Signal transduction via the histidyl-aspartyl phospho- relay

REVIEW

Signal transduction via the histidyl-aspartyl phospho-relay

Linda A. Eggera, Heiyoung Parkb and Masayori Inouye*Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is thepredominant mode of signal transduction in bacteria. Adaptation to environmental changesoccurs through a sensor histidine protein kinase and a response regulator. The histidine proteinkinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein.Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation eventsthat control downstream effectors. Following autophosphorylation at a conserved histidineresidue, the histidine kinase serves as a phospho-donor for the response regulator. Oncephosphorylated, the response regulator mediates changes in gene expression or cellularlocomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors externalosmolarity and responds by differentially modulating the expression of the OmpF and OmpCmajor outer membrane porins, will be described as a model system. While histidine kinases werethought to be present only in prokaryotes, they have recently been identified in eukaryoticsystems. Here, we review the unique and conserved features of this growing family of signaltransducers.

Bacterial signal transduction via thehistidyl-aspartyl phosphorelayBacteria thrive by instantaneously responding to rapidlychanging environments. Stressful situations involvingchanges in temperature, osmolarity, pH, nutrients andtoxins are all taken in stride as bacteria quickly managemany types of stress. Currently, it has been establishedthat the His-Asp phosphorelay, formerly described asthe two-component system which originated in 1986,is the predominant mode of signal transduction inbacteria that mediates responses to environmental stress(Inouye 1996; reviewed in Parkinson 1993; Stock et al.1989, 1990). In addition, these phosphorelay reactions

are required for the establishment and maintenance ofinfectious states in host organisms. An exception tothis rule, however, is the identification of serine/threonine kinases in several bacterial species including:Myxococcus xanthus, Anabaena sp. PCC7120, Yersiniapseudotuberculosis, Streptomyces coelicor, and Escherichia coli(reviewed in Cozzone 1993; Zhang 1996). A distin-guishing feature of these bacterial species is their abilityto undergo cell–cell interactions which may correlatewith multicellular eukaryotic organisms. In contrast,while protein tyrosine kinase activity has been reportedin bacteria, no tyrosine kinase genes have beenidentified (reviewed in Cozzone 1993).

In addition to the bacterial sensor histidine kinasesinvolved in the His-Asp phosphorelay, there are twoother classes of bacterial protein kinases: (i) classicalmetabolite regulated, ATP dependent, protein kinases(i.e. nucleoside diphosphate kinase) and (ii) phospho-enolpyruvate:sugar phosphotransferase systems (Saier1993). While these other bacterial protein kinasesinvolve phosphorylation of histidine, they do notinclude phosphotransfer to an aspartate residue on a

q Blackwell Science Limited Genes to Cells (1997) 2, 167–184 167

* Correspondence: E-mail: [email protected] address: aMerck & Co., Inc., Department ofImmunology & Inflammation, PO Box 2000, RY80 W-107,Rahway, NJ 07065; bDepartment of Medicine, Massachu-setts General Hospital, Harvard Medical School, LeukocyteBiology & Inflammation Program, Bldg. 149, 13th Street,Charlestown, MA 02129, USA.

cognate response regulator. The sensor histidine kinasesinvolved in the His-Asp phosphorelay will be the focusof this review.

There are over 100 examples of His-Asp phosphor-elay systems in bacteria, and 17 systems have beencharacterized in E. coli (Fig. 1) (Volz et al. 1993). Whilethe His-Asp phosphorelay system may have originatedas a two member sensor-kinase and response regulatorpathway in bacteria, in some cases the system hasevolved into a multi-component pathway. For example,

at least seven components are involved in chemotaxis, amulti-step phosphorelay is used in sporulation, and theHis-Asp phosphorelay can even reside within a singlehybrid kinase protein. In contrast to the cascade ofprotein phosphorylation and dephosphorylation eventsin eukaryotes using protein tyrosine or serine/threoninekinases and phosphatases, the His-Asp phosphorelaysystems in bacteria efficiently and reversibly regulategene expression or cellular locomotion. The multiplesteps in eukaryotes may have evolved to impart

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168 Genes to Cells (1997) 2, 167–184 q Blackwell Science Limited

Figure 1 Histidyl-aspartyl phosphorelays in Escherichia coli. The adaptive system and signal (if known) are indicated in the left column.In the centre column, the following pairs of E. coli sensor histidine kinases and responseregulators are shown: EnvZ/OmpR (Comeau etal. 1985); CpxA/CpxR (Albin et al. 1986; Dong et al. 1993); PhoR/PhoB (Makino et al. 1986a); PhoQ/PhoP (Kasahara et al. 1992);BaeS/BaeR (Nagasawa et al. 1993), NarX/NarL (Egan & Stewart 1990); NarQ/NarL (Chiang et al. 1992); BasS/BasR (Nagasawa et al.1993), CreC/CreB (Amemura et al. 1986), KdpD/KdpE (Walderhaug et al. 1992); UhpB/UhpA (Friedrich & Kadner 1987; Weston &Kadner 1988); HydH/HydG (Blattner et al. 1993); NtrB/NtrC (Miranda-Rios et al. 1987); EvgS/EvgA (Utsumi et al. 1994); RcsS/RcsB (Stout et al. 1990); ArcB/ArcA (Drury et al. 1985); BarA (Nagasawa et al. 1992; Zhang & Normark 1996); and CheA/CheY(Matsumura et al. 1977, 1984). The sensor histidine kinase has a conserved C-terminal kinase domain of 240 amino acids (red rectangle)which contains: the histidine residue (H) that is autophosphorylated, an asparagine, DXGXG motif, a phenylalanine, and a GXGXGmotif. Transmembrane domains are indicated by black vertical bars. In the response regulator, the conserved N-terminal domain of 120amino acids (green rectangle) contains the conserved aspartate (D) residue which receives the phosphate from the sensor histidine kinase.Hybrid sensor histidine kinases include (ArcB, BarA, RcsC, and EvgS) in which the aspartate (D) containing response regulator typedomain is located downstream from the histidine kinase domain. Additional histidine containing domains are indicated (open rectanglecontaining an H), and nonconserved domains are indicated (white rectangle). In the right column, the affected genes or elicitedresponses are indicated.

specificity, to coordinate the transfer of signals tomultiple cellular compartments, and for extremeamplification of the signal.

