocitrate lyase Eschenichia coli 311Pyruvate, phosphate dikinase Bacteroides symbiosus 360PTS enzymes Bacteria 323

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g ucose

regulation of <-motilityadenylate cyclasesugar transport

9 ucose-6-P

HPr HPrp c aar bohydrateHPr-P metabolism

I-P I

pyruvate phosphoenolpyruvateFIG. 6. Phosphoenolpyruvate:glucose PTS system in E. coli and S. typhimurium. A phosphoryl moiety from phosphoenolpyruvate is

transferred first to a histidine residue in enzyme I, then to a histidine in HPr, and then to a histidine in enzyme IIIc; then, via the membranereceptor-transport protein, enzyme llg'c, the phosphoryl group is passed to incoming glucose to yield glucose-6-phosphate in the cytoplasm.Levels of phosphorylation of the PTS proteins regulate motility, adenylate cyclase, and sugar transport in response to glucose.

mol (443). By using high concentrations of ATP and purifiedCheA or NR,,, it has been possible to obtain levels ofphosphorylation approaching one phosphoryl group permonomer (134, 164, 431, 443). In vivo, when ATP/ADPratios are maintained between 10 and 100 (40, 411), it maynot be possible to use ATP to convert a substrate protein toits phosphohistidine form stoichiometrically. Thus, thephosphohistidinyl groups in the histidine protein kinasesshould probably be considered as high-energy phosphopro-tein intermediates in the transfer of phosphoryl groups fromATP to the response regulator proteins.The bacterial histidine kinase phosphotransfer compo-

nents are not the only phosphohistidinyl proteins involved inthe regulation of gene expression and motility. Carbohydrateregulation in many bacteria is mediated by a phosphotrans-ferase system (PTS) (for reviews, see references 235, 297,298, and 321). In the PTS, phosphoryl groups are passedfrom a histidine in one protein component to a histidine inanother protein, the location of the group alternating be-tween N-1 and N-3 positions on the histidine side chains(323). The initial phosphodonor is phosphoenolpyruvaterather than ATP, and the terminal acceptor is the hydroxylgroup on a sugar molecule such as glucose. The overallreaction catalyzed by the PTS serves to transport andphosphorylate carbohydrates at the expense of phosphoe-nolpyruvate (Fig. 6). Levels of phosphorylation of the phos-photransfer proteins of the PTS serve to regulate numerouscell activities, including carbohydrate metabolism, motility,and gene expression. In E. coli, the PTS involves at least 10

different membrane receptor-transport proteins, togetherwith at least 6 cytoplasmic phosphotransfer proteins.

Phosphorylation at Aspartate Residues

The response regulators are phosphorylated at aspartateside chains. Because of the instability of acyl phosphates atboth acid and alkaline pH, it has not generally been possibleto demonstrate their presence within a protein directly. Byusing indirect methods such as rates of hydrolysis underdifferent conditions (15) or reduction and labeling with [3H]borohydride (79), it has been possible to show that bothCheY and NR, are acyl phosphates (377, 431). The stoichi-ometry of CheY phosphorylation may be >1 (133), and bothof two highly conserved aspartate residues in the active siteof the response regulators, corresponding to Asp-13 andAsp-57 in CheY, are likely sites of phosphorylation (Fig. 4and 5). It has been demonstrated by borohydride reductionand tandem mass spectrometry that Asp-57 is a site ofphosphorylation in CheY (Sanders et al., in press). AnAsp-57-Asn mutant protein is not significantly phosphory-lated, while an Asp-13-Asn mutant CheY protein is phos-phorylated, albeit at a reduced rate (B. Lee, J. Mottonen,and A. Stock, unpublished observation). Strains containingmutations in CheY at either Asp-13 (Asn, Thr, Ser, Met) orAsp-57 (Asn, Tyr) are defective in chemotaxis (Sanders etal., in press).

In all response regulators the aspartate corresponding to

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TABLE 3. Phosphoaspartyl proteins

Protein Source Refer-ence

Ca2+Mg2' ATPase Rabbit sarcoplasmic reticulum 79H+ ATPase Neurospora 72H+K+ ATPase Rat stomach 345K+ ATPasea Escherichia coli 135Na+K+ ATPase Guinea pig kidney 296

Squalus acanthias 268Nucleoside phospho- Carrot 365

transferasea The phosphoaspartyl residue has not been directly demonstrated in the

E. coli enzyme but is presumed to exist because of sequence similarities withhomologous ATPases from eucaryotic systems.

Asp-13 in CheY is preceded by an acidic residue, usually anaspartate but in some cases a glutamate, and in mostresponse regulators the conserved aspartate is followed byan acidic residue. In CheY these acidic side chains extendinto a cleft formed by loops connecting the C-terminal endsof two central 1-strands to a-helices on opposing sides of the13-sheet (Fig. 5). Transfer from a phosphoramidate involvesnucleophilic attack by the phosphoryl acceptor at the phos-phorus and a concerted electrophilic attack, generally by aproton, at the nitrogen (30). The acidic pocket in CheY couldprovide both the phosphoacceptor nucleophile, an aspartatecarboxylate anion, and a proton from an un-ionized aspar-tate or glutamate side chain (375). The active site thatappears to be conserved among all response regulators maytherefore reflect a common phosphoacceptor chemistry.There are relatively few examples of proteins phosphory-

lated at carboxylate side chains (Table 3). The best-docu-mented instances are the phosphoaspartyl groups in a num-ber of different ion-translocating adenosine triphosphatases(ATPases) when a phosphorylation-induced conformationalchange provides the crucial energy-coupling step for theactive transport of H+, Na+, K+, or Ca2+. Jencks (159) andTanford (395) have advanced thermodynamic arguments thathigh-energy aspartyl phosphate bonds are ideally suited forthe generation of the large changes in protein conformationrequired for the production of transmembrane electrochem-ical gradients. Studies of model compounds indicate thatacyl phosphates should have the highest free energies ofhydrolysis of any phosphorylated amino acid side chain, -10to -13 kcal (-42 to -54 kJ)/mol (158). However, thestandard free energy of hydrolysis of the phosphoaspartylgroup in the sarcoplasmic reticulum Ca2+-ATPase has beenestimated to be +2 kcal (+8 kJ)/mol (293). It is thought thatthe ATPase converts the high-energy aspartyl phosphate to alow-energy state through a compensatory change in proteinconformation:

ATP + E1 E1 - P + ADPEl - P E2 - PE2 - P <-+ E2 + P

where E1 and E2 represent high- and low-energy conforma-tions of the ATPase. Brandl et al. (50) have predicted astructure for the phosphorylated energy-coupling domain ofthe Ca2+-ATPase that is remarkably similar to the structureof CheY. Although there is no apparent sequence homologybetween phosphorylated ion-translocating ATPases (50, 135,208, 337, 345, 346) and the phosphorylated response regula-tors, it seems likely that a similar phosphorylation-activated

switching mechanism may be functioning in both types ofproteins. The structure of CheY determined from X-raycrystallographic studies represents the dephosphorylatedconformation of the protein. Further studies will be requiredto determine the activated structure and the conformationalchange that underlies the switching mechanism.

Phosphatase Reactions

Whereas the histidine kinase and phosphorylated responseregulator components are conserved in all of these regula-tory systems, phosphatase functions tend to be relativelyvariable. There are several alternative mechanisms thatcontrol the rate of dephosphorylation of the phosphorylatedresponse regulators.

First, some of the regulators exhibit an autophosphataseactivity. The phosphorylated regulators, when denatured indetergent, exhibit a half-time of hydrolysis of 1 to 5 h underphysiological conditions of pH and temperature (145, 377,431). Under nondenaturing conditions, P-OmpR (145), P-SpoOF (A. Ninfa and I. Smith, unpublished observation),and P-SpoOA (E. Ninfa, J. Stock, and P. Youngman, unpub-lished observation) also exhibit half-times of about 1 h.These stabilities are within the range of what would beexpected from hydrolysis rates of model compounds such asacetyl phosphate (177). Under nondenaturing conditions,P-CheY and P-CheB exhibit half-times of approximately 10 s(133, 377), while P-NRI (164, 431) and P-PhoB (Makino etal., in press) have half-times of several minutes, indicatingthat these proteins contain an autophosphatase activity.Studies with P-CheY (Sanders et al., in press) and P-NRI(431) indicate that this autophosphatase reaction requiresMg2 . Thus, each different phosphorylated response regula-tor exhibits its own characteristic inherent stability.A second route for dephosphorylation of the phosphory-

lated response regulators is mediated by their correspondingkinases. This has been demonstrated most convincingly inthe Omp system, in which it has been shown that EnvZcatalyzes both the phosphorylation of OmpR and the de-phosphorylation of P-OmpR (3, 145). EnvZ-dependent P-OmpR phosphatase activity requires ATP, ADP, or any of anumber of nonhydrolyzable ATP analogs. Presumably,when EnvZ is in its native conformation in the inner mem-brane, the balance between its kinase and phosphataseactivities is controlled by transmembrane signals from theperiplasmic domain.

In the case of the nitrogen regulation system, an auxiliarysignal transduction component, P,,, interacts with the ki-nase-phosphatase NRI, to stimulate the catalytic dephospho-rylation of P-NR, (262). As with the EnvZ phosphatase, thisactivity requires ATP (164). P,, is regulated by nitrogenavailability, functioning as a regulatory subunit of the kinase(for a review, see reference 216).

Unlike the kinases of the Omp and Ntr systems, there isno evidence that CheA can act as a CheB or CheY phos-phatase. This difference may reflect some of the unusualfeatures of the CheA sequence compared with the sequencesof most other histidine kinase proteins (374).

Auxiliary regulatory proteins can also function as phos-phatases to enhance the rate of dephosphorylation of theresponse regulators. The best-understood instance of this isthe CheZ protein that accelerates the dephosphorylation ofP-CheY. The sequence of CheZ does not appear to berelated to that of any other known protein sequence. Theisolated CheZ protein is a large homopolymer composed of24,000-molecular-weight subunits (376). Genetic results in-

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dicate that it interacts with the flagellar motor (288, 289,444). CheZ specifically facilitates the dephosphorylationof P-CheY. It has no effect on the stability of P-CheB(133) or P-NRI (A. Stock and A. Ninfa, unpublished obser-vation).

In the following sections, we provide a detailed account ofthe role of kinase-phosphatase enzymes and their substratephosphoaspartyl regulators in a few specific signal transduc-tion systems: Che, Ntr, Pho, and Omp. The analysis of theseparticular systems is the most advanced. All have beenextensively investigated in enteric bacteria such as E. coliand S. typhimurium. After discussing these specific systems,we briefly summarize other homologous systems that havebeen identified. Finally, we attempt to extrapolate to abroader view of the role of the phosphohistidine-phosphoas-partyl phosphotransfer circuits within the context of thewhole cell.

CHEMOTAXIS

Physiology and Genetics

Flagellated bacteria such as E. coli and S. typhimuriumswim by rotating long helical flagellar filaments that extendinto the surrounding fluid (for reviews of flagellar structureand function, see references 209 and 210). Reversals (212) orpauses (17, 118, 191) in flagellar rotation cause changes indirection so that cells run for a few seconds along one path,tumble or pause, and then resume swimming along a newpath. When cells sense that they are running towards attrac-tants or away from repellents, tumbles and pauses aresuppressed, and the cells tend to continue on course (33,211). The result is a net migration toward favorable environ-mental conditions.

Strategies have been devised to select mutants that areeither unable to change their direction or otherwise defectivein chemotaxis (18, 20). In both E. coli and S. typhimurium,only six che genes have been identified: cheA, cheB, cheR,cheW, cheY, and cheZ. Deletion of any of these che genesproduces a cell that is fully motile, but completely unable tomigrate in chemical gradients (287). All six che genes havebeen sequenced (252, 349, 372-374, 376), and the corre-sponding proteins have been purified and characterized (348,349, 372-374, 376). All of the proteins are soluble cytoplas-mic constituents that either directly participate in or modu-late signal transduction between chemoreceptors and theflagellar motor.Three flagellar proteins appear to function at the interface

between the Che proteins and the flagellar motor (27, 66, 94,141, 185, 406, 444, 445). Mutant strains completely lackingthese proteins exhibit a Fla- phenotype. The genes thatencode these components are fliG, fliM, and fliN (for areview of the revised flagellar gene nomenclature, see refer-ence 147). Some missense mutations in these genes causecells to be motile but nonchemotactic (Che phenotype),other mutations at these loci cause immotility by interferingwith flagellar rotation (Mot phenotype), and still others leadto a complete loss of flagellar synthesis (Fla phenotype).

All Che mutants exhibit extreme swimming behaviors:cheW, cheA, and cheY mutants run constantly withoutchanging direction (285, 427); cheR mutants run constantly,but tumble in response to large repellent stimuli (38, 121,290, 359, 383); cheB and cheZ mutants tumble constantly,but run in response to strong attractant stimuli (19, 38, 320,333, 334, 380, 448); missense mutations with Che phenotypesinfliG,fliM, andfliN and in genes that encode receptors such

as tsr either run or tumble incessantly depending on theparticular allele (75, 173, 286, 320, 406, 427, 444, 445).

Sensory Inputs

There are numerous sensory mechanisms that operate tocontrol motility. Research has focused on a family of homol-ogous receptor-transducer proteins, Tsr, Tar, Tap, and Trg,that mediate responses to amino acids, peptides, and somesugars (for reviews, see references 209 and 366). Theseproteins have the same transmembrane topology as histidinekinase receptors such as EnvZ (225), and limited sequencesimilarities suggest that they may be related to the kinasereceptors (176, 372). There is no evidence, however, that thechemotaxis receptors are themselves protein kinases. TheN-terminal periplasmic domains of the chemotaxis receptorstend to be variable, while the C-terminal cytoplasmic do-mains are highly conserved (41, 181). The extracellulardomain is the site where stimulatory ligands bind (65, 180,249, 417), and the intracellular domain acts in conjunctionwith CheW and CheA to control the rate of CheY phosphor-ylation (45, 275). The intracellular domain contains tworegions that are methylated and demethylated at glutamateresidues (166-168, 171, 398). The sequences in these meth-ylated regions indicate an ot-helical structure, one face ofwhich is methylated (382, 398). Circular-dichroic measure-ments suggest that the cytoplasmic domain is >50% ox-helical (249). Genetic data indicate that the region betweenthe methylated sequences is the site of interaction withCheA and CheW (14). Binding of an attractant to theextracellular domain has several effects: in the presence ofCheA and CheW, the rate of CheY phosphorylation isdrastically reduced (45), the activity of the esterase towardcertain methylglutamate residues is also reduced (43, 169),and the level of methylation is increased (120, 381). In-creased levels of methylation appear to counteract the effectof attractants on phosphorylation to cause receptor desensi-tization (356, 385).There are numerous types of stimuli that appear to func-

tion by mechanisms that do not involve the methylatedreceptors. Most notable among these are oxygen and glu-cose. In E. coli, responses to oxygen appear to be mediatedby the redox state of components of the electron transportsystem rather than by oxygen directly (343), and responsesto glucose appear to be regulated by the phosphorylationstate of the glucose:phosphoenolpyruvate PTS (1, 269). Howthe electron transport system and PTS are linked to chemo-taxis remains a mystery.

