Kidney International, Vol. 49 (1996), pp. 964—967

Signal transduction in the control of phosphate-regulated genes of Escherichia coli

BARRY L. WANNER

Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA

Bacteria such as Escherichia co/i regulate the synthesis of aunique proteins with signatures of a sensor kinase (transmitter) large number of proteins in response to extracellular inorganicdomain and 170 with signatures of a receiver domain have been phosphate (P) levels by activating transcription of co-regulatedidentified on the basis of sequence similarities at the protein level genes. More than 80 proteins are synthesized in increased(as determined from searching DNA databases for homologous amounts in response to P. limitation in the gram-negative bacte-proteins). Analogous signaling systems exist in eucaryotic cells rium E. co/i [1—3]. Many of these proteins are the products of a set including Saccharomyces cerevisiae [6—8], Arabidopsis thaliana [9], of co-regulated genes known as the phosphate (Pho) regulon.and Neurospora crassa [10]. In yeast, a two-component system Altogether 38 Pho regulon genes have now been characterized (by(SLN1 and SSK1) can provide signaling input to a MAP kinase cloning, sequencing, mutational analysis, and/or examination ofcascade that is responsive to small molecules [7, 11]. MAP kinase their gene products) in E. co/i, or the closely related bacteriacascades are extremely important in signaling; they are key Enterobacter aerogenes and Sa/monella typhimurium [4]. Thesecomponents of convergent signal transduction pathways in mam- genes are arranged in eight (or more) transcriptional units, andmalian cells [12, 13]. Although histidine phosphorylation has also their gene products probably all have a role in the assimilation ofbeen recently found in human cells [14], the phosphorylation alternative phosphorus (P) sources from the environment. Theirchemistry of that system is distinctively different from two- transcription requires an upstream activation site (called a Phocomponent regulatory systems. Box) and a DNA-binding protein (called PhoB) that acts as a transcriptional activator only when it is phosphorylated (Fig. 1). P, control is a paradigm of a signal transduction pathway in The level of expression of Pho regulon genes is regulated by the which occupancy of a cell surface receptor(s) regulates gene amount of phospho-PhoB. expression in the cytoplasm P control of the Pho regulon involves a "two-component P1 control of the Pho regulon involves two processes: inhibition when environmental P1 is in excess (greater than a few jIM) and regulatory system" activation under conditions of P1 limitation (Fig. 2). It is con- The activation of Pho regulon genes such as phoA (encodingtrolled by the extracellular P1 level. The phosphate-specific trans- bacterial alkaline phosphatase, Bap) under conditions of P1 limi-porter (the Pst system) plays a crucial role in the process of tation requires two proteins: PhoR and PhoB. These proteins areinhibition; yet, transportperse is not involved. The expression of members of the large family of two-component regulatory systemsgenes of the Pho regulon is inhibited when environmental P1 (the in bacteria. These systems are important in a variety of adaptivepreferred P source) is in excess (greater than Ca. 4 jsM). This responses and signal transduction pathways [5]. All of them areinhibition requires an inhibitor form of PhoR (called PhoR'), a likely to share a common biochemical signaling mechanism inprotein called PhoU, and all components of the Pst system. By which a sensor kinase (usually membrane-associated, like PhoR)analogy to other two-component regulatory systems, PhoR is is autophosphorylated on a conserved histidine residue in re-thought to act as the P1 sensor. Whether PhoR detects P. directly sponse to an environmental stimulus (the extracellular P1 level in(perhaps via a regulatory site on PhoR) or indirectly via an the case of the Pho regulon) and then transfers the phosphoiylinteraction with PhoU (or the Pst transporter) is unknown. The group to a conserved aspartate residue on its partner responseexpression of Pho regulon genes is activated 100-fold or more regulator (commonly a transcriptional activator, like PhoB). during growth after exhaustion of environmental P. This activa- As many as 50 pairs of these sensor and regulator proteins cantion requires the activator form of PhoR (called Ph0RA) and exist in a single bacterium. Twenty-five potential signaling kinasesPhoB. and 37 response regulator (receiver domain) proteins have been Therefore, P1 control involves the interconversion of PhoR' identified on the basis of the E. co/i genome sequence, which isand PhoR'. This interconversion is likely to involve protein- now more than 50% known. A few examples of two-componentprotein interactions between PhoR, PhoU, and Pst components regulatory systems shown to act by a common phosphorylationand conformational changes brought about in response to the mechanism are listed in Table 1. Altogether, more than 110extracellular P level. Accordingly, PhoRR predominates when p. is in excess and Ph0RA predominates under conditions of P limitation. Formation of PhoR' can result due to association of © 1996 by the International Society of Nephrology PhoR with PhoU and the Pst system upon full P occupancy of the