Major players and distinguishing features

Bacterial sensor histidine kinases

Bacterial sensor histidine kinases are characterized by aconserved C-terminal catalytic domain of 240 aminoacids. This catalytic domain contains several conservedfeatures: a histidine residue which is the site ofautophosphorylation, an asparagine residue (N), aphenylalanine residue (F), and two glycine-rich motifs(DXGXG and GXGXG) which may be important forATP binding. One exception is CheA, an unorthodoxtransmitter, since the site of autophosphorylation liesupstream of the conserved catalytic domain (Fig. 1).Once autophosphorylated, the histidine kinase serves asthe phospho-donor for its cognate response regulator.While cross talk can occur, phosphotransfer ratesbetween family members occurs at least two orders ofmagnitude slower. In addition, bacterial histidinekinases, unlike eukaryotic kinases, do not readilyphosphorylate exogenous proteins such as histones,casein, protamines, or phosvitin (reviewed in Stock et al.1995; Cozzone 1993).

When comparing the histidine kinase superfamilyto the more common serine/threonine/tyrosine

kinases superfamily in eukaryotes, conserved functionalsimilarities may include: (i) the glycine-rich loopGXGXG which is involved in MgATP binding ineukaryotic protein kinases and (ii) a DXG region wherethe aspartate coordinates a Mg2þ ion while the glycineforms a hydrogen bond with the aspartate. While thefunction of the GXGXG and DXG regions in bacterialsensor histidine kinases may be similar to theireukaryotic counterparts, the distinct location of theseconserved sequences may imply very different structuraland catalytic mechanisms (Fig. 2) (reviewed in Bosse-meyer 1994; Stock et al. 1995). In Ser/Thr/Tyr kinases,the GXGXG sequence is N-terminal to the proteinsubstrate binding subdomain, while in bacterial sensorhistidine kinases this subdomain is C-terminal to theputative protein substrate binding domain. The otherconserved motif, DXG, precedes the GXGXG regionin bacterial histidine kinases within the putative ATPbinding subdomain, but is located in a region associatedwith the substrate binding domain in serine/threonine/tyrosine kinases. In addition, the conserved lysines ineukaryotic kinases are not present in bacterial histidinekinases.

Other major distinctions between kinase super-families are the chemical stability of the phosphorylatedresidue and the reversibility of the phosphotransfer.While phosphoesters and phosphotyrosines are acidstable, phosphoramidates are base stable and acid labile.Thus, standard methods for the detection of cellular

Histidyl–aspartyl phosphorelay


Figure 2 Protein kinase catalytic domain organization and conserved features. Protein kinase catalytic domain organization of theeukaryotic serine/threonine/tyrosine kinase superfamily and the prokaryotic sensor histidine kinase family. The entire catalytic domainof the eukaryotic protein kinases is indicated (red) which contains the substrate binding (red diagonal), ATP binding domain (arrow).Conserved residues (indicated by black vertical bars) in the Ser/Thr/Tyr superfamily include: GXGXG, K, DXG, and the T and Sphosphorylation sites (i.e. in the case of PKA). Conserved residues in the sensor histidine kinase catalytic domain (red) include: H site ofphosphorylation, N, DXGXG, F and GXGXG.

phosphoproteins which involve acid treatment thatreleases phosphoryl groups from phosphoramidates andacyl phosphates would only detect proteins with acid-stable phosphohydroxyamino acids (serine, threonine,tyrosine) (Cortay et al. 1991). This certainly wouldcontribute to the under-representation of eukaryotichistidine and aspartyl phosphoproteins. While theserine/threonine/tyrosine kinase superfamily catalysesan irreversible phosphorylation reaction, the histidinekinase reaction is reversible. The phosphotransferpotential of a phosphoester formed by serine/threoninekinases is several kcal/mol lower than MgATP. Thephosphotransfer potential of phosphotyrosine is closerto MgATP, but the reaction is still thermodynamicallyfavoured to proceed in an irreversible fashion. Incontrast, the phosphotransfer potential of the phos-phoramidate bond formed by histidine kinases is 1–3kcal/mol higher than the MgATP orthophosphatebond. In vivo this reaction is driven by the high ATP/ADP ratio coupled by rapid transfer of the phosphorylgroup to the response regulator.

Bacterial response regulators

Bacterial response regulators are phosphotransferaseswhich contain a conserved N-terminal domain of 120amino acids. Conserved residues include: the aspartateresidue that is phosphorylated, a pair of aspartateresidues preceding this site, a threonine, and a lysineresidue which all contribute to the acidic pocket for thephosphorylation site. Bacterial response regulators canbe subdivided into five families based on sequencehomology in the less conserved C-terminal domain(reviewed in Volz et al. 1993). The five superfamilies arerepresented by:1 CheY (including: RegA, SpoOF, and Xcc1)2 OmpR (including: ArcA, BaeR, BasR, CopR,

CreB, CpxR, KdpE, NisR, PetR, PhoB, PhoP,ResD, SparR, SphR, TctD, ToxR, and VirG),

3 NtrC (including: AlgB, DctD, HydG, and PilR),4 FixJ (including: DegU, EvgA, FimZ, NarL, RcsB,

and UhpA) and5 Unclassified (including: BvgS, CheB, PleD, RcsC,

and VirA).The CheY superfamily of 125 amino acids contains

only a single conserved N-terminal domain while theOmpR superfamily of 240 amino acids and FixJsuperfamily of 220 amino acids also contain a C-terminal DNA binding domain. The NtrC familyincludes proteins of <460 amino acids that contain anATPase domain and a DNA-binding domain down-stream of the conserved N-terminal domain (Fig. 1).

The structure of the conserved N-terminal domainhas been determined for CheY, and the pattern ofconserved residues implies that all response regulatorswill have a similar structure. The N-terminal domainconsists of five a-helices that surround a five-strandedparallel b-sheet in which the conserved aspartate islocated in an acidic pocket surrounded by conservedaspartate, threonine and lysine residues (Stock et al.1989; Volz & Matsumura 1991). Recently, the crystalstructure for another response regulator, NarL, has alsobeen determined. The NarL structure confirms thestructural conservation in the N-terminal domain, andprovides the first structural determination for theC-terminal domain of the FixJ superfamily (Baikalovet al. 1996). Due to the instability of the phosphorylatedform of CheYand NarL, both crystal structures were inthe unphosphorylated state. Thus, the nature of preciseconformational changes which occur upon phosphory-lation remains elusive.

Hybrid histidine kinases

While most bacterial His-Asp phosphorelay systemsinvolve direct transfer from the phosphorylated histi-dine sensor kinase to the conserved aspartyl residue ofthe response regulator, there are many examples of Histo Asp phosphotransfer within the same protein. Hybridhistidine kinases (i.e. ArcB, BarA, EvgS and RcsC in E.coli) contain both a sensor histidine kinase domainlinked to a C-terminal response-regulator domain(Fig. 1). Some hybrid sensor histidine kinases alsocontain a second histidine containing domain locatedC-terminal to the response-regulator type domain. Thissecond histidine domain, however, does not contain theother conserved features of the histidine kinase domain(N, F and glycine-rich motifs).