Phosphorylation and Signal Transduction

An overview of signal transduction in the E. coli and S.typhimurium chemotaxis systems is outlined in Fig. 7. Themembrane chemoreceptor-transducer proteins stimulate theflow of phosphoryl groups through CheA to CheY and CheB(133, 377, 443). This process requires CheW, is inhibited bythe binding of attractant ligands to the receptors (45), and isstimulated by methylation of the receptors (E. Ninfa and J.Stock, unpublished observations). P-CheY interacts with theflagellar switching apparatus, presumed to be a complex ofFliG, FliM, and FliN, to cause clockwise rotation andthereby induce tumbly behavior (264, 289, 352, 444). CheZantagonizes CheY activity by facilitating the dephosphory-lation of P-CheY (133, 186). P-CheB is the active form of anenzyme that deamidates or demethylates glutaminyl and

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attractants and repe I I ents

I I I IH20 Iv

--->ICH30H I

Trg Tap|

I /

AdoMet

CheRAAdoHcy

(CheW )

-CheB-P CheB

H20 PI

CheY-P CheY

2 CheZ p

(FIIM FIiN FIiG)

tumbly behavior

FIG. 7. System that mediates chemotaxis in E. coli and S.typhimurium. Attractants such as aspartate, serine, maltose, ribose,galactose, and peptides interact either directly or through periplas-mic binding proteins with membrane receptor-transducer proteins,Tar, Tsr, Trg, and Tap, in the cytoplasmic membrane. The same

receptors also mediate responses to repellents such as leucine,indole, acetate, Co2", and Ni2+. The receptors, in a process thatrequires CheW, control the activity of the CheA kinase. P-CheApasses phosphoryl groups to CheB and CheY. Phosphorylationcauses CheY to interact with flagellar motor components, FliM,FiN, and FliG, to cause tumbly behavior. CheZ accelerates the rateof CheY dephosphorylation. P-CheB is activated to demethylatereceptor methylglutamate residues that are synthesized by theS-adenosylmethionine (AdoMet)-dependent CheR methyltrans-ferase.

-y-carboxylmethyl glutamyl residues in the membrane recep-tor-transducer proteins (205, 377). The -y-carboxylmethylgroups are produced by the CheR methyltransferase (349,359). Levels of receptor methylation act to control receptorsensitivity to the effects of stimulatory ligands (for reviews,see references 356 and 385; J. Stock, in W. K. Paik and S.Kim, ed., Protein Methylation, in press).CheA is the central regulator of the chemotaxis system. It

modulates flagellar switching through CheY and at the sametime feeds back through CheB to control receptor signaling.The purified protein is a homomultimer of several 73,000-molecular-weight subunits (374). Each monomer seems to becomposed of at least three domains: a central histidinekinase domain (374) flanked by an N-terminal domain (132)that contains the site of histidine phosphorylation, His-48,and interacts with CheY and CheB, and a C-terminal domain(274) that is thought to function as a receiver of informationfrom membrane chemoreceptor proteins and CheW (176,274).

The location of the phosphohistidine in CheA has impor-tant implications in terms of the genetics of CheA expres-sion. It has been known for some time that similar amountsof two forms of CheA are produced from the cheA gene: alarge 73-kilodalton (kDa) form designated CheA, and a short64-kDa form designated CheAs (77, 347). CheAs appears tobe produced by an alternative site of translational initiation(353). N-terminal analysis of the purified CheAs proteinindicates that the initiation codon corresponds to Met-98 inthe full-length form of the protein (Ninfa and Stock, unpub-lished observation). Thus, CheAs completely lacks the his-tidine phosphorylation site as well as a large portion of theregion that interacts with CheY and CheB. Molecular-sievechromatography indicates that the native CheAs protein is ahomomultimer of slightly lower molecular weight than theCheA homomultimer, but no mixed multimers of CheA plusCheAs were detected (374). Nonsense mutations in theregion of cheA proximal to the secondary site of initiationproduce only the short form of the protein (353). Cells withthis defect exhibit the typical nonchemotactic, constantlyrunning phenotype of cheA deletion mutants (274, 353).Clearly, the full-length CheA protein together with its kinaseactivity is an essential component of the chemotaxis system.It is not known whether CheAs is essential or what pheno-type would be exhibited by a cheA mutant that producedonly the full-length protein.CheY is the response regulator of the chemotaxis system.

CheY mutants exhibit the same behavior as CheA mutants:they run constantly. This effect is dominant over the inces-santly tumbly phenotype caused by mutations in cheB orcheZ so that cells deleted in cheY, cheB, and cheZ exhibitthe same constantly running phenotype as mutants defectiveonly in che Y (285). This contrasts with the effects of mutantsdefective in both cheY and a component of the flagellarswitching apparatus such as fliG or fliM when the finalphenotype represents a combination of the effects of eithermutation alone (78, 289, 444). Since the phenotypes of thefla-cheY double mutants depend on the particular allelesinvolved, it has been suggested that there is a direct inter-action between CheY and the flagellar switch. Similar resultshave been obtained when Che alleles of fliG or fliM arecombined with specific alleles of cheZ as well as with acombination between a specific fliG and a specific cheAallele. Although it is possible that all of these proteinsinteract directly with the flagella, the possibility of indirectinteractions via intermediary signal transduction reactionsseems more probable. For instance, cheR-cheB double mu-tants exhibit allele-specific phenotypes that are intermediatebetween the extremes of cheR and cheB point mutations(379), but the CheR methyltransferase and the CheB meth-ylesterase only interact through their opposing effects onlevels of receptor modification.Another line of evidence that CheY directly interacts with

the flagellar switch has come from experiments in whichpure CheY protein was introduced into vesicular envelopeswith rotating flagella (302). Flagella attached to envelopesprepared without CheY only rotated counterclockwise, cor-responding to constantly running behavior (301). Addition ofpure CheY during preparation of the vesicular envelopescaused flagella to rotate clockwise, corresponding to con-stantly tumbly behavior. Although neither ATP nor CheAwas added to the preparations, these requirements couldhave been supplied by residual cytoplasmic constituents thatremained associated with the envelope fraction. Alterna-tively, at the high concentrations of CheY used, sufficient

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CheY may be able to adopt an active conformation in theabsence of ATP-dependent phosphorylation. It should benoted that in these vesicle preparations rotation was neverobserved to switch from one sense to another even underconditions in which, within the population of vesicles,roughly half rotated clockwise and half rotated counter-clockwise.A requirement for ATP in the CheY-dependent generation

of tumbly behavior in living cells has been thoroughlydocumented. Flagellar rotation has been shown to require anelectrochemical proton gradient in E. coli, S. typhimurium,Bacteroides subtilis, and several other species (for a review,see reference 210) and an electrochemical sodium ion gradi-ent in some alkalophilic species (138). When cells are de-pleted of ATP by either incubation in arsenate (19, 192) ordepletion of adenine nucleotides (342), they only rotate theirflagella counterclockwise and therefore run constantly. Mu-tants with defects in a gene such asfliG orfliM that encodesflagellar switch components exhibit tumbly behavior that isunaffected by ATP depletion (301, 352). These fla mutationsalso cause tumbling in deletions that lack CheY, CheA, orCheW or all three (78, 441). From these observations, it hasbeen concluded that ATP acts with the Che proteins tocontrol the flagellar rotation switch rather than as an energysource for clockwise rotation or switching per se. Smith etal. (352) concluded from genetic reconstitution experimentsthat ATP is required for CheY activity independent of CheA.It is likely, however, that at very high concentrations ofintracellular CheY there is sufficient phosphorylation fromalternative histidine kinases to cause tumbly behavior. Areport that acetate causes tumbly behavior in CheA dele-tions raises the possibility that acetyl phosphate or someother product of acetate metabolism can act to phosphory-late CheY (440). It is interesting within this context thatphosphotransfer has previously been demonstrated betweenan acylphosphate-acetate kinase intermediate and a histidi-nyl residue in enzyme I of the glucose PTS (108).P-CheA readily transfers its phosphoryl group to CheY

(133, 443). The P-CheY produced in this reaction is rapidlyhydrolyzed under physiological conditions to CheY and Pi:half-life (t112), approximately 10 s. If P-CheY is denatured insodium dodecyl sulfate, it exhibits the characteristic stabilityof an acyl phosphate: t112, 1 to 2 h (377). Even a 10-s half-lifeis quite long in terms of the duration of chemotactic re-sponses, however; the tumbly episodes thought to be causedby CheY phosphorylation typically last only a fraction of asecond.CheZ enhances the rate of CheY dephosphorylation (133).

Mutant strains lacking CheZ tumble all the time, presumablybecause they have elevated levels of P-CheY. CheZ-defi-cient strains still run in response to strong attractant stimuli,but there is a latency in these responses that lasts a fewseconds (334). Attractants inhibit CheY phosphorylation(45), and the latent interval in CheZ attractant responsesprobably reflects the time required for spontaneous hydrol-ysis of P-CheY. Missense mutations in cheZ, like some cheYmutations, can be compensated by missense mutations ingenes that are presumed to encode the flagellar switch, fliGandfliM (288, 289, 444). These effects may be indirect, actingthrough interactions between P-CheY and the flagellarswitching apparatus. Another possibility is that CheZ inter-acts directly with the flagellar switch to provide a mechanismby which the state of the motor can feed back to control thelevel of P-CheY.P-CheA can also transfer its phosphoryl group to the CheB

protein (133, 377). The rates of phosphotransfer from CheA

to CheB measured with purified components are slightlyfaster than corresponding rates of transfer to CheY. NativeP-CheB is converted to Pi even more rapidly than P-CheY:t112, approximately 5 s rather than 10 s (Ninfa and Stock,unpublished observations). As with P-CheY, P-CheB dena-tured in sodium dodecyl sulfate is much more stable than thenative phosphoprotein. CheZ does not accelerate the rate ofCheB dephosphorylation (133).CheB is composed of two domains, an N-terminal domain

homologous to CheY, and a C-terminal domain that cata-lyzes the demethylation of y-glutamylmethyl esters in themembrane transducer-receptor proteins (348, 372). A cheBgene with a deletion that extends through the entire regionthat encodes the regulatory domain produces a truncatedCheB protein with methylesterase activity (348). The cata-lytic and regulatory domains are separated by a protease-sensitive hinge region (348). Cleavage at this position causesa severalfold increase in esterase activity. It is thereforeapparent that the CheB response regulator domain interfereswith esterase activity. CheB phosphorylation relieves thisinhibition, causing a severalfold increase in esterase activity(205, 377).There appear to be two levels of control of receptor

methylation. Stimulatory ligands induce conformationalchanges in the receptors that appear to change the accessi-bility of potential substrate residues to the methylating anddemethylating enzymes (358, 381). This type of regulationrequires neither CheA nor the CheB regulatory domain (43,357). The changes in methylation produced by this effect arespecific to the receptor that is stimulated, and they persistfor as long as the stimulus is present (120, 355). The secondlevel of regulation occurs through the methylesterase en-zyme and requires CheA and CheW (357, 366). This effect isnot receptor specific (324), is transient (169, 170), is depen-dent on intracellular ATP (352), and requires the N-terminalregulatory domain of the CheB protein as well as the C-terminal catalytic domain (205, 367). It is this level of CheBregulation that is thought to be due to phosphorylation ofCheB.CheB is unusual among response regulators in that the

associated C-terminal effector domain is an enzyme ratherthan a transcriptional activator. Although there is no evi-dence that CheB regulates transcription, the C-terminaldomain of CheB is, in fact, a DNA-binding protein (A.Borczuk, and J. Stock, unpublished observation). More-over, this region of CheB has a very good consensussequence for the helix-turn-helix motif common to manytranscriptional regulators.CheW has been purified as a 36-kDa homodimer (373). The

amino acid sequence of CheW suggests a nucleotide-bindingsite, but the sequence fingerprint that defines this site is notconserved within an homologous protein that functions toregulate gliding motility in Myxococcus xanthus (231). cheWmutants behave the same as cheA mutants as if CheW wererequired for CheA kinase activity in vivo. CheW dramati-cally stimulates the rate of CheY phosphorylation in areconstituted system containing CheA and a membranepreparation with high levels of the Tar receptor-transducerprotein (45). In the absence of receptors, the purified CheWprotein has no apparent effect on CheA autophosphoryla-tion, phosphotransfer from P-CheA to CheY or CheB, CheZphosphatase activity, or the inherent stability of P-CheA,P-CheY, or P-CheB (45; Ninfa and Stock, unpublishedobservations). The mechanism by which CheW mediates theeffects of receptors on CheY phosphorylation remains to bedetermined.

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lgiApl g%ALpZ gi sLp

WL-fI %A I L irSCGS

Tr

I1

NRI1 NRII

1.(NRII) PI \ v

2 UMP UTP A

NR-P NRI GIn UR/UT 2KG GS-AMP AT GS-H10 PP1 D

PII-UMPNRIz NR -PI__P

\ATP A P,l

G I u + NH,+ , io- I n

ATP ADP

I - rtranscriptIonaI activatIon of Ntr reguIonFIG. 8. System that regulates nitrogen utilization in E. coli and S. typhimurium. The activity of glutamine synthetase (GS) is regulated by

adenylylation and deadenylylation reactions catalyzed by an adenylyltransferase (AT) that is in turn regulated by uridylylation anddeuridylylation of the PI, protein. PI, uridylylation, catalyzed by a uridylyltransferase (UT) is stimulated by 2-ketoglutarate (2KG). PI,deuridylylation, catalyzed by a uridylyl-removing activity (UR), is stimulated by glutamine. The state of PI, uridylylation also controlsexpression of the glutamine synthetase gene g1nA. P-NRI activates transcription from glnAp2 by binding to sequences upstream of the glnAp2promoter, indicated by filled boxes. NR, is phosphorylated by the NRI kinase. The deuridylylated form of PI,, together with NRII, catalyzesthe dephosphorylation reaction. In the absence of P-NRI, the glnALG operon is transcribed at low constitutive levels from the glnApi andglnLp promoters. P-NRI binds to sites overlapping these promoters to repress these transcripts. At very high levels, P-NR, activatestranscription of the genes in the Ntr regulon. UMP, uridine monophosphate; UTP, uridine triphosphate.