964 Wanner: P1 regulation of Escherichia coli genes 965

Gene product/function Gene organization

01phoA (psiA)-psi? Bap, Unknown

cii phoBR Regulator, P1 sensor 01phoE—. Polyanion porin ni phoH(psih') ATP-binding protein

cIphnCDEFGHlJKLMNOP(psiD Carbon phosphorus lyase pathway

001pstSCAB-phoU P1 uptake and P1 inhibition

001ugpBAECQ(psiB,>C) Uptake sn-glycerol-3-phosphate Fig. 1. Organization of sequenced genes of the Pho Unknown regulon. (Adapted from Figure 5 in [31 Used with permission from the American Society for Phosphonatase pathway pnXWil1phnR>o,phnST13V Microbiology.)

Table 1. Examples of two-component regulatory systems The cytoplasmic domains of PhoR include an unusually large Sensor kinase "linker domain" of about 150 residues following its membrane Regulatory system Response regulator(s) domain and a C-terminal kinase domain beginning near residue Chemotaxis CheA CheY, CheB 159 that is highly conserved among family members. Further, Nitrogen control NtrB (NR11) NtrC (NR1) in-frame deletions of its linker domain (160 to 167, M23 to 167, Osmoregulation EnvZ OmpR P1 control PhoR PhoB M10 to 157, and M60 to 174) lead to low level activation, Catabolite control CreC (PhoM) CreB suggesting that it has a regulatory role [161. The linker domain can Aerobic respiratory control ArcB AreA interact with PhoU. A truncated form of PhoR lacking its N-terminal 83 amino acids is radiolabeled with y-[32P]-ATP in vitro on His-213 [18] and phospho-'PhoR rapidly transfers the phosphoryl group to PhoB. It is reasonable to suppose that PhoR Pst system, or binding of P. to a hypothetical regulatory site onfacilitates the dephosphorylation of phospho-PhoB, yet no such PhoR. In the latter case, P. saturation of the Pst system canactivity has been demonstrated. Numerous phoR missense facilitate P1 binding to the regulatory site. A conformationalchanges have been characterized, including partially dominant change leading to formation of Ph0RA can result due to release ofones (suggestive of oligomerization) and ones causing constitutive PhoR from the inhibition complex, low P1 occupancy of the Pstsignaling. All of these alter residues of its cytoplasmic domain(s) system, or low P. occupancy of a PhoR regulatory site. [3J. PhoR is a cytoplasmic membrane protein that acts as a The Pho regulon is subject to additional controls that can be sensor kinase forms of "cross regulation" PhoR is a cytoplasmic integral membrane protein of 431 amino PhoR is required for inhibition when P is in excess. In its acids in length. It appears to be composed of three domains: anabsence, two other signaling pathways—involving CreC (formerly N-terminal "membrane-binding domain" and two cytoplasmiccalled PhoM) or acetyl phosphate—lead to high level activation of domains. Its N-terminal domain of about 50 amino acids has twoPhoB in response to carbon sources [19—21]. Therefore, the Pho predicted a-helical transmembrane segments capable of forming aregulon appears to be subject to multiple controls, each of which helical hairpin with a few amino acids exposed to the periplasm. Inaffect the phosphorylation of PhoB or dephosphorylation of agreement, its N-terminal segment is capable of translocating aphospho-PhoB. Its control by P, involves detection of the extra- reporter protein to the cell surface; in-frame fusions of TnphoAcellular P1 level and is coupled to the first step in P metabolism, [15, 16] to residues 29, 37, and 40, but not to 54 or beyond,P, uptake. Its additional controls are P1 independent and can be produce PhoR'-'PhoA fusion proteins with Bap activity, indicatingforms of cross regulation. This is because Pho regulon control by localization of these hybrid proteins to the periplasm. Full-lengthCreC and acetyl phosphate (which are independent of each other) PhoR is localized to the cytoplasmic membrane, while a truncatedinvolves regulatory couplings between P1, carbon, and energy protein (denoted 'PhoR) lacking this domain is cytoplasmic.metabolism. Both are coupled to subsequent steps in P1 metabo- These 'PhoRs lead to low level activation ("constitutive signal-lism, the entry of P1 into ATP, the primary phosphoryl donor in ing") of PhoB in vivo [17; A. Haldimann and B.L. Wanner,cellular metabolism. CreC is the catabolite regulatory sensor, unpublished data]. PhoR is unusual among membrane-associatedwhich (like PhoR) can phosphorylate PhoB. Acetyl phosphate is sensor kinases, for it has no large extracellular (periplasmic)an intermediate in the phosphotransacetylase-acetate kinase (Pta- domain. This has been verified by its proteinase K inaccessibilityAckA) pathway for P. incorporation into ATP. Acetyl phosphate in spheroplasts [16]. Most similar sensor proteins appear to havecan activate Pho regulon gene expression either directly by acting periplasmic domains longer than ca. 125 residues, or to haveas a chemical phosphorylating agent or indirectly via an unknown multiple membrane-spanning segments. signaling histidine kinase. 966 Wanner: P regulation of Escherichia coli genes