Bacterial phosphatases

While the phospho-histidine is a high energy moleculewhich can be rapidly dephosphorylated by acidhydrolysis, the stability of the phosphorylated responseregulator is quite variable depending on the system (i.e.10 s for CheY; several hours for OmpR, Stock et al.1990). In the bacterial His-Asp phosphorelay, there are avariety of types of phosphatases. In the EnvZ/OmpRsystem, EnvZ histidine kinase can also act as aphosphatase (Comeau et al. 1985). In the NtrB/NtrCsystem, dephosphorylation is mediated by the com-bined action of histidine kinase, response regulator and aPII accessory protein (Ninfa et al. 1995). There are also

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examples of distinct bacterial phosphatases such asCheZ in E. coli (Blat & Eisenbach 1994) and a family ofindependent phosphatases in the sporulation phos-phorelay in Bacillus subtilus (Hoch 1995). In contrast, ineukaryotes, there are distinct classes of phosphatasesincluding: protein serine phosphatases (PP1, PP2 A,PP2B, PP2C, PP4, PP5), protein tyrosine phosphatases(receptor and non-receptor), and dual specificityphosphatases which ensure the reversibility of thephosphorylation reaction (reviewed in Hunter 1995).Interestingly, eukaryotic protein phosphatases 1, 2A and2C can also act as histidine phosphatases (Kim et al.1993).

Mechanism of signal transduction

Histidine kinase dimerization

While ligand-induced dimerization is a common themefor eukaryotic single transmembrane receptors, this doesnot apply to all bacterial receptors that have atransmembrane domain organization. In most eukary-otic transmembrane receptors (i.e. growth factor,platelet-derived growth factor, epidermal growthfactor), ligand binding induces receptor dimerizationwhich then contributes to signalling. One exception isthe insulin receptor which is in a preformed dimericstate cross-linked by disulphide bonds whichclearly indicates that signalling is not dependent on amonomer to dimer transition (reviewed in Lemmon &Schlessinger 1994; Stock 1996). Similarly in thebacterial chemoreceptor Tar, dimerization is indepen-dent of ligand binding (Milligan & Koshland 1988).Crystallization of the periplasmic domain of Tarchemoreceptor has established that ligand bindingoccurs between two monomers in a preformed dimericstate (Scott et al. 1993).

While Tar is not a histidine kinase, it is the mostthoroughly characterized transmembrane receptor inbacteria. In Tar, it has been demonstrated that slightmovement between a-helices of adjacent transmem-brane domains contributes to the regulation of thesignal transduction pathway. Thus, the importance ofthe transmembrane domains in signalling serves as amodel for other transmembrane receptors with similardomain organization (Pakula & Simon 1992). While itis known that Tar forms a stable complex with CheAsensor histidine kinase and CheW cytoplasmic proteinin a 2:1:2 ratio to mediate signalling (Gegner et al.1992), it has recently been proposed that Tar cantransduce its signal within a single cytoplasmic domain.However, it is unclear under what physiological

conditions this would be applicable (Tatsuno et al.1996; Gardina & Manson 1996). It is important toemphasize that the cytoplasmic domain of Tar has noenzymatic function. Therefore, the signalling state ofthe cytoplasmic domain of Tar, whether monomeric ordimeric, may not be directly applicable to theoligomerization of the cytoplasmic domain of bacterialsensor histidine kinases.

In bacterial sensor histidine kinases despite com-parisons with Tar, the role of the periplasmic andtransmembrane domains in dimerization and signallinghas not been well established. In contrast, it is wellknown that the conserved cytoplasmic histidine kinasedomains dimerize to undergo trans-autophosphory-lation between two monomers (i.e. EnvZ, NtrB andCheA) (Yang & Inouye 1991; Ninfa et al. 1993;Swanson et al. 1993). Thus, it is evident thatdimerization is a prerequisite for signalling by bacterialhistidine kinases.

Phosphotransfer

In the His-Asp phosphorelay, the sensor histidine kinasecan wear several hats including acting as an autokinaseand phosphotransferase which applies to all members,and the less common added role of phosphatase whichapplies to a few members (i.e. EnvZ). The responseregulator can act as phosphotransferase which applies toall response regulators, or also as an autophosphatasewhich applies to a few members (i.e. NtrC and CheZ)due to the extremely short t½ of the phosphorylatedstate. The general mode of phosphorelay involves thefollowing steps:1 autophosphorylation of the sensor histidine kinase at

a conserved histidine,2 the histidine kinase serves as a phospho-donor for the

response regulator which receives the phosphate ontoa conserved aspartate,

3 a phosphorylation induced conformational change ofthe response regulator which allows the regulation ofdownstream effectors (i.e. by mediating changes incellular locomotion in the chemotaxis system ormore commonly by mediating changes in geneexpression by acting as a transcription factor), and

4 down-regulation of the pathway by dephosphory-lation via phosphatases and/or adaptation via methy-lating proteins in the chemotaxis system.The rate of phosphotransfer depends on both the rate

of autophosphorylation and the specific protein–protein interactions between the sensor histidinekinase and the response regulator. The responseregulator has an active role in obtaining the phosphate



from either its kinase partner or alternatively fromsmall-molecule phosphodonors (i.e. acetyl phosphate,imidazole phosphate, carbamoyl phosphate, and phos-phoramidate) (Lukat et al. 1992; McCleary et al. 1993).Small-molecule phosphodonors, however, are muchless palatable because they have such a low affinity forthe response regulator. Following phosphotransfer, thesensor histidine kinase and response regulator dissociate,which has been demonstrated in the case of CheA andCheY (Schuster et al. 1993). The standard free energy ofhydrolysis for acylphosphate is ¹10 to ¹13 kcal/mol,which is higher than the standard free energy ofhydrolysis of any other phosphoryated residue. Thisallows the protein to undergo a large energeticallyfavourable conformational change induced by phos-phorylation and still maintain kinetic control of thephoshorylation and dephosphorylation reactions(reviewed in Stock et al. 1995).