NITROGEN REGULATION

Adaptive Response to Nitrogen Starvation

The preferred nitrogen source for most bacteria is ammo-nia. The assimilation of ammonia present in low concentra-tion is accomplished by the ATP-dependent reaction cata-lyzed by glutamine synthetase: glutamate + NH3glutamine. The glutamine amido group is the direct source ofnitrogen for the biosynthesis of nucleotides, amino sugars,and the amino acids histidine, tryptophan, and asparagine.The remaining organic nitrogen in the cell comes fromglutamate, formed by glutamate synthase from glutamineand 2-ketoglutarate. Since no other pathway for the forma-tion of glutamine other than the glutamine synthetase reac-tion exists (189, 230), ammonia can be considered to be anessential metabolite; if an environmental source of ammoniais lacking, ammonia must be derived by the catabolism of

nitrogenous compounds such as amino acids or urea or fromthe reduction of atmospheric N2. A wide range of nitroge-nous compounds can be used, and various bacteria differ inability to utilize particular compounds. For reviews of nitro-gen metabolism, see references 21, 86, 215-217, 307, and407.When cells are grown under conditions in which ammonia

is limiting, there is a dramatic increase in the transcription ofa number of genes. The Ntr regulon constitutes a subset ofthese nitrogen-regulated genes that are regulated by commonregulators (215, 217, 307). The individual Ntr operons arealso subject to Ntr-independent controls such as inductionby a specific nitrogen source and catabolite repression. Innitrogen-fixing bacteria, the Nif regulon is a subset of the Ntrregulon whose products, nitrogenase and associated pro-teins, facilitate the utilization of N2 as the sole nitrogensource.

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The adaptive response to nitrogen starvation occurs inseveral stages. A central role is played by the glnALGoperon (also known as the ginA ntrBC operon), whichencodes glutamine synthetase (the glnA gene product), thehistidine kinase of the Ntr regulon, NR,, (product of glnLIntrB), and the response regulator of the Ntr system, NRI(product of glnGlntrC) (24). NRI and NR,, regulate thetranscription of glnA and the other Ntr and Nif genes.The glnALG operon contains three distinct promoters

(Fig. 8): two are upstream from the ginA gene, ginApi andglnAp2; and the third is located in the glnA-glnL intergenicregion immediately upstream from the ginL gene (281, 305,408). Under conditions of nitrogen excess, a low rate oftranscription from glnApi produces a small amount of glu-tamine synthetase, and a low rate of transcription from theglnL promoter maintains a low intracellular concentration ofNR,, and NR,. The ginApi and glnL promoters are utilizedby the major form of RNA polymerase, Eu70 (142, 304), andtranscription from glnApl and glnLp is repressed by NRI,which binds to two high-affinity binding sites overlapping the-35 region and transcriptional start site of glnApi and to asingle high-affinity site overlapping the -10 region of glnLp(304, 408). A cyclic adenosine 3',5'-monophosphate receptorprotein (CRP)-binding sequence is located upstream fromthe ginAp] promoter, and transcription from this promoter isactivated by the combination of cyclic AMP and CRP (305).

Starvation for nitrogen results in an increase in the tran-scription of the glnALG operon from the glnAp2 promoter(178, 305), which when fully activated is one of the stron-gest promoters in the cell. This increased transcription ofglnALG results in increased intracellular concentrations ofthe products of that operon: glutamine synthetase, NRII, andNR,. Transcription from glnAp2 requires both NR1 (24, 187,206, 215, 282) and RNA polymerase containing a minorsigma factor, u54, the product of the rpoN gene, also termedglnF and ntrA (76, 110, 111, 142). Full activation of tran-scription from glnAp2 requires only the very low intracellu-lar concentrations of NRI from transcription initiated atglnLp, about 10 molecules per cell (281, 304, 305), and in facthigh intracellular concentrations of NR, prevent full activa-tion of transcription from glnAp2 (25, 63). Activation oftranscription from ginAp2 causes at least a 10-fold increasein NR, despite a rho-independent transcriptional terminationsignal at the 3' end of ginA (304). The burst of transcriptionalactivation at glnAp2 when nitrogen starvation conditions arefirst imposed is therefore moderated by the elevated intra-cellular concentration of NR, that accompanies it.Ntr promoters such as glnAp2 share highly conserved

sequence elements around positions -24 and -12 thatreflect the utilization of these promoters by RNA polymer-ase containing u54 (34, 54, 190, 280, 314). The rpoN gene isnot regulated in response to nitrogen starvation, however,(51), and a number of additional promoters have been shownto contain the J54 consensus sequence (TTGGCACA N4TTGCA), including DctD-dependent DctA promoter inRhizobium spp., certainfla promoters in Caulobacter cres-centus (250), and the xylABC promoter on the TOL plasmidof Pseudomonas putida (85, 152). Thus, although U54 is anessential component for transcription from Ntr promoters, itshould not be considered a nitrogen-specific sigma factor (fora review, see reference 189a).

If ammonia is added to a nitrogen-depleted bacterialculture, transcription from ginAp2 is quickly reduced (305)and active glutamine synthetase is inactivated by adenylyla-tion on tyrosyl residues (2, 64, 112, 116, 189, 363, 364). If, onthe other hand, starvation for nitrogen continues unabated,

the increase in transcription of the glnALG operon is fol-lowed by an increase in the transcription of a number ofother Ntr operons whose products facilitate the catabolismof various alternative nitrogen sources such as amino acidsor urea (215, 407). The expression of some Ntr operons isapparently activated directly by the high intracellular con-centration of NRI that results from prolonged nitrogenstarvation, acting in conjunction with RNA polymerase thatcontains U54 (13, 51, 137, 187, 188, 281, 283). In other cases,the expression of nitrogen-regulated operons is activated byproteins whose expression is controlled by NR,. One suchcase is the Nif regulon, found in Klebsiella pneumoniae andother nitrogen-fixing bacteria, but absent in strains that donot fix nitrogen such as E. coli and S. typhimurium (21). Theregulators of Nif gene expression are encoded by the nifLAoperon. The nifL promoter has a (J54 consensus sequencepreceded by a low-affinity NRI-binding site (442), and tran-scription from this promoter requires U54 and a high concen-tration of NR, (387). The products of the nifLA operon areNifA, which shares homology with NRI in its central domain(Fig. 3) (88) and, like NR,, activates transcription frompromoters that are utilized by the EU54 form of RNApolymerase, and NifL (136), which is a regulator that con-trols NifA activity in response to nitrogen and oxygenavailability. When anaerobic nitrogen starvation conditionsprevail, NifA activates the transcription of nif genes whoseproducts, nitrogenase and associated proteins, permit theutilization of N2 as the nitrogen source (21, 86, 87, 89, 123,238, 239, 279). NR, has very little ability, however, toactivate transcription directly from Nif promoters such asthe nijH promoter (54, 387), which are efficiently activatedby NifA.Another case in which the effect of NR1 on the expression

of Ntr operons is probably indirect is the subset of Ntroperons regulated by the nac locus (29). The prototypicalmembers of this class are the hut operons, whose productsfacilitate the utilization of histidine as the sole nitrogensource. Activation of transcription from hut operons re-quires the high concentrations of NR, that result from theactivation of transcription at glnAp2 (281). In addition,activation of transcription from the hut promoters requiresthe product(s) of the nac locus (29). Mutations in the naclocus that are probably null mutations have no effect on theregulation of the glnALG operon or the nif operons, buteliminate the ability of a subset of Ntr operons that includesthe hut operons to be activated by nitrogen starvation. It isnot known whether the nac product(s) alone is sufficient forthe activation of transcription from hut promoters. In thisregard, it is of interest that Mu dlacamp-generated lacZtranscriptional fusions in the nac locus are regulated bynitrogen availability, and the hutH promoter lacks the U54consensus sequence (R. Bender, personal communication).One explanation for these data is that a product of the naclocus activates hut transcription and the appearance of thisproduct requires a high intracellular concentration of NRI.

In summary, the Ntr-dependent adaptive response tonitrogen starvation involves, first, an increase in the level ofglutamine synthetase to enhance the ability of the cells to uselow concentrations of ammonia and then the induction of Ntroperon products such as histidase and nitrogenase to provideammonia from sources of organic nitrogen or from atmo-spheric N2 (for reviews, see references 217 and 307). Thisprogression of events is controlled by a cascade of transcrip-tional regulation that begins with the activation of NR,.

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Signal Transduction

The signal transduction cascades that regulate transcrip-tion of the glnALG operon and control the enzymatic activ-ity of glutamine synthetase are presented schematically inFig. 8. The balance between nitrogen and carbon metabolismas reflected by the intracellular ratio of glutamine and2-ketoglutarate appears to be the critical signal (363, 364).This ratio controls the activity of a bifunctional enzyme, theginD gene product, that catalyzes the uridylylation anddeuridylylation of tyrosine residues in another regulatoryprotein, PII (26, 56, 64, 112, 116, 363, 364). The opposingenzymatic activities of GlnD(UT/UR) are differentially sen-sitive to the presence of 2-ketoglutarate and glutamine: theuridylyltransferase (UT) activity is stimulated by ketoglut-arate and the uridylyl-removing (UR) activity is stimulatedby glutamine. Thus, when ammonia is plentiful and the ratioof ketoglutarate/glutamine is relatively low, PI, is predomi-nantly in its unmodified form; when ammonia is limiting andthe ratio of ketoglutarate/glutamine is relatively high, PI1 ispredominantly in its uridylylated form (26, 112, 364).The ratio of PI/P11-uridine monophosphate controls the

sensitivity of glutamine synthetase to the effects of allostericligands through its effects on the product of the glnE gene (2,26, 56, 64, 101, 102, 112, 116, 308, 363, 364). GlnE(AT),where AT is adenylyltransferase, is a bifunctional enzymethat catalyzes the adenylylation and deadenylylation oftyrosine residues in glutamine synthetase. Adenylylationinhibits glutamine synthetase activity. Unmodified PI, stim-ulates the adenylylation reaction, and P11-uridine monophos-phate stimulates the deadenylylation reaction (56, 364).Mutants deficient in PI1 retain an ability to control the levelof glutamine synthetase adenylylation in response to nitro-gen availability when the cell is otherwise wild type, butexhibit overadenylylation of glutamine synthetase whenintracellular concentrations of glutamine synthetase are ab-normally low due to defects in GlnD(UT/UR), NR1, or c54(56). From these observations it has been concluded thatGlnE(AT) acts slowly in the absence of PI, and has a naturalpropensity for the adenylylation reaction. The mechanismby which GlnE(AT) responds to nitrogen limitation in theabsence of PI1 is not known.

P11 also serves to control transcription of the glnALGoperon (39, 56, 102, 308). In ginD mutants, Ntr promotersare not activated in response to nitrogen starvation (39, 56,101, 102). Similarly, dominant mutations in PI1 have beenidentified that prevent activation of transcription fromglnAp2 (299). These mutations are thought to result in PI1variants that can no longer be converted to P,,-uridinemonophosphate by the uridylyltransferase activity. Finally,since glutamate synthase is the major glutamine-utilizingenzyme in the cell, mutants defective in this activity accu-mulate relatively high levels of glutamine which preventexpression from glnAp2 in a PI,-dependent manner (283).Mutants lacking P11, on the other hand, have increasedtranscription of the glnALG operon even in the presence ofexcess ammonia (56, 308). Since this level of increasedtranscription is not quite as high as that found under fullyderepressing conditions, it seems likely that additional sig-nals other than the presence or absence of unmodified PI,also affect glnALG transcription. Mutants lacking PI, retainthe ability to fully activate transcription from glnAp2 whenstarved for nitrogen. Thus, unmodified PI, is a negativeregulator of transcription from the nitrogen-regulated glnAp2promoter, and uridylylation inactivates this aspect of PI1function.

Genetic evidence indicates that unmodified PI, exerts itseffect on glnALG transcription by controlling the activity ofNRI, (25, 56, 63, 206, 213, 233). NRI, is not required foractivation of transcription from glnAp2 in vivo, and in factmutations in NR11 have little effect on steady-state levels ofglutamine synthetase expression (25, 56). NRI has a dra-matic effect, however, on the timing of responses to changesin nitrogen availability (306). In wild-type cells, transcriptionfrom glnAp2 rapidly adjusts to changes in nitrogen metabo-lism; in cells lacking NR11, the regulation of this transcriptionis extremely slow. In addition, missense mutations in NR11can produce essentially the same phenotype as null muta-tions in PI1 (63, 283), elevated expression of glnA in thepresence of ammonia, and full expression under conditionsof nitrogen deprivation. Since null mutations in NRI sup-press all of the effects of glnD (UT/UR) and glnB (PI,)mutations (56), it is apparent that the GlnD(UT/UR)/P1lsignal transduction pathway acts to regulate the expressionof Ntr genes entirely through NRII.

Studies of NR,,-deficient strains indicate an NR1I-indepen-dent mechanism for Ntr regulation. In wild-type cells, NRI1can override this alternative system. NR11 is both a positiveand a negative regulator of transcription. Its negative actionis demonstrated in glnD mutants; in that background NR1Imakes the cell Gln- Ntr-. Its positive action is seen inmutants lacking PII; these strains have increased expressionof glnA on nitrogen-rich media only when the cell alsocontains NR,,. Thus, both the positive and negative activi-ties of NRI can dominate the alternative regulatory mecha-nism. In cells that contain NR1,, but are deficient in GlnD(UT/UR) and PI1, a level of ginA expression intermediatebetween fully activated and fully repressed is found regard-less of nitrogen availability (56). It has been suggested that insuch cells NR1I catalyzes the futile interconversion of NR1between its active and inactive states (56).

NRI, is a histidine kinase that regulates transcription fromglnAp2 by controlling the phosphorylation state of the tran-scription factor NRI. In a cell-free transcription systemconsisting of purified components, phosphorylation of NRIby NRI, confers upon NR1 the ability to activate transcrip-tion from glnAp2 (262). The phosphorylation mechanism issimilar to that seen with CheA and CheY; NR,, is autophos-phorylated on a histidine residue and can transfer thisphosphate to NR1 in the absence of nucleotides (264, 431).NRI is phosphorylated within the N-terminal domain that isconserved in all response regulators (164). Partial trypticdigestion of NR1 gives a 12.5-kDa N-terminal fragment thatcan be phosphorylated by NRI1 just as well as the intactprotein (164). P-NR1 is rapidly hydrolyzed under physiolog-ical conditions to NR, and Pi: p112, approximately 4 min (164,264, 431). P-NRI, isolated free of NRI, by batch adsorptiononto heparin-Sepharose followed by washing and elutioninto buffers lacking divalent cation, has been shown to befully competent to activate transcription from glnAp2 (429,431).When PI, is added to reaction mixtures containing P-NRI,

NR,,, ATP, and divalent cation, a rapid dephosphorylationof P-NRI is observed (164, 262). This dephosphorylationcoincides with loss of the ability by the reaction mixture toactivate transcription from ginAp2 (262). The ability of PI, toeffect the dephosphorylation of P-NRI depends on NRI1. PI,does not activate P-NRI dephosphorylation in the presenceof a mutant NR,, protein, NR112302, that confers a constitu-tive phenotype on gInA expression in vivo. Moreover, PIIhas no effect on transcription from ginAp2 in the presence ofthis NR,1 mutant protein (262). Thus, P1l appears to activate

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an ATP-dependent NR,, phosphatase that hydrolyzes P-NRIand thereby prevents the activation of transcription fromglnAp2.

