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EnvZ-OmpR Interaction and Osmoregulation in Escherichia coli

2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês

10.1074/jbc.m110715200

1083-351X

Sheng Jian Cai, Masayori Inouye,

RNA and protein synthesis mechanisms

EnvZ, a histidine kinase/phosphatase inEscherichia coli, responds to the osmolarity changes in the medium by regulating the phosphorylation state of the transcription factor OmpR, which controls the expression levels of outer membrane porin proteins OmpF and OmpC. Although both ompRand envZ genes are located on the ompB locus under the control of the ompB promoter and transcribed as a single polycistronic mRNA, the expression of envZ is known to be significantly less than ompR. However, to date no accurate estimation for the amounts of EnvZ and OmpR in the cell has been carried out. Here we examined the levels of EnvZ and OmpR in the wild-type strain MC4100 by quantitative Western blot analysis using anti-OmpR and anti-EnvZc (cytoplasmic domain of EnvZ) antisera. It was observed that during exponential growth in L-broth medium there were ∼3500 and 100 molecules per cell of OmpR and EnvZ, respectively. The levels of OmpR and EnvZ in MC4100 cells grown in a high osmolarity medium (nutrient broth with 20% sucrose) were about the same as those grown in L-broth, whereas they were 1.7-fold higher than those in a low osmolarity medium (nutrient broth). With His10-OmpR, we also determined that theK d value for the EnvZc-OmpR complex formation is 1.20 ± 0.17 μm. On the basis of these results, the molecular mechanism of osmoregulation of ompF andompC is discussed. EnvZ, a histidine kinase/phosphatase inEscherichia coli, responds to the osmolarity changes in the medium by regulating the phosphorylation state of the transcription factor OmpR, which controls the expression levels of outer membrane porin proteins OmpF and OmpC. Although both ompRand envZ genes are located on the ompB locus under the control of the ompB promoter and transcribed as a single polycistronic mRNA, the expression of envZ is known to be significantly less than ompR. However, to date no accurate estimation for the amounts of EnvZ and OmpR in the cell has been carried out. Here we examined the levels of EnvZ and OmpR in the wild-type strain MC4100 by quantitative Western blot analysis using anti-OmpR and anti-EnvZc (cytoplasmic domain of EnvZ) antisera. It was observed that during exponential growth in L-broth medium there were ∼3500 and 100 molecules per cell of OmpR and EnvZ, respectively. The levels of OmpR and EnvZ in MC4100 cells grown in a high osmolarity medium (nutrient broth with 20% sucrose) were about the same as those grown in L-broth, whereas they were 1.7-fold higher than those in a low osmolarity medium (nutrient broth). With His10-OmpR, we also determined that theK d value for the EnvZc-OmpR complex formation is 1.20 ± 0.17 μm. On the basis of these results, the molecular mechanism of osmoregulation of ompF andompC is discussed. His-Asp phosphorelay signal transduction system nitrilotriacetic acid nutrient broth outer membrane porin proteins 5′-adenylyl-β,γ-imidodiphosphate Bacteria primarily rely on the His-Asp phosphorelay signal transduction system (HAP system),1 or the two-component signal transduction system, to acclimate in response to the changes of environmental conditions (1Hoch J.A. Curr. Opin. Microbiol. 2000; 3: 165-170Crossref PubMed Scopus (626) Google Scholar, 2Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2417) Google Scholar). The HAP system basically consists of a histidine kinase, which serves as a signal receptor, and its cognate response regulator, which regulates specific gene expression or mediates cellular locomotion (3Grebe T.W. Stock J.B. Adv. Microb. Physiol. 