Response regulator activation

In the absence of phosphorylation, the C-terminaldomain of response regulators is not usually active. It hasbeen proposed that the N-terminal domain exerts aninhibitory function, and when phosphorylated theconformation of the C-terminal domain is altered andcapable of activation (i.e. DNA binding). Geneticdeletion or proteolytic cleavage of the conservedN-terminal domain can constitutively activate theC-terminal domain in several response regulatorsincluding: CheB, FixJ, SpoOA, and PhoB (Simms etal. 1985; Kahn & Ditta 1991; Green et al. 1991; Makinoet al. 1994). In other response regulators (i.e. OmpR)removal of the N-terminal domain is not sufficient forin vivo activation (Tsung et al. 1989). Oligomerizationinduced activation has been proposed for severalresponse regulators including: DegU, BvgA, NtrC andOmpR (Podvin & Steinmetz 1992; Scarlato et al. 1990;Porter et al. 1993; Tsuzuki et al. 1994). Phosphorylationsite mutations generally block response regulatorfunction. This has been demonstrated in severalresponse regulators including: CheY, CheB, PhoB,VirG, and OmpR. Second-site mutations, however, canoften restore activity in the absence of phosphorylation(reviewed in Parkinson & Kofoid 1992). The preciseconformational change that occurs during phosphory-lation in response regulators is unclear.

Osmoregulation in E. coli via theEnvZ-OmpR His-Asp phosphorelayThe EnvZ-OmpR His-Asp phosphorelay in E. coli is a

model system to study signal transduction and generegulation. EnvZ and OmpR respond to osmoticchanges and regulate the expression of the OmpF andOmpC major outer membrane porins. Osmoregulationin E. coli is essential for survival in rapidly changingenvironments such as fresh water with low soluteconcentration as well as in the gut of an organism withhigh solute concentration (reviewed in Csonka &Epstein 1995; Burg et al. 1996). Enteric bacteria,E. coli and S. typhimurium, respond to osmotic stress in amanner similar to many higher organisms. Whenbacterial cells experience an increase in mediumosmolarity, their first response is to rapidly increaseintracellular potassium levels to restore negative turgorand counter osmotic stress (Sutherland et al. 1986). Inresponse to increased osmotic stress, secondaryresponses are activated to begin accumulating othercompatible solutes including: glycine betaine, taurine(an amino acid present in bile), trehalose (a disac-charide), and choline (a precursor of glycine betaine)which do not interfere with intracellular metabolism(Higgins et al. 1987). At this time, potassium effluxsystems are also activated. Within the first few minutesafter an osmotic shock, the OmpF and OmpC porincomposition in the outer membrane is dramaticallyaltered via activation of the EnvZ-OmpR His-Aspphosphorelay (van Alphen & Lugtenberg 1977).

While outer membranes in E. coli are rigid providingresistance to host defence factors, the inner membranewhich separates the periplasm from cytoplasm is flexibleand cannot support osmotic pressure. To ensure that theperiplasm and cytoplasm remain iso-osmotic, theexpression of the major outer membrane porins,OmpF and OmpC, is differentially regulated. OmpFand OmpC are present at 105 molecules per cell andexist as trimers that form nonspecific diffusion channelsto allow the reversible passage of small hydrophilicmolecules < 650 Da (Nikaido & Vaara 1985). Inresponse to low medium osmolarity, OmpF levelsincrease while OmpC levels decrease and reciprocalregulation is seen at high osmolarity. Despite 65%similarity at the amino acid level and in function, theompF and ompC porin promoter regions and hencetranscriptional regulation is very different for each porin(Hutsul & Worobec 1994). The diffusion rates for eachporin type are also distinct. OmpF has a slightly largerpore diameter (1.12 nm) than OmpC (1.08 nm) whichresults in a 10-fold faster diffusion rate that may providea selective advantage at low osmolarity to rapidlyscavenge scarce nutrients (Nikaido & Vaara 1987).

The regulation of the major porins in E. coli, OmpFand OmpC, is regulated by the EnvZ-OmpR phos-

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phorelay (reviewed in Forst & Roberts 1994; Pratt &Silhavy 1995b). envZ and ompR are transcribed from theompB locus (Verhoef et al. 1977). OmpR is a cyto-plasmic transcription factor and EnvZ is an innermembrane histidine kinase. The basic elements of thesignal transduction pathway which mediates the responseto osmolarity changes involves the following steps:1 activation of the inner membrane sensor histidine

kinase EnvZ,2 autophosphorylation of EnvZ at His243,3 phosphate transfer to OmpR at Asp 55,4 binding of OmpR-P to upstream sites on the ompF

and ompC porin promoters to differentially modulatetheir transcription, and

5 dephosphorylation of OmpR-P via the phosphatasefunction of EnvZ. (Fig. 3).

EnvZ osmosensor histidine kinaseEnvZ is an inner membrane spanning protein of 450amino acids consisting of an N-terminal cytoplasmic tail(residues 1–15), two transmembrane domains (16–47and 163–179), a periplasmic domain (48–162), and acytoplasmic C-terminal domain (Forst et al. 1987).Conserved amino acid residues that EnvZ shares withother members of the histidine protein kinase familyinclude: H243 which is the autophosphorylation



Figure 3 The EnvZ-OmpR phosphorelay. The major components involved in osmoregulation in E. coli by the His-Asp phosphorelayare shown. The OmpF (yellow) and OmpC (orange) porins exist as trimers which are located in the outer membrane (grey bar). EnvZ isan inner membrane histidine kinase (catalytic domain in red), and OmpR (N-terminal activation domain in green, C-terminal DNA-binding domain in blue) is located in the cytoplasm of the cell. In response to an osmotic signal, EnvZ is activated, autophosphorylated atH243 and serves as a phospho-donor for OmpR (Roberts et al. 1993). After OmpR is phosphorylated at D55, there is a conformationalchange which allows DNA binding of OmpR-P as dimers to the OmpR-P binding sites located upstream of the porin genes, ompF andompC. Phosphorylation and dephosphorylation of OmpR is regulated by the EnvZ kinase and phosphatase activities, respectively. Atlow levels of osmolarity, there are lower levels of phospho-OmpR (OmpR-P) resulting in transcriptional activation of ompF. In contrast,at high osmolarity, there are higher levels of OmpR-P resulting in repression of ompF and activation of ompC. In addition, at highosmolarity and high temperatures, micF antisense RNA is transcribed and micF antisense RNA binds to a complementary region ofompF mRNA to block its translation.

site, N347 (Dutta & Inouye 1996), DXGXG motif(373–377), F387, and GXGXG motif (401–405).