Activation of transcription from glnAp2 in intact cellsrequires NR, whether or not NRI, is present (25, 56); thus, itseems likely that additional histidine kinases can donatephosphoryl groups to NRI. This type of cross-talk has beendemonstrated in vitro with both CheA and EnvZ kinases(145, 264).

Mechanism of Transcriptional Activation

As discussed above, the evidence clearly indicates thatP-NR, activates transcription from the glnAp2 promoter.This raises the question as to whether the high-energyphosphate on P-NRI plays a direct role in transcriptionalactivation or whether it serves to induce an active confor-mation of the NR, protein. Several selection schemes havebeen used to isolate mutations in NR, that result in transcrip-tional activation in intact cells under conditions in whichactivation usually does not occur. For example, NR, mu-tants have been isolated that activate Ntr transcription atlow intracellular NR, concentrations (190), that activateglnAp2 transcription under strongly repressing conditions(429), and that suppress the Gln- Ntr- character of cellslacking GlnD(UT/UR) (429). In each case, the purifiedmutant NR, is able to drive transcription from glnAp2 invitro in the absence of phosphorylation (165, 429). Theactivation of transcription by these mutant NRI proteins isstill significantly less than that obtained with phosphorylatedwild-type NR,, however, and transcriptional activation bythe mutants is still dramatically stimulated by phosphoryla-tion. In one case, the characteristics of the mutant NRIprotein have been examined in detail (429). NR,316 has apoint mutation, Ser-160-Phe, within the conserved centraldomain that it shares with DctD and NifA (Fig. 3). PurifiedP-NR,316 catalyzes the same spontaneous rate of dephos-phorylation as wild-type NR1, and on hydrolysis the en-hanced ability to activate transcription provided by phos-phorylation is immediately lost. Even when it is completelydephosphorylated, however, NR1316 retains an ability toactivate transcription from the glnAp2 promoter. This resultsuggests that the response regulator domain functions as aphosphorylation-activated switch to regulate the activity ofthe central domain of NR1, and, unlike wild-type NR1,NR1316 can adopt the active conformation in the absence ofphosphorylation. Perhaps, by analogy with CheB, phosphor-ylation of NRI results in a conformational change thatunmasks a portion of NR, that activates transcription byinteracting with S4 polymerase holoenzyme. It should benoted, however, that although partial tryptic digestion ofNRI results in the formation of a 41-kDa C-terminal fragmentthat binds to DNA, this fragment does not activate transcrip-tion (V. Weiss, A. Ninfa, and B. Magasanik, unpublishedobservations). Furthermore, constitutive NR, mutants lack-ing the N-terminal domain have not been reported. Thus, thepossibility cannot be excluded that the N-terminal portion ofNR, plays a role in the activation process by interactingdirectly with DNA or polymerase.

In the absence of phosphorylation, pure wild-type NRI,even at very high concentrations, shows no ability to acti-vate transcription from ginAp2 (142, 262). In an S-30 systemmade from a strain lacking NR,,, NRI has been observed toactivate transcription to a small extent in the absence ofNR11 and to a greater extent upon addition of NR,, (165).This result has been advanced as evidence that wild-type

NR, is able to adopt an active conformation in the absence ofphosphorylation. It seems more likely, however, that theS-30 mixtures contained other histidine kinases that couldfunction in place of NRI to generate P-NR,. A very low levelof P-NR, would have been sufficient to account for the levelof transcription observed in these experiments.

E. coli a" RNA polymerase associates with the glnAp2promoter in the absence of other factors (265, 266, 303, 326).When linear templates are used, this interaction is entirelyunproductive (142). When supercoiled templates are used,the association of polymerase with the promoter results in asmall amount of transcription from glnAp2 in the absence ofany additional factors (142). These results suggest thatpolymerase is blocked in the pathway leading to the forma-tion of active transcription complexes at some point after theinitial recognition of the promoter by polymerase in solutionand that this block is alleviated by P-NR, or, to a much lesserextent, by supercoiling.

Association of polymerase with promoter in the absenceof NR1 results in formation of a closed complex, and thepolymerase and DNA readily dissociate when the complex isdiluted or challenged with heparin (261). When the polymer-ase and promoter are incubated together with NR1, NR11,and ATP, stable complexes that are insensitive to heparinchallenge or dilution are obtained. That these complexes canbe formed in the presence of ATP as the sole nucleotide andthe rifampin sensitivity of these complexes suggest that theycorrespond to the open complex (232, 261). These opencomplexes can be isolated by gel filtration chromatography,and upon addition of nucleotides they generate glnAp2transcripts. Finally, in intact cells grown under nitrogenstarvation conditions and subsequently treated with ri-fampin, opening of the DNA strands in the area of the site oftranscription initiation can be observed only when NR, ispresent (326). Similar results have been obtained with S.typhimurium components (295). All of these data are consis-tent with the notion that the role of P-NR, is to stimulate thetransition from a closed to an open transcription complex.There is no evidence as to whether or not P-NR, plays anyadditional role at later stages of the initiation process, forexample, by stimulating the initiation event or by allowingclearance of polymerase from the promoter.

Footprinting experiments indicate that there are two high-affinity NRI-binding sites located at positions -149 to -130and -121 to -100 relative to the site of glnAp2 transcriptinitiation (13, 129). At relatively high concentrations of NR1,three additional sites are detected between the high-affinitysites and the promoter (139, 266). Phosphorylation increasesthe affinity of NR, for DNA, but P-NR, has the same bindingspecificity as the unphosphorylated protein (266). The sitesare filled in the same order in the presence and absence ofphosphorylation (1 + 2, then 3, 4, and 5).

Experiments with intact cells and with purified compo-nents have indicated that the ability of low concentrations ofP-NR, to activate transcription from glnAp2 depends on thepresence on the template of at least one of the two high-affinity NR,-binding sites (265, 266, 306). These sites func-tion in a manner analogous to the enhancer elements ofeucaryotic cells in that they retain their ability to facilitatetranscription when moved to new positions much fartherupstream or downstream from the promoter. In vitro exper-iments indicate that the sites could only enhance transcrip-tion in cis (261).

nif promoters also contain upstream sites that function asenhancers in transcriptional activation (8, 55, 310). Theseupstream sequence elements are probably the site of NifA

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binding. Another potential enhancer has been identifiedupstream from a set of divergent C. crescentusfla promotersthat contain the U54 consensus sequence (251). Thus, en-hancer elements may be a general feature of cr54-requiringpromoters.

Since the effect of the high-affinity sites can be observedon linear templates at great distances upstream or down-stream from the promoter, it seems unlikely that NRI passesan activating signal via a topological alteration through theintervening DNA. It also seems unlikely that a "tracking"mechanism (300) is involved since the sites could be locatedon either side of the promoter. Because activation of tran-scription in the presence of the high-affinity sites requiresvery little NR, even when the sites are removed to positionsquite far from the promoter, an "oozing" mechanism (300)also seems to be excluded. The most likely model seems tobe one involving DNA looping, with P-NR, bound at high-affinity sites, making direct contact with RNA polymeraseand/or promoter DNA. Thus, the NR,-binding sites mayfunction to cause an increase in the local concentration ofP-NRI in the promoter region. This idea is supported by thefinding that the requirement for NRI-binding sites is notobserved at very high concentrations of P-NRI (265, 266).

Activation of transcription by P-NRI only occurs at pro-moters that use EU54 RNA polymerase holoenzyme. P-NR1does not activate Eu70-dependent transcription from the lacpromoter when high-affinity NR,-binding sites are intro-duced upstream (G. L. Ray and B. Magasanik, personalcommunication). Thus, some structure unique to the Cr54-RNA polymerase closed complex appears to be required forNR, activity.

PHOSPHATE REGULATION

Phosphate Uptake

In E. coli there are several proteins within the cell enve-lope that transport phosphate into the cytoplasm. Activetransport across the cytoplasmic membrane is mediated bylow- and high-affinity Pi transport systems (PIT and PST)and several sugar phosphate transport systems (Gpt, Pgt,and Uhp) (31, 53, 92, 162, 193, 254, 319, 388). An anion-specific porin (PhoE) mediates passage across the outermembrane into the periplasm (259, 278, 401), and within theperiplasm phosphatases such as E. coli alkaline phos-phatase, PhoA (402), and binding proteins, PstS and UgpB(331, 390), help deliver usable phosphate to the transportsystems in the cytoplasmic membrane (10, 53, 92, 193, 254,388, 391).The expression of many of the proteins involved in phos-

phate uptake are regulated by phosphates in the cell'senvironment. Pi represses the phoA and phoE genes as wellas the ugp and pst operons (10, 52, 53, 92, 122, 254, 341, 392,402, 403, 424), and sugar phosphates such as glycerol phos-phate and hexose phosphates induce genes that encode theirrespective transport systems, gptP (193) and uhpT (162). Anumber of different histidine kinases and response regulatorsact to control these genes. The PhoR kinase and PhoBregulator modulate the expression of phoA, phoE, the pstoperon, and the phoBR operon itself. This collection ofgenes, all of whose promoters require PhoB, have beentermed the Pho regulon (for a review, see reference 419). Aset of genes has recently been characterized that are thoughtto be the B. subtilis equivalents of phoR and phoB, termedphoR (336) and phoP (335), respectively.

Pho Regulon

The Pho regulon was originally characterized throughstudies of expression of the E. coli alkaline phosphatasegene, phoA (402). Addition of phosphate to wild-type cellsrepresses phoA transcription. Constitutive phoA mutantshave been isolated that map to two different loci, corre-sponding to pst and phoR (48, 403). Further studies haveshown that mutations in any of the five genes of the pstoperon (in order of transcription: pstS, pstC, pstA, pstB,phoU) cause high-level constitutive synthesis of alkalinephosphatase (10). The high-affinity PST system functions todetect high extracellular phosphate and uses this informationto cause the repression ofphoA as well as other genes of thePho regulon, including pst itself.The major histidine kinase of the Pho regulon is PhoR

(Table 1). This has been directly demonstrated with apurified C-terminal fragment of PhoR that contains the entireconserved histidine protein kinase domain (Makino et al., inpress). This PhoR fragment incubated with ATP is autophos-phorylated. The stability of the P-PhoR produced in thereaction is virtually identical to that of P-CheA and P-NRII,indicating that a 3-phosphohistidine is involved (cf. refer-ence 264 and Makino et al., in press).PhoB is the response regulator of the Pho regulon. This

protein is essential forphoA expression, and, in fact, the Phoregulon is defined as the set of genes whose expression isdependent on PhoB. PhoB protein is homologous over itsentire length to the subfamily of response regulators thatincludes OmpR, ArcA, PhoM-ORF2, VirG, PhoP, and TctD(Fig. 3). PhoB has been purified and shown to function as anacceptor for the phosphoryl group in P-PhoR (Makino et al.,in press). P-PhoB appears to have an inherent autophos-phatase activity; the half-time for hydrolysis under physio-logical conditions is approximately 10 min (Makino et al., inpress). PhoB phosphorylation stimulates in vitro transcrip-tion from the phoA promoter (Makino et al., in press).Pho promoters have -10 sequences typical for the major

u70 form of RNA polymerase holoenzyme, and P-PhoB-dependent pstS expression occurs with Eu70 (10, 175, 220,390; Makino et al., in press). The -35 regions of Phopromoters contain consensus sequences, Pho boxes, thatfunction as PhoB-binding sites (219). Phosphorylation ofpurified PhoB has been shown to result in enhanced bindingof PhoB to the Pho box upstream from the pstS promoter(Makino et al., in press).

Signal Transduction Pathways That Regulate PhoB

In the Pho system, the kinase (PhoR) appears to be atypical membrane receptor with an extracellular sensorydomain and an intracellular signaling domain (Fig. 1). Onemight suppose from this that PhoR activity is regulatedeither directly by phosphate in the periplasm or indirectly byan interaction of the receptor with the periplasmic phos-phate-binding protein, PstS. This does not appear to be thecase, however. An analysis of the genetics of Pho regulationindicates that all components of the pst operon are essentialfor Pho repression in high phosphate (10). PST is a typicalbinding-protein-dependent bacterial transport system (12,218, 236, 390); like homologous permeases for maltose,galactose, ribose, arabinose, histidine, oligopeptides, etc., itis composed of a periplasmic binding protein, PstS; a periph-eral membrane protein, PstC; and two integral membraneproteins, PstA and PstB. In addition, the pst operon containsa fifth gene, phoU, that encodes a 21-kDa cytoplasmic

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P(

< po 1yphosphates3)

ezt ernal ms I i eu

(PhoA)_ _~~MM Pi peripl asm

( PstS )

PST

I &

lP

PhoU

: transcrI ptIona I activatIon of Pho regulonFIG. 9. System that regulates phosphate uptake in E. co/i. Extracellular Pi diffuses across the outer membrane through porins, VhoE,

OmpF, and OmpC. Alternatively, phosphoesters in the periplasm are hydrolyzed to Pi through the action of alkaline phosphatase, PhoA. Atlow phosphate concentration, phosphate is transported via a phosphate-binding protein, PstS, through the PST system into the cytoplasm.The PST system in conjunction with PhoU regulates the activity of the transcriptional activator, PhoB, that regulates expression of all operonsin the Pho regulon, including phoE, phoA, pstSCABphoU, and phoBR. The active form of PhoB is P-PhoB, produced through the action ofthe PhoR kinase. We propose, by analogy with the role of P11 in nitrogen regulation, that PhoU, together with PhoR, acts to dephosphorylateP-PhoB.

constituent with no apparent homology to any other knownprotein (10, 389, 391). PhoU is not essential for phosphateuptake, but is required for phosphate repression of the Phoregulon (223). Based on these findings, it seems likely thatPhoU promotes the dephosphorylation of P-PhoB in re-sponse to a signal that reflects the activity of the PST system(Fig. 9). In terms of its effect on PhoR and PhoB activity,PhoU appears to be analogous to PI, in the nitrogen system,acting to regulate a phosphatase activity that is presumablyassociated with PhoR.