Inhibition Activation [Pd <4 ( (%[Pd > 4 tM (;j2 A A Periplasni

His® ®ASP Asp® Fig. 2. Transmembrane signal transduction in P, control of the Pho regulon. PhoR is thought to exist in two forms; Ph0RR refers to the inhibitor (formerly repressor) form of PhoR and Ph0RA PstA, C - Channel Proteins refers to the activator form of PhoR. These were PstB - ABC Permease (ATPase) named PhORR and Ph0RA long before it was Transcription understood how they can act. (Adapted from PstS - P1 binding protein Rho regulon Figure 4 in [3]. Used with permission of the promoters American Society for Microbiology.)

Many unanswered questions remain in regard to our References understanding of Pho regulon control in E. coli 1. WArERBL:Phosphorus assimilation and its control of gene expres- In spite of the wealth of information on the molecular genetics, sion in Escherichia coli, in The molecular basis of bacterial metabolism, molecular biology, and biochemistry of the Pho regulon and other edited by HAUSKA G, TI-IAUER R, Heidelberg, Springer-Verlag, 1990, signal transduction pathways in bacteria, many questions remain p 152 unanswered. Several Pho regulon genes are yet unidentified. Are 2. WANNER BL: Gene regulation by phosphate in enteric bacteria. J Cell Biochem 51:47—54, 1993 any of them involved in intracellular P1 metabolism? Both PhoR 3. WANNERBL:Phosphorus assimilation and control of the phosphate and the P1 binding protein PstS (see the article by F. Quiocho, this regulon (Chapt 84), in Escherichia coli and Salmonella yphimurium issue) are required for P sensing. Does PhoR detect P, directly? Cellular and Molecular Biology, edited by NEIDHAROT FC, CURTISS RI, Or, does PhoR "sense" full P1 occupancy of the Pst system only GROSS CA, INGRAHAM JL, LIN ECC, Low KB JR, MAGASANIK B, indirectly? An inhibition complex composed of PhoR, PhoU, and REZNIKOFF W, SCHAECFITERM,UMBARGER HE, RILEY M, Washing- ton, D.C., American Society for Microbiology, 1996 (in press) the Pst system has been proposed to control the Pho regulon. Do 4. JIANG W, METCALF WW, LEE K-S, WANNER BL: Molecular cloning, all of these proteins interact? If so, which ones interact directly? mapping, and regulation of Pho regulon genes for phosphonate How do they interact? Do the cytosolic domains interact? Or, do breakdown by the phosphonatase pathway of Salmonella typhimurium membrane spanning domains interact within the membrane? In LT2.JBacteriol 177:6411—6421, 1995 5. PARKINSON JS, KOFOID EC: Communication modules in bacterial any case, extracellular P1 is detected. How is the signal transmitted signaling proteins. Annu Rev Genet 26:71—112, 1992 across the membrane in order to regulate gene expression in the 6. OTA IM, VARSHAVSKY A: A yeast protein similar to bacterial two- cytoplasm? Cross regulation can be a form of global control component regulators. Science 262:566—569, 1993 resulting from phosphorylation of PhoB by a nonpartner sensor 7. MAEDA T, WURGLER-MURPHY SM, SAITO H: A two-component kinase(s) or acetyl phosphate. Is this kind of regulation of system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:242—245, 1994 fundamental importance, or does it merely reflect nonspecific 8. BROWN JL, BUSSEY H, STEWART RC: Yeast Skn7p functions in a biochemistry reactivities between highly similar proteins? eukaiyotic two-component regulatory pathway. EMBO J 13:5186— New studies are aimed towards elucidating the answers to these 5194, 1994 and many other questions. For the sake of brevity, citations are 9. CHANG C, KwoK SF, BLEECKERAB,MEYEROWITZ EM: Arabidopsis ethylene-response gene ETRI: Similarity of product to two-compo- given only for research reported within the past five years. Six nent regulators. Science 262:539—544, 1993 recent reviews [2, 3, 21—24] cover these and other aspects of P,10. SWANSON RV, ALEX LA, SIMON MI: Histidine and aspartate phos- regulated genes of the Pho regulon in more detail. They also phorylation: Two-component systems and the limits of homology. include additional citations of the primary research. Trends Biochem Sci 19:485—490, 1994 1. HERSKOWITZ I: MAP kiiiase pathways in yeast: For mating and more. Cell 80:187—197, 1995 Reprint requests to Bany L. Wanner, Ph.D., Professor, Department of 12. BLUMER KJ, JOHNSoN GL: Diversity in function and regulation of Biological Sciences, Purdue Universily, West Lafayette, Indiana, 47907 USA MAP kinase pathways. Trends Biochem Sci 19:236—240, 1994 Email: [email protected] 13. HILL CS, TREISMAN R: Transcriptional regulation by extracellular signals: Mechanisms and specificity. Cell 80:199—211, 1995 Acknowledgments 14. CROVELLO CS, FURIE BC, FURIE B: Histidine phosphosylation of This laboratory is supported by NIH grant GM35392 and NSF grant P-selectin upon stimulation of human platelets: A novel pathway for MCB9405929. activation-dependent signal transduction. Cell 82:279—286, 1995 15. TRAXLER B, BOYD D, BECKWITH J: The topological analysis of integral cytoplasmic membrane proteins. J Membr Biol 132:1—11, 1993 Wanner: P regulation of Escherichia coli genes 967