Genetic analysis

EnvZ null strains indicate that while ompC can still beweakly regulated presumably through cross-talk, EnvZis essential for the repression of ompF at high osmolarity(Forst et al. 1988). Mutational analysis of EnvZ hasdetermined that the most critical domains for receptorsignalling are centered at the linker region (beginningafter the second transmembrane domain and endingbefore the autophosphorylation region) and the autop-hosphorylation-site region. Most mutations that clusternear or at the site of phosphorylation result in a lockedhigh osmolarity phenotype. Mutations and deletions ofthe linker region exhibit various low and highosmolarity locked phenotypes and provide evidencethat this region is critical for signalling (Park & Inouye1997). Periplasmic deletions result in a locked highosmolarity phenotype or have no effect (reviewed inForst & Roberts 1994; Leonardo & Forst 1996). AnN-terminal truncation of 38 amino acids that preventsproper localization of EnvZ, retains its ability to beautophosphorylated, but cannot respond to osmolaritychanges. This implies that the stimulus is derived fromthe periplasm or the membrane rather that thecytoplasm (Igo & Silhavy 1988). Overexpression ofthe periplasmic domain in fusion with the maltosebinding protein, however, did not compete for anosmotic signal in the periplasmic compartment. Thisresult implies that the activation signal is in excess orthat additional domains (i.e. transmembrane) arerequired for titrating the activation signal (Egger &Inouye 1997a). Transmembrane mutations result invarious phenotypes which indicates their importance intransmembrane signalling. Mutations of the conservedasparagine residue N347 result in a locked lowosmolarity phenotype in which the kinase activity isdefective but the phosphatase activity is still intact (Yang& Inouye 1993; Dutta & Inouye 1996).

Activation signals

While it is clear that EnvZ regulates the phosphory-lation state of OmpR, the primary osmotic signal ornatural ligand for EnvZ has not been identified.Interestingly, of the 17 His-Asp phosphorelays thathave been characterized in E. coli, only six systems havea defined activation signal (Fig. 1). The EnvZ-OmpRpair of regulators respond to a variety of environmentalstresses. The high osmolarity response can be induced

by: increased medium osmolarity, acidic pH, increasedtemperature, or alteration of the chemical compositionof the medium (i.e. procaine) (van Alphen & Lugten-burg 1977). Previously proposed activation signals suchas membrane-derived oligosaccharides and DNAsupercoiling have been shown not to be directlyinvolved in osmoregulation (reviewed in Pratt et al.1996). While it is possible that a distinct ligand mayexist, it is more likely that EnvZ is activated by amechanical signal which may result in conformationalchanges or receptor dimerization. Alternatively, EnvZmay respond to a collective property of the periplasmiccompartment or cytoplasmic environment (i.e. macro-molecular crowding or changes in water reactivity)(reviewed in Wiggins 1990).

In the absence of a natural ligand for EnvZ, theactivation pathway has been effectively probed bygenerating chimeric receptors. Taz1, a chimericreceptor containing N-terminal Tar chemoreceptor1–256 and C-terminal EnvZ 223–450 can be activatedby aspartate which is the natural ligand for Tar (Utsumiet al. 1989). Tar and EnvZ share a common domainorganization consisting of two transmembrane domains,a periplasmic domain and a cytoplasmic domain. UsingTaz1, it has been shown that ligand binding to thereceptor domain regulates the ratio of kinase tophosphatase activities of the EnvZ signalling domainby primarily regulating the phosphatase activity (Jin &Inouye 1993). The signalling pathway of EnvZ has alsobeen investigated by constructing a similar hybridprotein containing the N-terminal portion of Trgchemoreceptor, and the C-terminal domain of EnvZ(Baumgartner et al. 1994). This hybrid was capable ofrecognizing the sugar-occupied ribose-binding proteinby its periplasmic domain and activating an ompC-lacZfusion via its C-terminal signalling domain. These hybridproteins provide evidence of physical and functionalreceptor coupling between heterologous domains thatcontain minimal sequence identity. This implies that thechemosensor and osmosensor share a common signaltransducing mechanism across the membrane.

Activation domain

While genetic evidence implies that the periplasmicdomain may contribute to signal detection, there is nodirect evidence that this domain senses the primaryactivation signal. Recently, an envZ homologue of 342amino acids has been identified in Xenorhabdusnematophilus insect pathogenic bacteria. This homo-logue shares 50% identity with the C-terminal domainof E. coli EnvZ, but does not contain a periplasmic

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domain. Instead, the N-terminal 40–50 amino acids arehighly hydrophobic. Interestingly, X. nematophilus envZcan complement an E. coli DenvZ strain to restore porinprofiles and the response to osmolarity (Tabatabai &Forst 1995). The major porins in X. nematophilus,however, do not respond to osmolarity (Forst et al.1995). While this complementation approach providesevidence that the C-terminal domain of EnvZ issufficient for porin regulation, one cannot excludethat the periplasmic domain of EnvZ has an importantregulatory function. The periplasmic domain in E. colimay sense a specific signal other than an osmotic signal,and may have a role in oligomerization, and henceactivation of EnvZ. Alternatively, although unlikely, theperiplasmic domain may only act to provide properspacing between the transmembrane domains. Replace-ment of portions of the periplasmic domain with a non-homologous domain of the PhoR histidine kinase ofBacillus subtilus has also been used to demonstrate thatthe periplasmic domain of EnvZ is not essential forsensing osmolarity signals.

Catalytic domain

Using truncated or soluble forms of EnvZ-C it has beenestablished that the conserved C-terminal domain is thesignalling domain which is autophosphorylated by ATPand serves as a phospho-donor for OmpR (reviewed inForst et al. 1994). Thus, the autokinase, phosphotrans-ferase and phosphatase activities are conferred by theconserved C-terminal histidine kinase domain. While ithas been established that the C-terminal domainsdimerize during the autophosphorylation reaction, therole of the periplasmic and transmembrane domains indimerization have not been established (Roberts et al.1993). It has also been speculated that dimerizationcontrols the kinase and phosphatase activities of EnvZ.Complementation of an autophosphorylation mutant(kinase¹phosphatase¹) with a DXGXG domain mutant(kinase¹phosphataseþ) completely restores all enzy-matic activities (Yang & Inouye 1993). In contrast, twoEnvZ phosphatase mutants cannot complement eachother which implies that the kinase and phosphataseactivities may be regulated as a dimer to monomertransition, respectively. The Ka for EnvZ dimerizationhas been estimated at 105

M¹1 (Hidaka et al. 1997).

OmpR response regulatorOmpR is a cytoplasmic phosphotransferase of 239amino acids which receives the phosphate fromEnvZHis243 onto a conserved aspartate residue,

Asp55 (Kanamaru et al. 1990). OmpR is characterizedby a conserved N-terminal activation domain (1–120),a flexible Q linker domain conserved in bacterialresponse regulators (121–136), and a C-terminal DNA-binding domain (137–239). OmpR-phosphate(OmpR-P) can thus serve as both a negative andpositive transcription factor for ompF, and as a positiveregulator for ompC (reviewed in Forst & Roberts 1994).It has been established that the level of phosphorylatedOmpR increases as the medium osmolarity increases.