In phoR mutants, phoA is constitutively expressed atabout one-third the maximal level observed in phosphate-starved wild-type cells or about one-sixth the level detectedin pst mutants (223, 399, 420, 421, 424). This expression

depends on a gene designated phoM. The sequence of thePhoM product indicates that it is a typical membrane recep-tor-histidine kinase (Fig. 1). Presumably, PhoM can act inplace of PhoR to provide phosphoryl groups for PhoBactivation. phoM is not part of the Pho regulon (420, 424),and the PhoM kinase does not appear to be regulated by PST(424). It appears to be part of a distinct regulatory systemwith sufficient cross-specificity for PhoB so that in PhoRmutants it can function to a limited extent as a phosphodonorin place of PhoR (11, 202, 222, 400, 420, 421, 426).These observations emphasize the fact that the PhoR/PST

system exerts both positive and negative control of PhoB. Inlow-phosphate, PhoR and PhoM probably both function toactivate Pho expression by donating phosphoryl groups to

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PhoB. The pst products appear not to be involved in thisprocess. In high phosphate, PhoR with PST functions torepress the Pho regulon; probably PST activates a PhoR-dependent phosphatase that dephosphorylates P-PhoB. APhoR-dependent phosphatase activity would explain thelack of effect of PhoM on Pho expression at high phosphatein wild-type cells.ph'oM is the third open reading frame (ORF3) within an

operon that encodes three additional proteins: ORF1, ORF2,and ORF4 (11). The predicted sequence of ORF2 indicatesthat it is a phosphorylated response regulator that belongs tothe PhoB-OmpR subfamily (Fig. 3 and 4). Presumably, thePhoM-ORF2 protein accepts phosphoryl groups from PhoMto regulate a separate set of target genes. The predictedORF1 and ORF4 proteins do not appear to be related to anyother proteins of known sequence. By analogy with the Che,Ntr, and Pho regulatory systems, it would seem likely thatthe ORF1 or ORF4 protein or both function like PI,, CheZ,and possibly PhoU to regulate a PhoM-ORF2 dephosphory-lation reaction. By using phoM::lacZ fusions, it has beenshown that the phoM promoter is not regulated by PhoB,PhoR, PST, or levels of phosphate (424).One very interesting aspect of Pho regulation is the

phenomenon of clonal variation. This was first observed in astrain with a mutation at the psst locus, pho463, that causedan unstable constitutive expression ofphoA (423). Cells froma pho463 PhoA+ colony growing at high phosphate onnutrient agar gave mixtures of PhoA+ and PhoA- cloneswhen streaked on the same medium, and each of theseclones gave PhoA+ and PhoA- clones, etc. This type ofclonal variability has also been detected in phoR mutants inthe presence of a mutant variant that maps to the phoMoperon in the region of ORF1 and ORF2 (422, 426). Thefrequency of variation depends on the nutritive conditionsused to grow the cells as well as on other environmentalfactors. The switching process is recA independent, andthere is no evidence that a genetic rearrangement is in-volved.

OSMOREGULATION

Solute Relationships across the EnvelopeE. coli cells are composed of two compartments, peri-

plasm and cytoplasm, delineated by the cell wall-outermembrane and the inner cytoplasmic membrane (70, 260,273, 284). The cytoplasmic membrane is not rigid and cannotsupport an osmotic pressure gradient. This function is sup-plied instead by the cell wall-outer membrane complex.Because this rigid outer structure is impermeable to solutesof molecular weight >-600 (259, 291), proteins and otherpolymers are trapped within the periplasm. Among thesemolecules, polyanions such as the membrane-derived oli-gosaccharides support the osmotic strength of the periplasmthrough their attraction of high concentrations of cationssuch as Na+ and K+; i.e. the osmotic strength of theperiplasm reflects the Donnan equilibrium between the peri-plasm and extracellular medium (172, 384). Since the cyto-plasmic membrane cannot sustain a pressure gradient, theosmotic strength of the periplasm and cytoplasm must re-main equal through movements of water between the twocompartments (for a recent review of osmoregulation inbacteria, see reference 71).The inner membrane contains active transport systems

and receptors that move material in and out of the cytoplasmand detect environmental signals (70). The permeability

properties of the outer membrane are largely determined bychannels formed from porin homotrimers (259). There aretwo major porins in E. coli, OmpF and OmpC. OmpF makesa slightly wider pore than OmpC (128). OmpF and OmpCprovide relatively nonspecific pores that facilitate move-ments of small hydrophilic solutes across the outer mem-brane. There are also specialized porins that function pri-marily to facilitate the diffusion of specific macromolecules;PhoE is an anion-specific porin that facilitates the influx ofpolyphosphates (32), and LamB mediates the influx of mal-todextran polysaccharide chains (200, 201).

Regulation of Porin Expression

E. coli alter the porin composition of their outer mem-branes in response to changing environmental conditions,thereby changing their overall permeability towards solutesin the surrounding medium (for reviews, see references 71and 106). Under certain conditions specialized porins areexpressed, such as PhoE (phosphate starvation) (16, 401)and LamB (growth on maltose) (330). OmpF and OmpC arethe major porins present under most culture conditions. Therelative levels of these porins are regulated in response to anumber of environmental parameters: medium osmolarity(163, 409), temperature (203), carbon source (332), etc. (28).Levels are also affected by the composition of the outermembrane itself, such as alterations in the lipopolysaccha-ride core sugars (203) and the membrane lipid structure (84,153). OmpF and OmpC tend to be regulated in a reciprocalmanner such that the total level of porin protein remainsapproximately constant (61, 203, 328).The regulation of porin expression in relation to osmolar-

ity and temperature has been rationalized in relation to thedifferent environmental niches E. coli occupies in the wild.In the intestinal tract of animals, these cells are exposed torelatively high temperatures and high osmolarity. These areconditions that favor the expression of OmpC in preferenceto OmpF. The smaller pore size of OmpC significantlyreduces the diffusion of larger hydrophobic and negativelycharged molecules (259). Thus, OmpC may offer protectionagainst inhibitory compounds such as the negatively chargedbile salts while still allowing for sufficient diffusion of nutri-ents in the relatively rich environment of the interior of ananimal. In contrast, in the external environment which haslower temperatures, lower osmotic strength, and lowerconcentrations of both toxic substances and nutrients, thelarger channel of OmpF may provide a distinct advantage.The porins also act as receptors for bacteriophage and

colicins, and the genetic loci involved in OmpF and OmpCexpression were originally identified by selection of phage-or colicin-resistant mutants (28, 74, 107). These mutantsdisplayed a variety of phenotypes, lacking OmpF, OmpC, orboth. The mutations mapped to three regions of the chromo-some: ompF at 21 min, ompC at 48 min, and ompB at 74 min(28, 61, 74, 325, 412). Biochemical and genetic studies (143,327, 410, 412) established that ompF and ompC encoded themajor E. coli porins and that the ompB locus encoded apositive activator required for ompF and ompC expression.Using fusions of /acZ to the ompC promoter, Hall andSilhavy (125) demonstrated that oinpB-mediated porin regu-lation occurs at the level of transcription. Fine-structuremapping, complementation studies, and DNA sequenceanalysis have shown that the ompB locus is comprised oftwo genes, designated ompR (outer membrane protein reg-ulation) and envZ (pleiotropic defect affecting expression ofenvelope proteins) (68, 126, 127).

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Roles of EnvZ and OmpR

EnvZ is a 450-amino-acid inner membrane protein (103,197) thought to function as an osmosensor (126). It iscomposed of a 115-amino-acid periplasmic domain and a270-amino-acid cytoplasmic histidine kinase domain (Fig. 1).EnvZ controls the activity of OmpR, the response regulatorof the porin expression system. OmpR is a 239-amino-acidprotein that is homologous over its entire length to the familyof transcriptional regulators that includes PhoB, ArcA,PhoM-ORF2, VirG, PhoP, and TctD (Fig. 3). EnvZ is auto-phosphorylated when incubated with ATP, and from thestability of the product, it is apparent that the modificationinvolves a phosphohistidine linkage (3, 104, 146). Mutationof the histidine in EnvZ that appears to be conserved amongmost histidine kinase proteins, His-243-Val, produces anEnvZ variant that is no longer autophosphorylated (104).The phosphoryl group in EnvZ is readily transferred to

OmpR (3, 104, 144). This phosphorylation event, whichoccurs within the N-terminal region of OmpR, enhances theability of the C-terminal region to bind to sequences up-stream of the ompF and ompC promoters (161, 245, 272,396). Phosphorylation of OmpR also stimulates the ability ofOmpR to activate Ec70-dependent ompF transcription invitro (144). Unlike NRI, in which, for the wild-type protein,phosphorylation plays an essential role in transcriptionalactivation, at sufficiently high concentrations OmpR is ableto activate transcription in the absence of phosphorylation(144, 272). There is a caveat, however, in that the purifiedP-OmpR protein is over 20 times more stable than P-NR,(145), and it is possible that preparations of OmpR that havenot been phosphorylated in vitro contain low levels ofresidual P-OmpR that had been generated in vivo. Residuallevels of OmpR phosphorylation may explain some of thedifferences in DNA binding that have been observed be-tween preparations of OmpR obtained from various differentenvZ mutants (106).There is no indication of a gene that encodes a specific

phosphatase function in osmoregulation analogous to CheZin chemotaxis, PI1 in the nitrogen regulation system, orpossibly PhoU in phosphorus regulation. Studies with puri-fied components indicate that the cytoplasmic domain ofEnvZ, by itself, catalyzes the dephosphorylation of P-OmpR(3, 145). This activity resembles the P-NR, dephosphorylat-ing activity of NR1l in that the reaction requires ATP (cf.references 3, 145, and 164). In the case of EnvZ, it has beenshown that the phosphatase reaction does not require ATPhydrolysis since ADP as well as several nonhydrolyzableATP analogs, such as adenylyl-imidodiphosphate (AMP-PNP) and adenylyl (3,-y-methylene)-diphosphonate (AMP-PCP), can act in place of ATP in the EnvZ-dependentphosphatase reaction (3, 145).The role of OmpR phosphorylation in ompC expression

has not been assessed by in vitro transcription assays, andlittle is known of the effect of environmental signals onOmpR phosphorylation and dephosphorylation. A great dealof information is available from genetic studies, however,concerning the effects of a variety of different envZ andompR mutations on regulation of porin gene expression.Mutant strains with ompR deleted are completely unable toexpress either ompF or ompC (127). This phenotype, termedOmpRi, is also produced by a variety of defects in both theN-terminal phosphorylated regulatory domain and the C-terminal DNA-binding domain. For instance, deletion of theentire N-terminal domain (L. Appleman, J. Slauch, and A.Stock, unpublished observation) or insertion of Ala-Leu-Gly

in place of Val-53 (148) as well as a large deletion within theC terminus (255) or insertion of a Cys in place of Arg-209(106) all produce the OmpRl phenotype.A variety of missense mutations in ompR have been

isolated that show altered patterns of porin expression underdifferent environmental conditions (for a review, see refer-ence 106). Wild-type cells exhibit a continuous spectrumfrom OmpF+/OmpC- at low temperature and osmotic pres-sure to OmpF-/OmpC' at high temperature and osmoticpressure. Many ompR mutants appear to be shifted withinthis spectrum so that they cause either an OmpF+/OmpC-bias, designated the OmpR2 phenotype (127), or an OmpF-/OmpC+ bias, the OmpR3 phenotype (255). OmpR2 mutantstend to have defects within the C-terminal domain thatprobably cause deficiencies in DNA binding, e.g., Arg-220-Cys (198), while OmpR3 mutants, which are much rarer,are caused by mutations within the N-terminal domain, e.g.,Arg-15-Cys (255). Deoxyribonuclease I footprinting studieswith a purified OmpR2 mutant protein (Val-203-Met) showedit to be defective in binding to sites around -50 in the ompFpromoter and -90 in the ompC promoter (245). In a parallelstudy, an OmpR3 mutant protein (Arg-15-Cys) exhibited thesame pattern of binding as wild type.The interaction between OmpR and the ompF and ompC

promoters has been extensively investigated both in vitrowith purified OmpR protein and promoter DNA and in vivoby a variety of genetic analyses. The ompF promoter,located approximately 110 bases upstream from the ompFATG initiation codon, has a conventional Pribnow box at-12 to -7 and a -35 region that has weak homology to theconsensus sequence where +1 corresponds to the firstnucleotide of the transcribed messenger RNA as defined byS1 mapping and primer extension (151, 397). Deletion anal-yses have defined an essential upstream region extending to-90 that is required for expression from the oinpF promoter(151). DNA footprinting studies with purified OmpR proteinhave established multiple sites of OmpR binding between-40 and -95 (161, 245, 272). In addition to this essentialupstream activator binding region, there is some evidencethat sequences between -240 and -1400 may be necessaryfor maximal expression of the ompF promoter under condi-tions of low osmotic strength (276). A similar genetic analy-sis as well as footprinting studies with OmpR indicate thatthe ompC promoter has a structure that is similar, but notidentical, to that of the ompF promoter, with sites for OmpRbinding and activation localized within a region extendingfrom -35 to -95 base pairs 3' of the site of transcriptionalinitiation (161, 214, 245, 246, 272).DNA sequence analysis of the region proximal to the

ompC promoter revealed a gene, micF, that encodes a174-base-pair RNA that is complementary to a region nearthe 5' end of the ompF message, including 44 bases of theuntranslated leader sequence and 28 bases of the codingregion (14a, 244). micF is transcribed in the opposite direc-tion from ompC from a promoter that appears to share someregulatory elements with the ompC promoter. Transcriptionfrom the micF and ompC promoters is similarly affected byOmpRl and OmpR2 mutations as well as by mutations inenvZ that produce an OmpR3-like phenotype (244). It hasbeen shown that high levels of the micF transcript expressedfrom multicopy plasmids repress OmpF synthesis (228, 244).From these data, it was hypothesized that micF functions toturn off ompF when the ompC gene is turned on (i.e., at hightemperatures and osmotic pressure). This conclusion wasdisputed, however, when it was found that ompF repressionat high osmotic strength was unaffected by deletion of the

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micF gene (228). Data have also been obtained that clearlyshow ompF repression at high osmolarity in a strain deletedfor the entire ompC/micF locus (329). Thus, there must be amechanism for repression at the ompF promoter that isindependent of events occurring at the ompC locus. ThisompC-independent repression probably acts in conjunctionwith the repressive effects of the micF transcript to facilitatethe switch from OmpF to OmpC. Recent results indicate thatmicF plays an important role in thermal regulation of ompFexpression (14b).