1999; 41: 139-227Crossref PubMed Google Scholar).One of most extensively studied HAP systems is the EnvZ/OmpR system mediating signal transduction in response to environmental osmolarity changes in Escherichia coli (4Egger L.A. Park H. Inouye M. Genes Cells. 1997; 2: 167-184Crossref PubMed Scopus (154) Google Scholar, 5Pratt L. Silhavy T.J. Hoch J. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 105-107Google Scholar, 6Forst S.A. Roberts D.L. Res. Microbiol. 1994; 145: 363-373Crossref PubMed Scopus (113) Google Scholar). EnvZ, a histidine kinase, undergoestrans-autophosphorylation on the highly conserved His-243 residue (7Yang Y. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11057-11061Crossref PubMed Scopus (144) Google Scholar, 8Yang Y. Inouye M. J. Mol. Biol. 1993; 231: 335-342Crossref PubMed Scopus (65) Google Scholar). The high energy phosphoryl group is subsequently transferred to the conserved Asp-55 residue of OmpR, a response regulator. Phosphorylated OmpR (OmpR-P) serves as a transcription factor differentially modulating the expression of the major outer membrane porin genes, ompC and ompF. OmpC and OmpF form channels in the outer membrane, which allow passive diffusion of small hydrophilic molecules of less than 650 Da in size (9Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar). EnvZ also possesses the OmpR-P phosphatase activity to dephosphorylate OmpR-P. It has been proposed that osmotic signal regulates the ratio of the kinase to the phosphatase activity of EnvZ to modulate the level of cellular OmpR-P by primarily altering the phosphatase activity (10Jin T. Inouye M. J. Mol. Biol. 1993; 232: 484-492Crossref PubMed Scopus (72) Google Scholar). The cellular OmpR-P levels reciprocally regulate the transcription ofompF and ompC. At low medium osmolarity a reduced level of OmpR-P, due to the decreased kinase/phosphatase ratio of EnvZ, favors the transcription of ompF. At high medium osmolarity an elevated OmpR-P level, resulting from the increased kinase/phosphatase ratio of EnvZ, allows the activation ofompC transcription. On the other hand, more OmpR-P molecules bind to the ompF promoter upstream region causing repression of ompF expression.EnvZ, consisting of 450 amino acid residues, exists as a dimer located in the inner cytoplasmic membrane of E. coli. The cytoplasmic domain of EnvZ (residues 180–450), EnvZc, also exists as a dimer and consists of the linker region (residues 180–222), domain A (or DHp domain, residues 223–289) and domain B (or CA domain, residues 290–450). It possesses both kinase and phosphatase activities similar to the intact EnvZ (11Park H. Saha S.K. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6728-6732Crossref PubMed Scopus (94) Google Scholar, 12Dutta R. Qin L. Inouye M. Mol. Microbiol. 1999; 34: 633-640Crossref PubMed Scopus (201) Google Scholar). The linker region is considered to play an important role in transducing the signal from the periplasmic receptor domain to the cytoplasmic catalytic domain (13Park H. Inouye M. J. Bacteriol. 1997; 179: 4382-4390Crossref PubMed Google Scholar). The NMR solution structures of both domain A and domain B have been solved (14Tomomori C. Tanaka T. Dutta R. Park H. Saha S.K. Zhu Y. Ishima R. Liu D. Tong K.I. Kurokawa H. Qian H. Inouye M. Ikura M. Nat. Struct. Biol. 1999; 6: 729-734Crossref PubMed Scopus (207) Google Scholar,15Tanaka T. Saha S.K. Tomomori C. Ishima R. Liu D. Tong K.I. Park H. Dutta R. Qin L. Swindells M.B. Yamazaki T. Ono A.M. Kainosho M. Inouye M. Ikura M. Nature. 1998; 396: 88-92Crossref PubMed Scopus (223) Google Scholar). Recently it has been demonstrated that domain A by itself can serve as the OmpR-P phosphatase, and it has been proposed that the phosphatase activity of EnvZ is modulated by the spatial arrangement between domain A and domain B (16Zhu Y. Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7808-7813Crossref PubMed Scopus (102) Google Scholar).The response regulator OmpR consists of an N-terminal CheY-like receiver domain containing the highly conserved Asp-55 phosphorylation site and a C-terminal DNA-binding domain (17Kato M. Aiba H. Tate S. Nishimura Y. Mizuno T. FEBS Lett. 1989; 249: 168-172Crossref PubMed Scopus (44) Google Scholar). The two domains are connected by a flexible linker (18Kenney L.J. Bauer M.D. Silhavy T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8866-8870Crossref PubMed Scopus (85) Google Scholar). The x-ray structure of the DNA binding domain shows a winged helix-turn-helix motif structure (19Martinez-Hackert E. Stock A.M. Structure. 1997; 5: 109-124Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar,20Kondo H. Nakagawa A. Nishihira J. Nishimura Y. Mizuno T. Tanaka I. Nat. Struct. Biol. 1997; 4: 28-31Crossref PubMed Scopus (83) Google Scholar). The phosphorylation of OmpR at Asp-55 greatly enhances its binding affinity for the regulatory sequences upstream of the ompFand ompC promoters (21Head C.G. Tardy A. Kenney L.J. J. Mol. Biol. 1998; 281: 857-870Crossref PubMed Scopus (122) Google Scholar, 22Forst S. Delgado J. Inouye M. J. Bacteriol. 1989; 171: 2949-2955Crossref PubMed Google Scholar, 23Aiba H. Nakasai F. Mizushima S. Mizuno T. J. Biochem. (Tokyo). 1989; 106: 5-7Crossref PubMed Scopus (98) Google Scholar, 24Huang K.J. Igo M.M. J. Mol. Biol. 1996; 262: 615-628Crossref PubMed Scopus (90) Google Scholar). The in vitro andin vivo footprinting studies have demonstrated that OmpR-P binds to the −100 to −38 region (C1, C2, and C3 sites) ofompC and the −380 to the −361 region (F4 site) and the −100 to −39 region (F1, F2, and F3 sites) of ompF(24Huang K.J. Igo M.M. J. Mol. Biol. 1996; 262: 615-628Crossref PubMed Scopus (90) Google Scholar, 25Tsung K. Brissette R.E. Inouye M. J. Biol. Chem. 1989; 264: 10104-10109Abstract Full Text PDF PubMed Google Scholar, 26Rampersaud A. Harlocker S.L. Inouye M. J. Biol. Chem. 1994; 269: 12559-12566Abstract Full Text PDF PubMed Google Scholar, 27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar). These OmpR binding sites consist of 20 base pairs each sharing a consensus sequence (28Harlocker S.L. Bergstrom L. Inouye M. J. Biol. Chem. 1995; 270: 26849-26856Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). It has been shown that two OmpR-P molecules bind to each site in a head-to-tail manner (28Harlocker S.L. Bergstrom L. Inouye M. J. Biol. Chem. 1995; 270: 26849-26856Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 29Harrison-McMonagle P. Denissova N. Martinez-Hackert E. Ebright R.H. Stock A.M. J. Mol. Biol. 1999; 285: 555-566Crossref PubMed Scopus (44) Google Scholar). The binding affinities of OmpR-P to these sites are in a hierarchical order such that OmpR-P independently binds to F1 and C1 sites whereas OmpR-P cooperatively binds to other sites only after the upstream F1 and C1 sites are occupied by OmpR-P (21Head C.G. Tardy A. Kenney L.J. J. Mol. Biol. 1998; 281: 857-870Crossref PubMed Scopus (122) Google Scholar, 27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar).The ompB operon of E. coli is comprised of two genes, ompR and envZ, which are co-transcribed as a polycistronic mRNA from a promoter located 5′ to theompR gene (30Hall M.N. Silhavy T.J. J. Mol. Biol. 1981; 151: 1-15Crossref PubMed Scopus (159) Google Scholar). It has been shown that significantly fewer EnvZ molecules are produced compared with OmpR molecules in a cell, and it has been estimated that there are about 1000 molecules of OmpR whereas there are only 10 molecules of EnvZ per cell (5Pratt L. Silhavy T.J. Hoch J. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 105-107Google Scholar). In this report, we reexamined the cellular OmpR and EnvZ levels under different growth conditions by quantitative Western blot analysis. We also determined the dissociation constant of OmpR and EnvZ by using His10-OmpR and Ni-NTA-agarose. These findings provide new insights into the mechanism by which the EnvZ/OmpR system regulates the osmosensory pathway in E. coli.DISCUSSIONIn the present study, OmpR and EnvZ levels in E. colicells grown in L-broth at mid-log phase were determined to be 3500 and 100 molecules per cell, respectively. OmpR and EnvZ levels were almost the same from cells grown in L-broth medium or in a high osmolarity medium (NB + 20% sucrose). However, when cells were grown in a low osmolarity medium (NB), both OmpR and EnvZ levels were about 60% of those in the high osmolarity medium. The mechanism for the differential expression of the ompB locus under different osmolarities remains to be elucidated. The autoregulation of theompB locus has been reported, where the ompBexpression is regulated by OmpR, the first product of theompB operon (35Liljestrom P. Laamanen I. Palva E.T J. Mol. Biol. 1988; 201: 663-673Crossref PubMed Scopus (46) Google Scholar). It should be noted that under all the different growth conditions used, the ratio of OmpR to EnvZ was almost constant, and OmpR molecules existed in 30–35-fold excess over EnvZ monomers or in 60–70-fold excess compared with EnvZ dimers. It has been reported that translation of OmpR mRNA is ten times more efficient than that of EnvZ using lacZ as a reporter inSalmonella typhimurium (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar). Because bothenvZ and ompR genes are transcribed as a single polycistronic mRNA (30Hall M.N. Silhavy T.J. J. Mol. Biol. 1981; 151: 1-15Crossref PubMed Scopus (159) Google Scholar), the expression of the two genes are differentially regulated at the level of translation initiation (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar).The apparent K d value of EnvZc and His10-OmpR interaction was 1.20 ± 0.17 μm under the conditions used. Assuming the cell volume is 10−15 liters (37Wanner B.L. Neidhardt F.C. Escherichia coli and Salmonella. 1. American Society of Microbiology, Washington, D. C.1996: 1359Google Scholar), the cellular concentrations of OmpR and EnvZ were calculated to be 6 and 0.18 μm, respectively. Therefore on the basis of the K d value thus obtained, ∼85% of EnvZ molecules in a cell are estimated to be occupied with OmpR. It is important to note that as OmpR molecules exist in concentrations 60–70-fold higher than EnvZ dimers, OmpR-P molecules formed on EnvZ dimers are always competed with free OmpR molecules in the cytoplasmic fraction. It has been demonstrated that OmpR-P has a much higher affinity for the ompF andompC promoter regions than OmpR, so that once the OmpR-P molecules released into the cytoplasm are bound to the ompFand ompC promoter regions they are readily sequestered from the EnvZ phosphatase activity (38Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 908-913PubMed Google Scholar).Previous reports (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar) have proposed that at low osmolarity, OmpR-P binds to the C1 site and cooperatively to F1, F2, and somewhat loosely to F3 sites resulting only in ompF expression, whereas at high osmolarity, the concentration of OmpR-P increases allowing C2, C3, and F4 sites (the repressor site for the ompF expression) to be also occupied by OmpR-P, resulting in the induction ofompC expression and repression of ompFexpression. In this fashion, the reciprocal expression ofompF and ompC is achieved depending upon the medium osmolarity. According to the footprinting experiment, F1, F2, and a significant fraction of the F3 site were protected when the OmpR-P concentrations were between 108 and 135 nm whereas all C1, C2, and C3 sites became protected only when the OmpR-P