Genetic analysis

Numerous OmpR mutants that have previously beenidentified can be broadly classified as activation or DNAbinding mutants (reviewed in Pratt & Silhavy 1995b).Activation mutants are derived from changes of aminoacids in either the N or C-terminal domain of OmpR,resulting in a null phenotype OmpF¹OmpC¹. DNAbinding mutants, derived from changes in amino acidsin the C-terminal domain of OmpR, either fail to bindand activate the porin regulon or inefficiently bind andfail to completely repress the ompF porin at highosmolarity.

Activation domain

As in other bacterial response regulators. the conservedN-terminal domain contains the aspartate residuewhich receives the phosphoryl group from its upstreamhistidine kinase partner. Phosphorylation of OmpR atAsp55 in the N-terminal activation domain results in aconformational change that greatly enhances its bindingaffinity for porin promoter sites which have been wellcharacterized (reviewed in Forst & Roberts 1994;Harlocker et al. 1995).

DNA-binding domain

Previously, it had been reported that the C-terminaldomain of OmpR or members of its subclass (PhoB,VirG, ToxR, ArcA and CpxR) does not contain anypreviously known DNA binding motifs. More recently,however, following crystallization of histone H5, aeukaryotic DNA binding protein with a helix-turn-helix (HTH) motif, the classification of OmpR haschanged based on its sequence identity and putativestructural similarity (Ramakrishnan et al. 1993; Suzuki& Brenner 1995). In addition, homology modelling hasbeen used to further support the notion that OmpR is aHTH DNA binding protein (Egger & Inouye 1997b).Preliminary crystallization of the C-terminal domain of



OmpR has been reported (Kondo et al. 1994; Martinez-Hackert et al. 1996), and the crystal structure of theDNA binding domain of OmpR has recently beensolved to confirm that OmpR is in fact a HTH DNAbinding protein (Martinez-Hackert & Stock 1997).

OmpR-P binding sites

In vitro and in vivo footprinting has identified OmpRbinding sites in the ompF (¹380 to ¹360 and ¹100 to¹40) and ompC promoter regions (¹100 to ¹40)(Rampersaud et al. 1994; Ikenaka et al. 1988; Maeda& Mizuno 1988; Huang & Igo 1996). While the

OmpR-P binding sites have been previously describedas 10-bp F or C boxes (reviewed in Pratt & Silhavy1995a), this terminology has recently been replaced bythe identification of 20-bp binding sites which are eachbound as direct 10-mer repeats by two molecules ofOmpR-P (Fig. 4) (Harlocker et al. 1995; Huang & Igo1996). There is no well defined consensus motif forOmpR-P, but by alignment, the sequencetTtaCTTTtTG-aACAT-tt represents the existing 20bp consensus (lowercase letters represent 57% identity,uppercase letters represent at least 71% identity). Amore refined consensus had been determined for the F1binding site by using a random selection and PCR

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Figure 4 Differential regulation of ompF and ompC by OmpR-P. The level of phosphorylated OmpR fluctuates according to the levelof medium osmolarity. At low osmolarity, there are lower levels of OmpR-P (blue) which bind primarily to the ompF upstreampromoter region (F1 and F2 binding sites) resulting in transcriptional activation. At high osmolarity, there is an increased level ofOmpR-P which now binds to all of the ompF promoter sites resulting in transcriptional repression, and binds to the ompC promoter sitesresulting in activation. The proposed ompF promoter repression loop that forms at high osmolarity is indicated. OmpR-P binding siteshave been determined by in vitro and in vivo footprinting (Rampersaud et al. 1994; Ikenaka et al. 1988; Maeda & Mizuno 1988; Huang &Igo 1996). The conserved cytidine residues located at 10-bp intervals which are critical for OmpR-P binding are indicated by a closedcircle above each residue. The 20-bp consensus for all seven OmpR-P binding sites is indicated. Residues conserved in four out of sevensites are indicated in lowercase, and residues conserved in >five out of seven sites are indicated in uppercase. The 20-bp binding siteswhich are bound by two molecules of OmpR-P as direct repeats (Harlocker et al. 1995) are indicated by open rectangles containing thedesignated binding sites (F1, F2, F3, F4) or (C1, C2, C3). Intervening sequences between F2 and F3 (¹60 to ¹59) and between C1 andC2 (¹78) are indicated by a black bar. The transcriptional complex that forms between a OmpR-P and the a-CTD of RNAP isindicated. The RNA polymerase (RNAP) holoenzyme consists of two b subunits, two a subunits and a j 70 subunit. The a subunitsform a dimer and contain an N-terminal domain and a C-terminal domain (CTD) which is connected by a flexible linker domain. Theb subunit represents the catalytic site of RNA polymerization and the b 0 subunit binds in a nonspecific fashion to the DNA.

amplification method to determine that TTA-CATNTN represent the critical base pairs within thelast 9-bp of the F1 site (Harlocker et al. 1995).

Within the 20-bp consensus sequence, the import-ance of the cytidine-guanosine residues located atpositions 5 and 15 has been demonstrated by mutationalanalysis of the ompF and ompC promoter regions(Harlocker et al. 1995; Rampersaud et al. 1989; Pratt& Silhavy 1995a). Interestingly, the cytidine residuesand their spacing is conserved in all of the OmpR-Pbinding sites at the ompF promoter and 4 of the 6 halfsites at the ompC promoter (Fig. 4). The ompCpromoter has also been investigated by random PCRmutagenesis to determine that the central AC residueslocated at positions 4, 5 and 14, 15 within a 20-bp siteare the most critical nucleotides for OmpR-P binding(Pratt & Silhavy 1995a). In addition to the 20-bpbinding sites, there is a 2-bp intervening sequencelocated at ¹60 to ¹59 between F2 and F3 in the ompFpromoter region, and a 1-bp intervening sequence at¹78 located between C1 and C2 at the ompC promoterregion. It has been proposed that the 2-bp interveningsequence located between F2 and F3 may play a role inthe formation of a proposed repression loop, or maycompensate for the spacing between sites in the DNAhelix where 10.5 bp represents one full helical turn(Russo & Silhavy 1991).