It seems likely that the osmotically regulated switch fromOmpF to OmpC is due to a change in the level of P-OmpR.To understand the switching mechanism, it is thereforeessential to understand the role of the corresponding histi-dine kinase-phosphatase, EnvZ. A mutant strain totallydeficient in EnvZ was shown to produce very low levels ofOmpF and no detectable OmpC (114). Subsequent studieswith the same strain under slightly different conditionsindicated much more substantial levels of ompF expressionin low-osmotic-strength media and switching towardOmpC+ at high osmotic strength (105, 414). Levels of OmpFwere not dramatically repressed at high osmotic strengthunless OmpR levels were increased by using a multicopyplasmid expression vector. At elevated levels of OmpR,osmoregulation of porin expression in the absence of EnvZwas similar to that seen in wild-type cells. One explanationfor these results is that alternative histidine kinases can actin place of EnvZ to phosphorylate OmpR. The possibility ofcross-talk has been demonstrated in vitro by showing OmpRphosphorylation and transcriptional activation of the ompFpromoter, using either the CheA or NR,, kinases in place ofEnvZ (145). In the few systems that have been studied, it isapparent that phosphorylation is turned on by stress, so it isnot difficult to imagine that one or more cross-reactingkinases might be activated by transition of cells to extremelyhigh-osmotic-strength media.Numerous additional types of env'Z mutants have been

selected with phenotypes essentially spanning the samerange of OmpF+/OmpC- to OmpF-/OmpC+ phenotypesexhibited by OmpR mutants (106). The OmpR2-like EnvZphenotype (OmpF+/OmpC-) corresponds to the phenotypeof an EnvZ-deficient strain and probably results from adecrease in the level of P-OmpR. Thus, envZ null mutationsthat presumably cause lowered levels of OmpR phosphory-lation produce essentially the same phenotype as OmpRmutants with lowered DNA-binding affinity. This result maybe understood in terms of a model for the ompFlompCswitch in which P-OmpR in low intracellular concentrationactivates ompF transcription by binding to high-affinity sitesin the -60 to -100 region of the ompF promoter; higherintracellular concentrations of P-OmpR repress ompF tran-scription by binding to a low-affinity site between -40 and-60 in the ompF promoter region and activate ompC tran-scription by binding to low-affinity sites between -75 to-105 in the ompC promoter region (Fig. 10).EnvZ mutants with an OmpR3-like phenotype have also

been obtained (413, 416). These are rare dominant mutantsthat cause a constitutive OmpF-/OmpC+ phenotype. Itseems likely that they represent an activated form of theEnvZ kinase or a phosphatase-deficient EnvZ protein orboth, so that in these strains levels of P-OmpR are elevatedcompared with the wild type. One of these envZ mutations,envZII, has been sequenced and shown to result from aThr-247-Arg mutation (227). This is in the vicinity of thepredicted phosphohistidine site in EnvZ, His-243 (Fig. 2).The envZI I defect could be corrected by a second-site

mutation in ompR (227), ompR77, that results from a Leu-16-Gln conversion, just four residues from Asp-12, one ofthe two totally conserved aspartates in the OmpR responseregulator domain that are thought to constitute the phos-phoacceptor site (Fig. 4). This set of mutant proteins appearsto provide one of the clearest cases of allele-specific sup-pression between two interacting proteins. The ompR77mutation has no apparent phenotype in the absence ofeni'ZII, and it fails to suppress the OmpR3-like phenotype ofanother envZ allele, envZ160, that results from a mutation,Leu-35-Gln, within the first hydrophobic membrane-span-ning sequence of the EnvZ protein. The envZJ I and envZ160alleles represent the two types of mutation one might expectwould lead to a hyperactive kinase: one, envZII, directlyaffecting the site of interaction between the kinase andresponse regulator; and the other, envZ160, affecting thesensory input domain of the kinase, presumably causing aconformational change that mimics the change induced byhigh osmotic strength.The effects of the several ompR and envZ mutations on

phosphorylation have been directly assessed with purifiedcomponents (5). The primary effect of the Arg-15-CysOmpR3 mutation is to inhibit the EnvZ-mediated P-OmpRdephosphorylation reaction, and the EnvZll protein exhib-ited an analogous defect in phosphatase activity while re-taining greater than wild-type kinase activity. The OmpR77mutant exhibited essentially wild-type activity with wild-type P-EnvZ, but was poorly phosphorylated by P-EnvZll.These results are consistent with the model for osmoregula-tion of porin expression outlined in Fig. 10. They argueagainst an alternative scheme proposed by Forst et al. (104)in which P-OmpR activates ompF and the dephosphorylatedform of OmpR activates ompC. The latter hypothesis isbased on the finding that an EnvZ mutant completely defi-cient in phosphorylation, His-243-Val, causes enhancedOmpC expression when it is overproduced from a plasmidexpression vector. The result is difficult to interpret, because(i) under the conditions used in the experiment there appearsto be considerable ompF expression even in the absence ofEnvZ, suggesting a high degree of cross-talk; and (ii) thephosphatase activity of the mutant EnvZ was never as-sessed.Other genes besides ompF and ompC are regulated by

OmpR, including the tripeptide permease, tppB (115), and anouter membrane protease, opr (60). In the case of thepermease, although expression is greatly reduced in strainslacking OmpR or EnvZ or both, expression is unaffected bydominant OmpC-constitutive envZl1-like mutations or bychanges in osmotic strength. Thus, it seems likely that thetppB promoter is saturated by a level of P-OmpR sufficient toturn on ompF. Interestingly, tppB expression is induced byleucine and anaerobiosis (157), so it seems likely that multi-ple regulatory inputs function to control this locus.Another class of genes are repressed by an EnvZ/OmpR-

dependent mechanism. The repressed genes include phoAand phoE of the Pho regulon, but not the phoBR orpstSCABphoU operon (58, 325). In addition, genes involvedin maltose utilization are repressed because the positive Malregulator, malT, is repressed (416), and genes involved iniron transport are repressed (204). Repression has beenobserved, however, only in the presence of a specific class ofenvZ mutants such as eni'ZII or in wild-type cells in thepresence of the local anesthetic procaine (58). In either case,repression requires OmpR (350). The mechanism of repres-sion is not understood but could simply involve binding ofP-OmpR to very low-affinity sites in the promoter regions of

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estetrnal ilieu

outer membr5ane

cell wal I

peripl ssm

cyt opl asm

OmpR-P OmpR

H t0 P,

IWfxl o mn p ZIMt oF

ompC

ItFIG. 10. System that regulates expression of the porins OmpF and OmpC in E. coli and S. typhimurium outer membranes. The EnvZ

receptor-kinase catalyzes the phosphorylation of the transcriptional regulator OmpR in response to pressure on the cell wall. We propose thatlow levels of P-OmpR activate transcription of ompF by binding to high-affinity sites upstream from the ompF promoter (open box). At higherlevels of P-OmpR, ompF expression is repressed and ompC expression is activated by binding of P-OmpR to low-affinity sites upstream ofboth genes ( ). Expression from the ompC promoter activates transcription of micF RNA. micF can act at the level of translation todecrease synthesis of OmpF.

the regulated genes. Suppressors of the envZII phenotypehave been mapped to rpoA, the gene that encodes theox-subunit of RNA polymerase (113, 229). There may there-fore be a direct interaction between P-OmpR and polymer-ase at the repressed promoters.

OTHER SYSTEMS

SporulationThe process of endospore formation in response to nutri-

ent deprivation by B. subtilis is a paradigm for developmen-tal regulation in procaryotes, and well over 100 genes

affecting this process have been identified (for a review, seereference 199). Sporulation and the development of compe-tence (see below) represent two alternative developmentalpathways available to B. subtilis entering stationary phase.The initiation of sporulation usually occurs only at the onsetof stationary phase when cells are limited for nutrients (for areview, see reference 351). In addition, the initiation ofsporulation may require intercellular interactions via se-creted factors.A number of mutants have been identified that fail to

initiate the sporulation process; among these are spoOA,spoOB, spoOE, spoOF, and spoOH. Each of these genes has

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been cloned and sequenced; spoOA and spoOF encode pro-teins containing the characteristic N-terminal domain of thephosphorylated response regulators (Fig. 3 and 4). spoOHencodes a sigma factor, a"30.The predicted spoOF product is similar to CheY in that it

consists only of the conserved domain. Overproduction ofSpoOF from a multicopy plasmid effectively inhibits sporu-lation (195). Mutants lacking SpoOF fail to initiate sporula-tion. This phenotype is suppressed by secondary mutationsaltering the N-terminal domain of SpoOA. Examples includethe sof-i, surOB20, and coi-J alleles (140, 344; G. Olmedoand P. Youngman, personal communication). These muta-tions have all been localized to SpoOA sequences that wouldbe expected to be adjacent to the acidic phosphoacceptorsite: Asn-12-Lys, Glu-14-Val, and Pro-60-Ser, respectively(Fig. 4 and 5). SpoOA is a DNA-binding protein (150) andprobably functions as a transcriptional regulator (351).SpoOA is also required for the development of competence,whereas SpoOF and several other spoO gene products are notrequired for competence. It seems possible that differentforms of SpoOA (phosphorylated and nonphosphorylated) ordifferent levels of P-SpoOA are required for sporulation andcompetence, and SpoOF has a role in modulating the level ofP-SpoOA. SpoOF could conceivably act by competing for acommon histidine kinase. Nucleotide sequence analysis ofthe spoIIJ gene has revealed that it encodes a member of thehistidine protein kinase family (P. Stragier, personal com-munication). Although the role of this gene is not welldefined, it could serve as a regulator of SpoOA and SpoOF.This idea is supported by the finding that overproduction ofspoIIJ can compensate for the Spo- phenotype caused byoverproduction of SpoOF (351).

Competence

In B. subtilis, the development of competence, the abilityto take up foreign DNA, involves an environmentally in-duced and temporally regulated series of events whosecontrol overlaps with the control of sporulation and theregulation of certain genes that are usually expressed only instationary-phase cultures. At least seven different compe-tence (com) genes have been identified, and their regulationhas been studied by using Tn9171ac operon fusions (6, 124).The expression of these com genes requires the sporulationregulator SpoOA and, to a lesser extent, the spoOH producta30. Other sporulation regulators, including SpoOB, SpoOF,SpoOE, SpoOJ, and SigB, are not required for competence,indicating that SpoOA can act independently of these geneproducts. The development of competence is also affectedby mutations in degS and degU (see below), which regulatethe expression of secreted proteins (131, 183). One compe-tence regulatory gene, comA, has been sequenced; thepredicted product contains the N-terminal domain charac-teristic of the response regulators and a C-terminal domainhomologous to proteins of the UhpA subfamily (D. Dubnau,personal communication).

Secretion of Degradative Enzymes

B. subtilis secretes a number of enzymes, including levan-sucrase, amylases, and proteases. Some of these enzymesare expressed only during vegetative growth, for example,levansucrase, and some are expressed only in stationary-phase cultures, such as the amylases and proteases. Theexpression of secreted proteins is globally controlled by theproducts of at least four genes: degQ, degT, degS, and degU

(23, 184, 253, 340, 394, 446). Regulation is at the level oftranscription (340, 394). Missense mutations in the regula-tory genes either pleiotropically abolish the expression ofsecreted enzymes or greatly increase their expression. Mu-tations of the latter class in degS and degU [formerly calledsacU(Hy) rnutations] also lack flagella, are unable to developcompetence, and sporulate efficiently in the presence ofglucose (23). The degS and degU genes are adjacent, but it isnot known whether they are cotranscribed. Both genes havebeen sequenced, and the deduced products have character-istic histidine kinase and response regulator homologies(131, 183, 393). One of the degU mutations resulting in theSacU(Hy) phenotype alters His-11 to Leu. This is adjacentto the putative site of phosphorylation within the acidic cleftat a position similar to that where mutations in OmpR thatcause an OmpR3 phenotype have been identified. AnotherdegU mutation with similar phenotype results from thealteration of Glu-107 to Lys adjacent to the completelyconserved Lys-108. Two sacU(Hy)-type degS alleles havebeen sequenced: the alterations map in the conserved C-terminal histidine kinase domain between regions I and II(Fig. 1 and 2). The site at which DegU acts to stimulatetranscription is located far upstream from the site of tran-scription initiation in a number of the regulated promoters(22, 130); these far-upstream sites may be analogous to theenhancers of gln and nif promoters.

Alginate Production

In P. aeruginosa, the production of the exopolysaccharidealginate, resulting in mucoidy, is almost exclusively associ-ated with the adventitious infection of cystic fibrosis patients(for a recent review, see reference 82). Infecting cultures areinitially nonmucoid, but develop to a mucoid state soon afterinfection. The critical developmentally regulated gene ap-pears to be algD, which encodes guanine diphosphate-mannose dehydrogenase; the algR gene encodes a positiveregulator of the algD gene. AlgR is a member of the responseregulator family (80). A good candidate for a gene thatencodes a kinase involved in alginate regulation, algQ, hasbeen identified in the vicinity of algR (81). However, sinceunlinked mutations have been isolated that may play aregulatory role (99, 100), the location of the kinase compo-nent as well as the number of regulatory elements remain tobe determined.

Exoprotein Synthesis

In Staphylococclus aurelus and other bacteria, certainproteins are expressed only in stationary-phase cultures. InStaphylococcus aureus, this class of proteins includes aserine protease, a nuclease, a lipase, fibrinolysin, ox-hemo-lysin, ,B-hemolysin, 5-hemolysin, enterotoxin B, and toxicshock syndrome toxin. Most of these proteins are secreted.A number of different mutations that pleiotropically affectthe expression of these proteins have been isolated; agrA isa pleiotropic regulatory gene that was originally identified bya Tn55I insertion that eliminated at-hemolysin expression(224). The agrA gene encodes a protein homologous to theresponse regulators. The agrA gene is apparently expressedfrom a weak constitutive promoter and from a much strongerupstream promoter. Expression from the strong upstreampromoter requires AgrA and is developmentally regulated(292). A second open reading frame, agrORF2, encoding ahistidine kinase homolog has also been identified (R. Nov-ick, personal communication).

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Symbiotic Nitrogen Fixation

Rhizobium meliloti is one of several strains of bacteriacapable of symbiotic association with plants in a specializedroot structure known as the nodule. Within the nodule and inthe free-living state, R. meliloti is capable of producingnitrogenase and thus can carry out the reduction of atmo-spheric dinitrogen to ammonia. As in K. pneumoniae,expression of nitrogenase requires the transcriptional acti-vator NifA and a". Unlike the situation in K. pneumoniae,in which the expression of NifA is controlled by P-NR1 andthus responds to nitrogen starvation, in R. meliloti theexpression of NifA is controlled by FixL and FixJ andresponds to oxygen limitation (73). In addition to the expres-sion of NifA, FixL and FixJ also regulate the expression ofgenes in the FixN cluster which are actively transcribedduring symbiosis.FixL has a histidine kinase domain at its C terminus (Fig.

1 and 2) and contains two putative transmembrane se-quences within the nonconserved N-terminal domain. Pre-sumably, this protein has a membrane location and topolog-ical organization similar to those of EnvZ and most otherhistidine kinases. FixJ contains the characteristic N-terminaldomain of the phosphorylated response regulators (Fig. 3and 4) and probably functions as the activator of nifA andfixN transcription. FixJ shares considerable homologythroughout its entire length with members of the UhpAsubfamily of response regulators (Fig. 3).