16. SCHOLTEN M, TOMMASSEN J: Topology of the PhoR protein of DANIELS LL: Are the multiple signal transduction pathways of the Pho Escherichia coli and functional analysis of internal deletion mutants. regulon due to cross talk or cross regulation? (Chapt 15), in Regulation Mol Microbiol 8:269—275, 1993 of Gene Expression in Escherichia coli, edited by LIN ECC, LYNCH AS, 17. YAMADA M, MAKINO K, SHINAGAWA H, NAKATA A: Regulation of the Austin, R.G. Landes Co., 1995, p. 297 phosphate regulon of Escherichia coli: Properties of phoR deletion 22. WANNER BL: Multiple controls of the Escherichia coli PHO regulon mutants and subcellular localization of PhoR protein. Mo! Gen Genet by the P sensor PhoR, the catabolite regulatory sensor CreC, and 220:366—372, 1990 acetyl phosphate (Chapt 3), in Phosphate in Microorganisms, Cel- 18. SHINAGAWA H, MAKINO K, YAMADAM,AMEMURA M, SAT0 T, lular and Molecular Biology, edited by TORRIANI-GORINI A, YAGIL NAKATA A: Signal transduction in the phosphate regulon of Esche- richia coli: Dual functions of PhoR as a protein kinase and a protein E, SILVER 5, Washington, D.C., American Society of Microbiology, phosphatase, in Phosphate in Microorganisms: Cellular and Molecular 1994, p 13 23. WANNER BL: Signal transduction and cross regulation in the Esche- Biology, edited by TORRIANI-GORINI A, YAGIL E, SILVER S. Washing- ton, D.C., American Society for Microbiology, 1994, p 285 richia coli phosphate regulon by PhoR, CreC, and acetyl phosphate 19. WANNER BL, WILMES-RIESENBERG MR: Involvement of phospho- (Chapt 12), in Two-Component Signal Transduction, edited by HOCH transacetylase, acetate kinase, and acetyl phosphate synthesis in the JA, SILHAVY TJ, Washington, D.C., American Society for Microbiol- control of the phosphate regulon in Escherichia coli. J Bacteriol ogy, 1995, p 203 174:2124—2130, 1992 24. WANNER BL: Phosphate-regulated genes for the utilization of phos- 20. WANNER BL: Minireview. Is cross regulation by phosphorylation of phonates in members of the family Enterobacteriaceae (Chapt 36), in two-component response regulator proteins important in bacteria? J Phosphate in Microorganisms, Cellular and Molecular Biology, edited by Bacteriol 174:2053—2058, 1992 TORRIANI-GORINI A, YAGIL E, SILVER S, Washington, D.C., American 21. WANNER BL, JIANG W, KIM S-K, YAMAGATA5,HALDIMANN A, Society for Microbiology, 1994, p 215