Differential regulation of ompF and ompC byOmpR-P

Regulation of porin gene expression involves bindingof OmpR-P to upstream porin promoter sites as well asan interaction between OmpR-P and the a C-terminaldomain (CTD) of RNA polymerase (RNAP) (Fig. 4)(Bowrin et al. 1994; reviewed in Pratt & Silhavy 1994).The ¹35 and ¹10 regions of the ompF and ompC geneshave low identity (50%) with the consensus sequencewhich necessitates binding of OmpR-P, a j70 typeDNA binding protein, for transcriptional regulation(Mizuno et al. 1983a,b). It is the level of phospho-OmpR that determines which porin will be preferen-tially expressed. At low osmolarity, there are low levelsof phospho-OmpR resulting in activation of ompF,through binding to high affinity sites in the promoterregion. At high osmolarity there are higher levels ofphospho-OmpR resulting in repression of ompFthrough binding to both low and high affinity sitesresulting in the formation of a proposed repression loop,and activation of ompC through binding of phospho-OmpR. The affinity of OmpR-P binding to these sitescan be correlated with the consensus motifs found in

the promoter regions (Fig. 4). While the regulation ofthe ompC promoter is relatively straightforward (bind-ing of OmpR-P at high osmolarity results in activation),the regulation of the ompF promoter is more complex.ompF promoter region mutations and OmpR mutantscorrelate binding of OmpR-P at F1 and F2 withactivation and binding at F3 and F4 with repression, butthe individual or cooperative function of each site inactivation or repression of ompF is unclear (Mizuno et al.1988; Tsung et al. 1989).

Other factors which affect ompF regulation

In addition to the transcriptional regulation byOmpR-P, the ompF promoter region is also regulatedby the presence of four sets of T-rich tracts separated byone turn of the helix that contribute to the intrinsicbending of the promoter region and repression of ompFat high osmolarity (Mizuno 1987). In contrast, theompC promoter region is not intrinsically bent. Bindingof integration host factor (IHF), a non essential histone-like protein that binds and sharply bends DNA by 1308,to consensus sites in the ompF promoter region (¹177,¹68) may also contribute to the negative regulation ofompF. While IHF¹ strains are OmpFc, they also have apleiotropic phenotype which makes it difficult to assessthe direct contribution of IHF to the regulation of ompFtranscription (Tsui et al. 1988; Slauch & Silhavy 1991;Huang et al. 1994). In addition, the lack of ompFrepression in the IHF- strain was most striking atmoderate shifts in osmolarity which indicates that athigh osmolarity, IHF may not be essential for mediatingrepression.

Features which contribute to the translationalregulation of ompF include micF antisense RNA, thepresence of a downstream box (DB) (Sprenghart et al.1996), and indirect regulation of translation by otherprotein factors. A putative downstream box (DB),located downstream from the Shine–Dalgarnosequence of ompF, may interact with the 30 end regionof the 16S rRNA and contribute to the efficiency oftranslational initiation. Translational regulation of ompFby micF antisense RNA occurs at high temperatures,high osmolarity, or in the presence of ethanol when themicF gene, which is located upstream of ompC, istranscribed in the opposite direction from the ompCgene. A 92 bp micF transcript is complementary to aportion of the ompF promoter region from þ74 toþ166 and hence blocks translation of OmpF (Mizuno etal. 1984). Binding of the leucine response protein (Lrp)and the histone-like DNA-binding protein H-NS hasalso been implicated in the regulation of ompF



(reviewed in Pratt et al. 1996). Lrp and H-NS are bothglobal regulators that bind between the ompC and micFpromoter region resulting in positive regulation of bothmicF and ompC transcription that negatively regulatesompF translation via binding of micF antisense RNA(Ferrario et al. 1995; Suzuki et al. 1996).

While osmoregulation involves many regulatoryproteins, the EnvZ-OmpR His-Asp phosphorelay iscritical for directly monitoring the external osmolarityand eliciting the signal transduction pathway to regulatethe porin regulon. Osmoregulation in E. coli is aparadigm for his-asp phosphorelays in prokaryotes andeukaryotes.

Eukaryotic His-Asp phosphorelaysWhile histidine kinases were thought to be presentonly in prokaryotes, they have recently been identi-fied in eukaryotic systems including Saccharomycescerevisiae, Schizosccharomyces pombe, Arabidopsis thaliana,Neurospora crassa and Dictyostelium discoideum (reviewedin Alex et al. 1996). Similar to bacterial hybridhistidine kinases, the majority of the eukaryotichistidine kinases which have been characterized todate include both a conserved histidine kinase domainand a response regulator type domain in the sameprotein. Examples of this domain organization includethe Sln1p protein from S. cerevisiae and the ETR1protein from A. thaliana.

In the flowering plant, Arabidopsis thaliana, anethylene response gene has been identified that encodesa transmembrane histidine kinase ETR1. A Raf-kinaselike protein, CTR1, has been identified that actsdownstream of ETR1 which implies that ETR1 isalso involved in regulating a MAP kinase cascade.Interestingly, there is remarkable structural and func-tional similarity between bacterial response regulatorsand eukaryotic Ras-type signalling proteins (reviewedin Parkinson & Kofoid 1992; Zhang 1996).

Other eukaryotic examples of the his-asp phospho-relay are present in S. cerevisiae, Neurospora crassa, andDictyostelium discoidium. In S. cerevisiae, the Skn7 proteinhas a response regulator type domain that is homo-logous to heat shock transcription factors. While theupstream histidine kinase remains elusive, the functionof Skn7 has recently been determined to be involved inoxidative stress resistance (Krems et al. 1996). InNeurospora crassa, a light-dependent system involvingan aspartyl phosphoprotein (WC-1/WC-2), and a geneencoding a hybrid histidine kinase, NIK, has beenidentified. In Dictyostelium discoidium, a hybrid histidinekinase with homology to bacterial histidine kinases has

also been identified. Identification of these proteins aresuggestive that his-asp phosphorelay systems also exist inthese organisms (reviewed in Swanson et al. 1994), andprovides evidence that the his-asp phosphorelaymechanism is a universal mode of signal transductionin both prokaryotes and eukaryotes.