Dicarboxylate Transport

The primary sources of carbon for R. meliloti and R.trifolii in the symbiotic state are dicarboxylic acids providedby the plant (316). The uptake of succinate, fumarate, andmalate in the symbiotic and free-living states requires apermease encoded by dctA; the expression of this permeaseis induced by succinate (93). The importance of dicarboxylicacid transport in the symbiotic state is underscored by theobservation that, although detA mutants are able to nodulateplants, the nodules they produce are unable to fix nitrogen(42).The dctA promoter contains a C-4 consensus sequence,

and mutants lacking &54 are Dct- (314). In addition, theexpression of dctA is regulated by the products of dctB anddctD (313). The predicted DctB protein contains a histidinekinase domain at its C terminus and two hydrophobicpotential membrane-spanning segments within its noncon-served N-terminal domain (Fig. 1 and 2). DctD protein isbelieved to be a transcriptional activator required for dctAexpression. The predicted protein has an N-terminal domaincharacteristic of the response regulators (Fig. 3 and 4); thecentral portion of DctD is highly homologous to similarportions of NifA and NRI, which are also transcriptionalactivators that act in concert with Ea54 RNA polymeraseholoenzyme.

Tricarboxylate Transport

S. typhimurilum contains three periplasmic binding-pro-tein-dependent transport systems for the uptake of citrateand other tricarboxylic acids, designated TctI, TctII, andTctIII. The TctI system consists of the products of threestructural genes arranged in an operon, tctCBA. tctCBAexpression requires the product of tctD, a gene upstreamfrom the tctCBA promoter that is transcribed in the oppositedirection from tctCBA. The tc tD sequence predicts a protein

that belongs to the PhoB/OmpR subfamily of responseregulators (435). Studies with gene fusions indicate that tctDtranscription is repressed by glucose through a crplc ya-dependent mechanism, presumably involving a cyclic AMP-CRP-binding site located approximately 100 residues up-stream from the site of transcriptional initiation (435). Thesestudies also indicate that TctD does not regulate its owntranscription.TctD works in trans to activate transcription of the

tctCBA operon. No associated histidine kinase has beenidentified, however. Moreover, despite the fact that the TctItransport system is missing in E. coli, the system can beexpressed in these cells from plasmids containing the Sal-inonella genes (434). This raises the possibility that relativelynonspecific kinases present in both E. coli and S. typhimu-rium may be responsible for the TctD phosphorylationreaction.

Oxygen Regulation

The arcA gene of E. coli (also known as dye, fexA, seg,msp, cpxC, and sfrA) is perhaps the most pleiotropic of allresponse regulator genes. Mutations in arcA result in avariety of phenotypes, including loss of F-plasmid expres-sion, sensitivity to certain dyes, altered levels of some outermembrane proteins, and lack of repression of enzymes ofaerobic metabolism under anaerobic conditions. The latterphenotype has provided the gene designation arc for "aero-bic respiration control" (156). When growing under anaero-bic conditions, cells repress synthesis of many enzymes ofaerobic metabolism, including components of the tricarbox-ylic acid cycle, the glyoxylate shunt, the fatty acid degrada-tive pathway, and the electron transport system. The signalfor this repression is not known, but there is evidenceagainst the direct action of molecular oxygen.ArcA is a 26-kDa cytoplasmic protein that is homologous

over its entire length to OmpR (90). Like OmpR, ArcAappears to act in both a negative and a positive fashion.ArcA functions as a repressor in expression of aerobicenzymes and as an activator in expression of F-plasmidgenes. Parallels with the osmoregulation system suggest theinvolvement of a kinase in the regulation of ArcA activity.There are two candidates for the role of an ArcA-specifickinase: CpxA and ArcB.The cpxA gene is one of three independently transcribed

genes, cpxA, cpxB, and cpxC (arcA), that were originallydefined by mutants exhibiting reduced conjugal DNA donoractivity due to a reduction in the level of F-plasmid tra geneexpression. CpxA is a 52,000-molecular-weight inner mem-brane protein with an N-terminal periplasmic domain and aC-terminal cytoplasmic domain that belongs to the family ofhistidine kinases (7, 428). Little is known of CpxB.The arcB gene has recently been identified as a second

gene involved in the global regulation of enzymes of aerobicmetabolism (154). Like mutations in arcA, mutations in arcBrelieve anaerobic repression of aerobic enzymes. High-levelexpression of arcA from multicopy plasmids can restoreanaerobic repression in arcB mutants. No sequence data areyet available for arcB, but mapping studies indicate that thegene is distinct from arcA and all of the cpx genes. It istempting to speculate that ArcB is a kinase that transducesinformation concerning the redox state of the cell (154). Ifso, the Arc/Cpx system may provide an example of twokinases that transduce distinct signals through a singleresponse regulator.

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Nitrate Reductase

Nitrate reductase activities play two distinct roles inbacterial physiology: nitrogen assimilation proceeding fromnitrate to nitrite and ultimately to ammonia, and oxidativemetabolism with nitrate acting in place of oxygen as anelectron acceptor for reduced metabolic intermediates suchas reduced nicotinamide adenine dinucleotide (for a review,see reference 368). Enzymes serving the latter function maybe coupled to processes that eventually lead to ammonia andnitrogen assimilation; alternatively, the nitrite produced maybe further reduced to gaseous nitrogen oxides (NO or N20)or dinitrogen (N2) and eliminated. Whatever the outcome interms of nitrogen metabolism, the primary function may beviewed as a substitution of nitrate for oxygen in respiratorymetabolism. In keeping with this role, these enzymes aregenerally induced by nitrate and repressed by oxygen.

Nitrate reductase in E. coli is a membrane-associatedrespiratory complex that couples the oxidation of reducedquinones to nitrate reduction (368). This complex is com-posed of at least three protein subunits, ox, 3, and y, encodedby the narGHJI operon (234, 354). The -y subunit (Narn orNarJ or both) appears to be a cytochrome b that acceptselectrons from quinol, while the ax subunit (NarG) containsthe active site for nitrate reduction as well as a binding sitefor a Mo cofactor that is required for the reaction. Theelectron transfer is coupled to proton pumping so that nitratereductase activity may be linked by a chemiosmotic mech-anism to the synthesis of ATP.Two loci have been implicated in the regulation of nitrate

reductase expression. One, the fnr locus, controls theexpression of formate and nitrate reductases as well asseveral other activities associated with anaerobic metabo-lism (368). Fnr is a global transcriptional regulator thatappears to control gene expression in response to the state ofrespiratory metabolism just as CRP controls gene expressionin response to carbohydrate metabolism (339).The second locus involved in nitrate reductase regulation

is located adjacent to the narGHJI operon. Beginning ap-proximately 2 kilobases upstream from the narGHJI pro-moter are two genes that are transcribed in the oppositedirection from narGHJI, narX and narL (271, 370). Althoughthere is a 5-base overlap between the end of narX and thebeginning of narL (271, 370), the two genes can be tran-scribed independently (369). Neither gene appears to beautoregulated, however (369). From the sequence of narXL(271, 370), it is apparent that NarX is a histidine proteinkinase and NarL is a response regulator of the UhpAsubfamily (Fig. 3). The NarX sequence indicates two trans-membrane domains (Fig. 1), suggesting that the protein is amembrane receptor, presumably acting to control the levelof NarL phosphorylation in response to nitrate in the peri-plasm. From genetic evidence, it seems likely that P-NarLinduces nitrate reductase by binding to sequences approxi-mately 200 base pairs upstream from the site of transcrip-tional initiation of the narGHIJ operon (196). P-NarL alsoprobably acts to repress other loci such as that encodingfumarate reductase (155). Thus, the combined effects of Fnrand NarX/NarL may function to establish a heirarchy for theutilization of terminal electron acceptors, with oxygen usedin preference to nitrate and nitrate preferred over fumarate.

Hydrogenase

E. coli contains three hydrogenases, one of which loses itsactivity during electrophoresis on nondenaturing acrylamide

gels and has therefore been designated the "labile" hydrog-enase. Expression of this hydrogenase is dependent on a54and the products of the hyd-17, hydL, and hydHG loci (35,386). The nucleotide sequence of hydHG predicts a histidinekinase, HydH, and a response regulator of the NR, subfam-ily, HydG (386). Presumably, P-HydG acts with Eu54 toactivate transcription of the labile hydrogenase gene(s).Genetic studies suggest that the N-terminal 70 amino acids ofHydG, constituting most of the putative phosphorylateddomain, are not absolutely required for transcriptional acti-vation (386). The environmental and/or cytoplasmic signalsthat regulate labile hydrogenase expression remain to bedetermined.

Phosphoglycerate TransportThe pgt genes of S. typhimuriurm encode a specific trans-

port system for phosphoenolpyruvate, 2-phosphoglycerate,and 3-phosphoglycerate that is induced in the presence ofextracellular phosphoglycerate (322). Four pgt genes havebeen identified. The pgtP gene encodes the transporter, a37,000-molecular-weight membrane protein that has se-quence homology with UhpT and GlpT, the transporters forhexose-6-phosphate and glycerol-3-phosphate (117). Theproducts of pgtA, pgtB, and pgtC regulate the expression ofpgtP.

PgtA is a 46-kDa protein with an N-terminal responseregulator domain. Yu and Hong (449) identified severalpotential translational initiation sites within the pgtA ORF.The downstream initiation site was proposed as the begin-ning of the coding region based on a complementationanalysis of deletion mutants that indicated only a partial lossof activity in mutants containing deletions through the twoupstream sites. It seems likely that the pgtA initiation sitehas been misidentified. Translation from the upstream initi-ation site predicts a protein with an N-terminal domainhomologous to the other response regulators (Fig. 3 and 4),while translation from the downstream initiation site predictsa truncated N-terminal domain lacking 24 residues. TheC-terminal region of PgtA exhibits some sequence similar-ities to corresponding portions of members of the NRIsubfamily of response regulators (Fig. 3). The region up-stream of pgtP (117) contains no obvious EU54 recognitionsite, however. Although PgtA as well as PgtB and PgtC havebeen localized to membrane fractions of sonicated cellextracts (447), the sequence of PgtA contains no stretches ofhydrophobic residues that would provide likely transmem-brane regions. PgtB, a 69-kDa protein with one potentialtransmembrane sequence, has sequence homology to thefamily of histidine kinases. PgtC is a 45-kDa membraneprotein.

Results from genetic analysis of the pgt system (160)provide evidence for the roles ofpgtA, pgtB, and pgtC in theregulation ofpgtP expression. The response regulator (PgtA)is required for expression of the regulated gene (pgtP).Deletion of pgtB or pgtBpgtC results in constitutive expres-sion of the phosphoglycerate transporter, whereas deletionof pgtC results in no expression of the transporter. Thesedata can be understood in terms of a model in which PgtCregulates a PgtB kinase-phosphatase activity in response toextracellular phosphoglycerate, in a manner somewhat anal-ogous to the regulation of NR,, by PI, in the nitrogenregulatory system.

Uptake of Hexose PhosphatesHexose phosphate uptake in E. coli is mediated by a sugar

phosphate-phosphate antiporter (9), the product of uhpT

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(432). Expression of the UhpT transporter is induced byextracellular glucose-6-phosphate (83, 439). Transcription ofuhpT is regulated by the products of three genes, uhpA,uhpB, and uhpC, and is independent of UhpT itself (433).UhpA, a 21-kDa protein, and UhpB, a 59-kDa protein,

belong to the families of response regulators and histidinekinases, respectively. The N-terminal half of UhpB is ex-tremely hydrophobic, with numerous potential transmem-brane regions that suggest a domain embedded within theplane of the membrane bilayer (109). UhpC, a 22-kDamembrane protein, is homologous to the UhpT transporter.The two proteins have similar hydropathy profiles and share33% sequence identity in a 185-amino-acid region corre-sponding to the middle of the 51-kDa UhpT transporter(109).

Genetic analyses have indicated that UhpA functions asan activator of uhpT transcription. Although mutations inany of the four uhp genes results in a transport-deficientphenotype, constitutive expression of transporter activitycan be obtained in the absence of luhpB and uhpC when theresponse regulator, UhpA, is expressed from a multicopyplasmid (432). Under these conditions, UhpB increases luhpTtranscription, consistent with its role in activating UhpA.Based on the UhpC/UhpT homology and the requirement ofUhpC for induction, it seems likely that UhpC functions asthe sensory receptor for external glucose-6-phosphate (109,433).

UvrC-ORF2

The uvr genes of E. coli are required for excision repair ofultraviolet (UV)-damaged DNA (415). Three genes, uvrA,uvrB, and uvrC, encode the endonuclease involved in repair.The recA-lexA-dependent SOS system regulates uiMrA andlivrB expression; the regulation of uv,rC transcription is lessclear.

Recent nucleotide sequence analysis of the region up-stream of iuvrC has revealed two ORFs designated ORF1 andORF2 that produce polypeptides of 28,000 and 24,000 mo-lecular weights (248, 338). The ORF2 protein has an N-terminal domain homologous to those of the response regu-lators and a C-terminal domain that places it within theUhpA/FixJ subfamily. The ORF2 coding region overlaps theiarC structural gene by 4 bases and contains P3, one of thepromoters that contributes to uvrC transcription in vivo.Another uvrC promoter, P2, is located upstream of ORF2and produces a transcript containing both ORF2 and uvrC(248).

Despite the location of ORF2 within the uv,rC region, aninsertion mutation that blocks transcription of ORF2 yields astrain with wild-type UV sensitivity. Thus, it has beenconcluded that ORF2 does not play an essential role in repairof UV-damaged DNA (248). The location of ORF2 within aregion potentially regulated by UV damage, however, in-vites speculation that ORF2 may provide a link between theSOS system and a presently undefined set of genes periph-erally involved in responses to UV damage. To date, nocorresponding histidine kinase has been identified.

Agrobacterium Virulence

Agrobacteriiim turnefaciens induces crown gall disease indicotyledonous plants by integrating a part of a tumor-inducing (Ti) plasmid into the nuclear DNA of the plant(257). The bacteria induce the tumor to synthesize opinesthat are subsequently utilized by the bacteria as sources of

carbon, nitrogen, and energy. The early stages of tumorinduction involve over 20 Oir genes encoded by the Tiplasmid as well as 4 genes located on the bacterial chromo-some. Two of the plasmid-encoded genes, virA and virG, areexpressed in vegetative cells, and both are required forinduction of the other vir genes upon exposure of thebacteria to exudates of wounded plant tissues.VirA and VirG are members of the histidine kinase and

response regulator families. The sequences of two 'irAgenes have been determined from plasmids that confer eitherwide-host-range or limited-host-range specificities (194).Both VirA proteins have molecular weights of approxi-mately 92,000 and exhibit 45% identity over their entirelengths. The VirA proteins each contain two potential trans-membrane sequences, and the proteins have been localizedto the inner membrane. This result, together with the obser-vation that the limited-host-range virA is unable to induce virgene expression in response to inducer molecules that func-tion with the wide-range-host virA gene, supports the hy-pothesis that VirA functions as a receptor that signals thepresence of inducer molecules (194). VirA has a responseregulator domain at its C terminus (Fig. 1); however, thesequence is somewhat less conserved than the correspond-ing domains at the N termini of most other response regula-tors (Fig. 4). VirG, a 29-kDa cytoplasmic protein, homolo-gous over its entire length to members of the PhoB/OmpRsubfamily, provides another response regulator in the virsystem (Fig. 3 and 4).The OirA and OirG genes are themselves targets of regula-

tion. The genes are constitutively transcribed in vegetativecells, induced by plant hosts via an autoregulatory mecha-nism, and induced by low pH or phosphate starvation via achromosomal virulence gene, chvD (362, 438).