concentration reached 540 nm (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar), assuming that the OmpR added was fully phosphorylated. Assuming that these results may be applied in vivo, at low osmolarity the phosphorylation of only 3.5% (120 nm or 70 OmpR-P molecules/cell) of total OmpR molecules in a cell (2024 OmpR molecules per cell) would be enough to activate the expression of ompF, whereas at high osmolarity the phosphorylation of about 10% (580 nm or 350 OmpR-P molecules/cell) of total OmpR molecules in a cell (3500 molecules per cell) would be sufficient to promote the expression ofompC and to repress the expression of ompF. Clearly the 60 EnvZ monomers or 30 EnvZ dimers per cell at low osmolarity and 100 EnvZ monomers or 50 EnvZ dimers per cell at high osmolarity are sufficient to fulfill the effective regulation ofompF and ompC. On the basis of OmpR amounts it has been shown that the OmpR-P level at high osmolarity was about 4-fold higher than that at low osmolarity (39Forst S. Delgado J. Rampersaud A. Inouye M. J. Bacteriol. 1990; 172: 3473-3477Crossref PubMed Google Scholar). Considering that the cellular OmpR level is increased by more than 70% at high osmolarity than that at low osmolarity, the actual OmpR-P level could be increased more than 7-fold at high osmolarity in comparison with the level of OmpR-P at low osmolarity. In addition, the cell volume increases at low osmolarity whereas it decreases at high osmolarity resulting in even more dramatic changes in the OmpR-P concentration. This sharp change of the OmpR-P concentration appears to account well for the expression patterns of ompF and ompC at high and low osmolarity. However, the regulation of ompF andompC in the cell may be more complex, as there are cross-talks among the His-Asp phosphorelay systems (40Wanner B.L. J. Bacteriol. 1992; 174: 2053-2058Crossref PubMed Google Scholar), and other genes besides ompF and ompC are known to be regulated by OmpR-P (41Verhoef C. Lugtenberg B. van Boxtel R. de Graaff P. Verheij H. Mol. Gen. Genet. 1979; 169: 137-146Crossref PubMed Scopus (44) Google Scholar, 42Shin S. Park C. J. Bacteriol. 1995; 177: 4696-4702Crossref PubMed Google Scholar, 43Mao W. Siegele D.A. Mol. Microbiol. 1998; 27: 415-424Crossref PubMed Scopus (12) Google Scholar).A similar quantitative Western blot analysis has been carried out with the proteins involved in chemotaxis signal transduction. The cellular contents of the total MCPs (methyl-accepting chemotaxis protein), CheA, CheY, and CheW were estimated to be 6000 molecules each per cell inE. coli (44Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 45Kuo S.C. Koshland D.E., Jr. J. Bacteriol. 1987; 169: 1307-1314Crossref PubMed Google Scholar). However it is important to know that CheA belongs to Class II histidine kinases, whereas EnvZ belongs to Class I, the major signal transducing kinases in E. coli as 29 of 30 histidine kinases in E. coli belong to Class I (46Mizuno T. DNA Res. 1997; 4: 161-168Crossref PubMed Scopus (298) Google Scholar). Class II kinases are involved in regulating flagella rotation whereas Class I kinases regulate transcription of specific genes (47Yamaguchi S. Aizawa S. Kihara M. Isomura M. Jones C.J. Macnab R.M. J. Bacteriol. 1986; 168: 1172-1179Crossref PubMed Google Scholar). The present report is the first to accurately estimate the cellular contents of a Class I kinase and its cognate response regulator, suggesting that the Class I kinase system is substantially different from the Class II kinase system in their regulatory mechanisms.The interaction between EnvZ and OmpR-P is not discussed in the present study. A recent report concerning this issue raised a question about the role of EnvZ phosphatase activity in osmoregulation (48Mattison K. Kenney L. J. Biol. Chem. 2002; 277: 11143-11148Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We have reexamined the OmpR-P interaction with EnvZc, and these results will be presented elsewhere. The characterization of the OmpR-P-EnvZ interaction provides important insights into the bi-functional roles of EnvZ in the osmoregulation of ompC and ompF. Bacteria primarily rely on the His-Asp phosphorelay signal transduction system (HAP system),1 or the two-component signal transduction system, to acclimate in response to the changes of environmental conditions (1Hoch J.A. Curr. Opin. Microbiol. 