Osmoregulation in S. cerevisiaeCurrently, the most thoroughly characterized exampleof his-asp phosphorelays in eukaryotes is the S. cerevisiaehigh osmolarity response pathway. In S. cerevisiae, amultiple his-asp phosphorelay responds to high osmo-larity stress and lies upstream of a MAP kinase cascade tomediate changes in glycerol accumulation (Ota &Varshavsky 1993; Albertyn et al. 1994; Posas et al.1996). The high osmolarity response pathway inS. cerevisiae involves a phosphorelay between Sln1p,Ypd1p, and Ssk1p proteins (Ota & Varshavsky 1993;Maeda et al. 1994; Posas et al. 1996). As in the E. coliosmosensing pathway, the primary osmotic signalremains elusive. Sln1p is an essential transmembranehybrid histidine kinase that contains both a conservedhistidine kinase domain and an aspartate containingresponse regulator type domain. Phosphotransfer fromHis to Asp within Sln1p is followed by transfer toYpd1p on His followed by transfer to the Ssk1presponse regulator on aspartate. Ypd1p binds to bothSln1p and Ssk1p to mediate the multistep phosphorelay.Interestingly, unlike bacterial response regulators thathave been characterized, phosphorylation of theresponse regulator Ssk1p in yeast results in down-regulation of the pathway. This has been demon-strated by the lethality of Dsln1p which results inaccumulation of unphosphorylated Ssk1p, and hyper-phosphorylation of HOG1p which can be rescuedby overexpression of a tyrosine phosphatase (Swansonet al. 1994).

In the budding yeast osmosensing pathway, a MAPkinase cascade is located downstream of the responseregulator and consists of Ssk2/Ssk22p (MEKK), Pbs2(MEK serine/threonine kinase), and HOG1p (MAPKtyrosine kinase). Specific phosphatases that act at thelevel of the MAP kinase cascade have also beenidentified. Effectors immediately downstream of theMAP kinase cascade may include a stress responsetranscription factor (STRF) such as Mcm1 which bindsto a stress response DNA-binding element (STRE) withthe consensus sequence of AGGGG or CCCCT hasbeen identified (reviewed in Ruis & Schuller 1995). InS. cerevisiae there is also a second high osmolarityosmosensor Sho1p which is nonessential and is not a

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histidine kinase. Instead, Sho1p has an SH3 domain forinteraction with the N-terminal proline-rich domainof Pbs2. These two osmosensors have differentconcentration-dependence and response kineticswhereby Sln1p is inactivated and Sho1p is activated inresponse to increased osmolarity.

Other osmosensing pathways in yeast are emerging. Alow osmolarity pathway in S. cerevisiae has beenidentified and members of the MAP kinase cascade(i.e. Bck1, Mkk1/Mkk2 and Mpk1) are currently beingcharacterized (Fig. 5) (reviewed in Ruis & Schuller1995). A similar osmosensing pathway is also emergingin Schizosaccharomyces pombe fission yeast (Ohmiya et al.1995). In fission yeast, the His-Asp phosphorelay hasnot been identified, but members of the MAP Kinase

cascade (MEK, Wis1 and MAPK, Sty1) as well asphosphatases PP2C acting on Wis1 and Pyp1/2 actingon Sty1 are currently being characterized (Shiozaki &Russell 1995; Millar et al. 1995).

Perspectives

His-Asp phosphorelays as antimicrobialtargets

Since his-asp phosphorelay systems are fundamental tomany adaptive responses in bacteria and are essential forvirulence in host organisms, they are suitable targets forantimicrobial action (Miller et al. 1989; DiRita &Mekalanos 1989; Deretic et al. 1991; reviewed in Volz



Figure 5 Prokaryotic and eukaryotic osmosensors. The bacterial osmosensing pathway includes the sensor histidine kinase (EnvZ),response regulator (OmpR) and gene regulation of the porin regulon (ompF and ompC). In S. cerevisiae, homologous His-Aspphosphorelay proteins have been identified. The high osmolarity pathway involves the His-Asp-His-Asp phosphorelay (Sln1p, Ypd1p,Ssk1p) (Ota & Varshavsky 1993; Maeda et al. 1994; Posas et al. 1996) and a MAP Kinase cascade (blue rectangle) consisting of MEKK(MAPKKK), MEK (MAPKK serine/threonine kinase), and MAPK (tyrosine kinase). The MAP kinase members in the yeast highosmolarity pathway are indicated (Ssk2/22p, Pbs2p, Hog1p). Phosphatases which downregulate the MAP kinase cascade include Ptc1p,Ptc3p, and Ptp2p (Maeda et al. 1994). In addition to Sln1p, there is another osmosensor, Sho1p, that mediates responses to highosmolarity. Sho1p is a transmembrane protein which does not contain a histidine kinase domain, but rather has an SH3 domain forinteraction with the N-terminal proline rich domain of Pbs2. In the yeast high osmolarity pathway, downstream effectors may include astress response transcription factor (STRF) such as Mcm1, and a stress response DNA-binding element (STRE) which contribute to thetranscriptional regulation of cytosolic glycerol-3-phosphate dehydrogenase (g3pdh) to regulate glycerol accumulation. In S. cerevisiae,members of the low osmolarity pathway which have been identified include: the MAP Kinase cascade (Bck1, Mkk1/Mkk2, Mpk1)(reviewed in Ruis & Schuller 1995).

1995; reviewed in Dziejman & Mekalanos 1995).Systems involving the EnvZ-OmpR phosphorelayunit include the following:1 In Pseudomonas auruginosa, OmpR has been impli-

cated in the AlgR1/AlgR2 pathway which mediatesexcessive alginate synthesis (mucous) that contributesto respiratory distress in cystic fibrosis patients;

2 In Shigella flexneri, an invasive pathogen thatcauses bacillary dysentery, mutations in envZ decreasevirulence;

3 In Salmonella, which is the causative agent for food-borne infections, the EnvZ-OmpR system isrequired for virulence.Other his-asp phosphorelay systems essential for

virulence include: VanR/VanS which mediates vanco-mycin resistance in Enterococcus faecium, PilA/PilB whichmediates pilin production in Neisseria gonorrhoeae, andNtrA/NtrC which mediates urease production inKlebsiella pneumoniae.

Since these systems are ubiquitous in bacteria,cumulative antimicrobial action could be effectivelyachieved by simultaneously blocking many pathways.Bacterial histidine kinase inhibitors and inhibitors ofaspartyl response regulator DNA-binding have beenidentified (Roychoudhury et al. 1993). It has also beendemonstrated that histidine kinases from S. cerevisiae canbe inhibited by genistein and staurosporin which havepreviously been classified as tyrosine kinase inhibitors(Huang et al. 1992; Bongue-Bartelsman et al. 1994).An important consideration, however, is the recentidentification of several his-asp phosphorelay systemsin eukaryotes. Thus, it will be critical to characterizeand identify additional eukaryotic members to assessthe usefulness of a multi-target approach and todetermine whether additional target specificity will berequired.

AcknowledgementsWe are grateful to Dr Ujwal Shinde, Dr Lisa Bergstrom, andRinku Dutta for critical reading of this manuscript. Spacelimitations required the omission of many relevant primaryreferences, and we apologize to the authors. Research from ourlaboratory was supported by an NIH grant (GM 19043).

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