Mutations in OirG abolish vir gene induction, while muta-tions in virA have been reported to either decrease (362) orabolish (438) induction. The difference between these re-ports may represent either the sensitivity of the assay for virgene induction or the contribution of cross-talk from otherkinases, a factor that might be extremely dependent onspecific assay conditions.

Salmonella Virulence

S. typhimluriutn is an intracellular pathogen of mice and acause of gastroenteritis in humans. The disease in mice hasbeen used as an experimental model for typhoid fever,caused in humans by S. typhoid. Pathogenicity depends onthe ability of the bacteria to survive within macrophages;genetic evidence indicates that this ability depends in part onthe resistance of the bacterium to macrophage defensinpeptides within phagalolysosomes (97, 98). Avirulent mu-tants that are specifically susceptible to defensins have beenisolated (98, 241). One of the loci involved is phoQP (98,241), which had previously been identified as a regulatorylocus for acid phosphatase, phoN, expression (174). Thepredicted PhoQ protein belongs to the histidine proteinkinase family, and PhoP appears to be its associated re-sponse regulator. Mutations in phoQP define a regulon thatincludes, in addition to phoN, several loci identified byoperon fusions, designated pagA, pagB, and pagC (241). Ofthese loci, only mutations inpagC result in avirulence. Fromresults with pagC::TnphoA fusions, it has been suggestedthat pagC is localized to the cell envelope (241), but the roleof PagC in virulence remains to be determined.From its sequence (241), PhoQ appears to be a typical

membrane receptor, with an N-terminal periplasmic sensory

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domain and a C-terminal histidine kinase signaling domain(Fig. 2). Preliminary evidence suggests that its signalingactivity may be regulated by pH as well as by nutrientdeprivation (241). Since the environment in the phagaloly-sosome is acidic, it is logical that loci required for virulenceare regulated by phoQP. PhoP appears to be a typicalresponse regulator of the PhoB/OmpR subfamily (Fig. 4),and by analogy with PhoB and OmpR, it probably bindsDNA sequences upstream from the phoN and pag promotersto regulate transcription in response to phosphorylation anddephosphorylation signals from PhoQ.

Frizzy

M. xanthus is a gliding bacterium that aggregates andforms fruiting bodies under conditions of nutrient depriva-tion (318). Frizzy mutants, frz, exhibit altered gliding motil-ity and, although capable of aggregating, these mutants failto form fruiting bodies (36, 450). Five genes,frzA,frzB,frzC,frzE, and frzF, have been identified (37). Recent nucleotidesequence analysis has revealed homologies among three ofthe Frz proteins and components of the E. coli and S.typhimurium chemotaxis systems (231).FrzA is a 17-kDa protein with homology to the chemotaxis

signal transduction protein CheW that couples the mem-brane receptors to the histidine kinase, CheA. FrzC, a44-kDa protein, has a 300-amino-acid region at its C terminusthat is homologous to the cytoplasmic domain of the meth-ylated chemotaxis receptors; an approximately 100-amino-acid region at the N terminus of FrzC is homologous to aregion of the B. subtilis sigma factor RpoD. FrzE is an86-kDa protein, homologous to CheA and other members ofthe histidine kinase family (W. McCleary and P. Zusman,personal communication). Like CheA, FrzE contains noextensive hydrophobic sequences and appears to be a cyto-plasmic protein (Fig. 1). The C terminus of FrzE is homol-ogous to CheY and other members of the response regulatorfamily (Fig. 4). Thus, FrzE, like VirA, appears to containboth kinase and regulator domains within a single polypep-tide.

Flagellar Biogenesis

The cell cycle of the dimorphic gram-negative bacteriumC. crescentus is characterized by the sequential formation ofdistinct morphological structures and the appearance of twodifferentiated cell types at division: motile non-replicativeswarmer cells and sessile replicative stalked cells (reviewedin reference 258). Approximately 10% of all cell proteins inthis organism are developmentally regulated and appear atcharacteristic periods of the cell cycle. The best-studied ofthe developmentally regulated systems in Caulobacter isthat which controls flagellar biogenesis. As in enteric bacte-ria, flagellum biosynthesis and motility require the productsof many (over 50) genes, and these genes are organized intoa regulatory cascade in which expression of genes at lowerlevels of the cascade depends on gene products expressed athigher levels of the regulatory hierarchy. In C. crescentus,unlike E. coli, expression of the lowest level of the regula-tory hierarchy, including the two major flagellin genes andthe hook gene, requires Err54 RNA polymerase (250, 251,263). A number of factors are required for the expression ofthis lowest level of the fla regulatory cascade, including theproducts offlbD, flaO, flbO, flaS, flaW, and flbF (258a).The nucleotide sequence offlbD predicts a protein that is

related to the NR, subfamily of response regulators (G.

Ramakrishnan and A. Newton, personal communication).The N-terminal domain of FlbD is unusual among responseregulators in that the highly conserved residues correspond-ing to Asp-13 and Lys-109 in CheY are absent. Nevertheless,the pattern of hydrophobic residues and other conservedfeatures indicate that this N-terminal domain is closelyrelated to that of the other response regulators and probablyhas a similar structure. The protein has an aspartate residueat a position which corresponds to the phosphorylated aspar-tate in CheY, Asp-57. There is no direct evidence that FlbD isphosphorylated, and an associated histidine kinase remains tobe identified. When produced in E. coli from a multicopyplasmid, FlbD partially suppresses the inability of strainslacking NRI to activate transcription from glnAp2 (Rama-krishnan and Newton, personal communication). In suchstrains, ginA expression is nitrogen regulated, which suggeststhat FlbD may be recognized and phosphorylated by NRII.

CROSS-TALK AND GLOBAL REGULATION

It seems likely that, to some degree, any histidine kinasecan act as a phosphodonor for any response regulator. Thishypothesis has been tested with the kinases and regulatorsthat are available as pure proteins in milligram quantities(CheA, NRII, and EnvZ and CheY, CheB, NR,, OmpR,SpoOA, and SpoOF), and in every case some cross-specificityhas been observed (145, 264; E. Ninfa, G. Olmedo, P.Youngman, A. Ninfa, I. Smith, U. Bai, and J. Stock,unpublished observations). Evidence for cross-talk in vivohas consistently been seen in mutants that lack a givenkinase. Such strains typically exhibit low-level constitutiveexpression that appears to depend only on the responseregulator. These effects are generally enhanced by overpro-duction of the regulator from a multicopy expression vector.Thus, although in many cases a specific kinase-regulator pairmay function together as the primary regulators of a partic-ular response (e.g., NR,,/NR,, PhoR/PhoB, and EnvZ/OmpR), each of these systems could potentially be influ-enced by other kinases and regulators. These peripheralregulatory effects could be quite subtle under some circum-stances, but this is not to say they are unimportant.

It should also be noted that there are kinases that do notappear to be coupled to only a single regulator. The best-defined instance is CheA, which is clearly involved in thephosphorylation of both CheY and CheB. SpoOA and SpoOFprobably provide another example, although the kinase thatphosphorylates these proteins has not been established. Aninteresting variation on this theme is provided by the Agro-bacterium Vir system, in which the kinase homolog, VirA,contains a response regulator domain and at the same timeseems to function to regulate the activity of a distinctresponse regulator, VirG. There are also several instances inwhich the kinase-regulator specificity remains to be deter-mined. The preferred substrates of CpxA and SpoIIJ havenot been established, nor is it clear which kinases act tophosphorylate SpoOA, SpoOF, ArcA, TctD, UvrC-ORF2,and AlgR.The features of protein structure that determine the spec-

ificity of a given kinase-response regulator interaction areprobably quite subtle. CheB and CheY accept phosphorylgroups from P-CheA with approximately the same kinetics,yet they have fewer sequence similarities than pairs ofregulators selected at random from the family. Rates ofphosphotransfer from P-CheA to CheY, NRI, and OmpRdiffer by less than a factor of 100. Much larger differences inaffinities between proteins have been observed with single-

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amino-acid substitutions, so there is no reason, in terms ofprotein structure, that the kinases should not have a muchgreater selectivity for their respective regulators. Thus, thecross-talk that can be measured in vitro is either importantenough to have been selected for, or sufficiently unimportantso as not to have been selected against.

In a sense, the histidine kinases and response regulatorswithin a cell may be considered as a phosphotransfer net-work comprised of two layers, the kinase layer and theregulator layer. Each protein within a layer exhibits aspectrum of connectivities with proteins in the opposinglayer. The sign and magnitude of the connection betweenany two components would be expected to depend onnumerous factors, including the concentration of kinase, theautophosphorylation activity of the kinase, the concentra-tion of the response regulator, the rate of phosphotransferbetween the phosphorylated kinase and the response regu-lator, the phosphatase activity of the kinase componenttoward the phosphorylated response regulator, and the ac-tivities of any other phosphatases that catalyze the dephos-phorylation reaction. Competition between kinases for agiven response regulator, or between regulators for a givenkinase, may also enter into the equation. Lateral interactionsbetween components within a layer do not appear to play asignificant role, however. Kinases do not accept phosphorylgroups from other kinases, and regulators do not passphosphoryl groups among themselves (264).

Connectivities between kinases and regulators are modu-lated to produce an appropriate output response to a giveninput stimulus. The input can apparently be simply a ligand-binding domain that is attached to the histidine kinasedomain (as proposed for EnvZ [144]), or it can be a signaltransduction cascade that involves several additional pro-teins (e.g., the Che, Ntr, or Pho system). Similarly, theoutput generally involves numerous additional proteins,such as the flagellar motor components that mediate theeffects of CheY, and the complicated promoter architectureand transcriptional machinery involved in systems that reg-ulate gene expression.

In principle, regulatory inputs could be exerted througheither the kinases or the regulators, through a complex of thetwo proteins together, or from auxiliary signal transductionproteins. For instance, in chemotaxis, CheZ facilitates de-phosphorylation of P-CheY and could thereby contribute tothe control of CheY activity; in nitrogen regulation, the levelof P-NRI is controlled by an interaction among NR,, theNR,, kinase, and Pi,; and in both chemotaxis and nitrogenregulation, a complex signal transduction mechanism func-tions to control the phosphotransfer reactions.The phosphatase activities associated with the kinase

proteins allow connections between phosphotransfer ele-ments to be either positive or negative so that the net flux ofphosphoryl groups can be directed into or out of a particularregulator. Negative effects are particularly relevant withinthe context of a network of phosphotransfer components inwhich a phosphatase acting on a regulator can drain phos-phoryl groups from peripheral kinase proteins. Moreover,activation of a specific phosphatase could function to blockeffectively the effects of nonspecific kinases by rapidlydephosphorylating any small amounts of phosphorylatedregulator they might produce.

In many systems specificity may be determined in part bypositive genetic feedback. The best-understood examplesare the Ntr and Pho systems. When P-NRI activates tran-scription at glnAp2, the resulting increase in the intracellularconcentration of NR, facilitates the phosphorylation of NR,

by additional cellular kinases, maintaining expression of thesystem. It is not difficult to imagine that, under conditions ofnitrogen deprivation in mutants lacking NR11, a differentkinase could become activated to trigger the system.A similar argument applies for the Pho regulon in which

transcription of the phoBR operon appears to be activated byP-PhoB. In PhoR-deficient strains, if cross-talk from PhoMcan produce small amounts of P-PhoB, the positive loop thatleads to increased PhoB expression comes into effect, andthe Pho regulon is turned on. Given the underlying positivefeedback control, it is not difficult to understand how clonalvariation could occur. Once a threshold level of P-PhoB isproduced in a given cell, the cell will switch to a Pho+ statethat could remain on almost indefinitely. If, on the otherhand, the level of P-PhoB were to drop below a criticalvalue, the same feedback mechanism could act negatively tocause the cell to switch back to a Pho- state. This sort ofepigenetic mechanism would help explain why clonal varia-tion switching frequencies depend so heavily on environ-mental conditions and the precise genetic background of thestrains involved.

Individual diversity may play a central role in the gener-ation of a response. This type of epigenetic phenomenonwould be expected to be especially important under condi-tions in which a critical regulatory protein is present at verylow levels within the cell. In wild-type E. coli, the EnvZkinase is present at an average level of only about 10molecules per cell (247). Assuming random partitioningbetween cells, a typical culture with 2 x 109 cells per mlwould be expected to contain between 103 and 106 cells perml with no EnvZ at all and a corresponding number withtwice the normal complement of EnvZ. Thus, individualswithin a population would be expected to show a wide rangeof responses to a given stimulus. If a particular type ofresponse is advantageous, individuals who happen to yieldthat response will be selected within a population. Suchindividual stochastic variation could function in conjunctionwith positive and negative feedback circuits to help cellsevolve more effective response strategies. Within this con-text, relatively rare cross-talk events could provide fortu-itous connections that might allow a few individuals within apopulation to produce a particularly advantageous responseto a given set of environmental signals.Under conditions in which a response regulator is fully

activated or inactivated, relatively small contributions fromcross-talk may have little significance. One would expectcross-talk to have its most profound effects on a givenregulatory system when that system is poised at a threshold.Thus, the role of cross-talk may only be fully appreciated aswe gain a greater understanding of the decision-makingprocesses that underlie transitions between regulatorystates.

ACKNOWLEDGMENTS

We thank the following individuals for helpful discussions and forproviding us with unpublished information: P. Bertics, V. Deretic,D. Dubnau, S. Forst, M. Igo, M. Inouye, D. Koshland, A. Lupas,B. Magasanik, J. Mottonen, A. Newton, E. Ninfa, R. Novick, G.Ramakrishnan, L. Reitzer, S. Roseman, M. Saier, M. Schmid, T.Silhavy, J. Slaugh, I. Smith, P. Stragier, B. Wanner, P. Weglenski,V. Weiss, P. Youngman, and D. Zusman.

LITERATURE CITED1. Adler, J., and W. Epstein. 1974. Phosphotransferase system

enzymes as chemoreceptors for certain sugars in Escherichiaeoli chemotaxis. Proc. Natl. Acad. Sci. USA 71:2895-2899.

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