2000; 3: 165-170Crossref PubMed Scopus (626) Google Scholar, 2Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2417) Google Scholar). The HAP system basically consists of a histidine kinase, which serves as a signal receptor, and its cognate response regulator, which regulates specific gene expression or mediates cellular locomotion (3Grebe T.W. Stock J.B. Adv. Microb. Physiol. 1999; 41: 139-227Crossref PubMed Google Scholar). One of most extensively studied HAP systems is the EnvZ/OmpR system mediating signal transduction in response to environmental osmolarity changes in Escherichia coli (4Egger L.A. Park H. Inouye M. Genes Cells. 1997; 2: 167-184Crossref PubMed Scopus (154) Google Scholar, 5Pratt L. Silhavy T.J. Hoch J. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 105-107Google Scholar, 6Forst S.A. Roberts D.L. Res. Microbiol. 1994; 145: 363-373Crossref PubMed Scopus (113) Google Scholar). EnvZ, a histidine kinase, undergoestrans-autophosphorylation on the highly conserved His-243 residue (7Yang Y. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11057-11061Crossref PubMed Scopus (144) Google Scholar, 8Yang Y. Inouye M. J. Mol. Biol. 1993; 231: 335-342Crossref PubMed Scopus (65) Google Scholar). The high energy phosphoryl group is subsequently transferred to the conserved Asp-55 residue of OmpR, a response regulator. Phosphorylated OmpR (OmpR-P) serves as a transcription factor differentially modulating the expression of the major outer membrane porin genes, ompC and ompF. OmpC and OmpF form channels in the outer membrane, which allow passive diffusion of small hydrophilic molecules of less than 650 Da in size (9Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar). EnvZ also possesses the OmpR-P phosphatase activity to dephosphorylate OmpR-P. It has been proposed that osmotic signal regulates the ratio of the kinase to the phosphatase activity of EnvZ to modulate the level of cellular OmpR-P by primarily altering the phosphatase activity (10Jin T. Inouye M. J. Mol. Biol. 1993; 232: 484-492Crossref PubMed Scopus (72) Google Scholar). The cellular OmpR-P levels reciprocally regulate the transcription ofompF and ompC. At low medium osmolarity a reduced level of OmpR-P, due to the decreased kinase/phosphatase ratio of EnvZ, favors the transcription of ompF. At high medium osmolarity an elevated OmpR-P level, resulting from the increased kinase/phosphatase ratio of EnvZ, allows the activation ofompC transcription. On the other hand, more OmpR-P molecules bind to the ompF promoter upstream region causing repression of ompF expression. EnvZ, consisting of 450 amino acid residues, exists as a dimer located in the inner cytoplasmic membrane of E. coli. The cytoplasmic domain of EnvZ (residues 180–450), EnvZc, also exists as a dimer and consists of the linker region (residues 180–222), domain A (or DHp domain, residues 223–289) and domain B (or CA domain, residues 290–450). It possesses both kinase and phosphatase activities similar to the intact EnvZ (11Park H. Saha S.K. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6728-6732Crossref PubMed Scopus (94) Google Scholar, 12Dutta R. Qin L. Inouye M. Mol. Microbiol. 1999; 34: 633-640Crossref PubMed Scopus (201) Google Scholar). The linker region is considered to play an important role in transducing the signal from the periplasmic receptor domain to the cytoplasmic catalytic domain (13Park H. Inouye M. J. Bacteriol. 1997; 179: 4382-4390Crossref PubMed Google Scholar). The NMR solution structures of both domain A and domain B have been solved (14Tomomori C. Tanaka T. Dutta R. Park H. Saha S.K. Zhu Y. Ishima R. Liu D. Tong K.I. Kurokawa H. Qian H. Inouye M. Ikura M. Nat. Struct. Biol. 1999; 6: 729-734Crossref PubMed Scopus (207) Google Scholar,15Tanaka T. Saha S.K. Tomomori C. Ishima R. Liu D. Tong K.I. Park H. Dutta R. Qin L. Swindells M.B. Yamazaki T. Ono A.M. Kainosho M. Inouye M. Ikura M. Nature. 1998; 396: 88-92Crossref PubMed Scopus (223) Google Scholar). Recently it has been demonstrated that domain A by itself can serve as the OmpR-P phosphatase, and it has been proposed that the phosphatase activity of EnvZ is modulated by the spatial arrangement between domain A and domain B (16Zhu Y. Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7808-7813Crossref PubMed Scopus (102) Google Scholar). The response regulator OmpR consists of an N-terminal CheY-like receiver domain containing the highly conserved Asp-55 phosphorylation site and a C-terminal DNA-binding domain (17Kato M. Aiba H. Tate S. Nishimura Y. Mizuno T. FEBS Lett. 1989; 249: 168-172Crossref PubMed Scopus (44) Google Scholar). The two domains are connected by a flexible linker (18Kenney L.J. Bauer M.D. Silhavy T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8866-8870Crossref PubMed Scopus (85) Google Scholar). The x-ray structure of the DNA binding domain shows a winged helix-turn-helix motif structure (19Martinez-Hackert E. Stock A.M. Structure. 1997; 5: 109-124Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar,20Kondo H. Nakagawa A. Nishihira J. Nishimura Y. Mizuno T. Tanaka I. Nat. Struct. Biol. 1997; 4: 28-31Crossref PubMed Scopus (83) Google Scholar). The phosphorylation of OmpR at Asp-55 greatly enhances its binding affinity for the regulatory sequences upstream of the ompFand ompC promoters (21Head C.G. Tardy A. Kenney L.J. J. Mol. Biol. 1998; 281: 857-870Crossref PubMed Scopus (122) Google Scholar, 22Forst S. Delgado J. Inouye M. J. Bacteriol. 1989; 171: 2949-2955Crossref PubMed Google Scholar, 23Aiba H. Nakasai F. Mizushima S. Mizuno T. J. Biochem. (Tokyo). 1989; 106: 5-7Crossref PubMed Scopus (98) Google Scholar, 24Huang K.J. Igo M.M. J. Mol. Biol. 1996; 262: 615-628Crossref PubMed Scopus (90) Google Scholar). The in vitro andin vivo footprinting studies have demonstrated that OmpR-P binds to the −100 to −38 region (C1, C2, and C3 sites) ofompC and the −380 to the −361 region (F4 site) and the −100 to −39 region (F1, F2, and F3 sites) of ompF(24Huang K.J. Igo M.M. J. Mol. Biol. 1996; 262: 615-628Crossref PubMed Scopus (90) Google Scholar, 25Tsung K. Brissette R.E. Inouye M. J. Biol. Chem. 1989; 264: 10104-10109Abstract Full Text PDF PubMed Google Scholar, 26Rampersaud A. Harlocker S.L. Inouye M. J. Biol. Chem. 1994; 269: 12559-12566Abstract Full Text PDF PubMed Google Scholar, 27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar). These OmpR binding sites consist of 20 base pairs each sharing a consensus sequence (28Harlocker S.L. Bergstrom L. Inouye M. J. Biol. Chem. 1995; 270: 26849-26856Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). It has been shown that two OmpR-P molecules bind to each site in a head-to-tail manner (28Harlocker S.L. Bergstrom L. Inouye M. J. Biol. Chem. 1995; 270: 26849-26856Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 29Harrison-McMonagle P. Denissova N. Martinez-Hackert E. Ebright R.H. Stock A.M. J. Mol. Biol. 1999; 285: 555-566Crossref PubMed Scopus (44) Google Scholar). The binding affinities of OmpR-P to these sites are in a hierarchical order such that OmpR-P independently binds to F1 and C1 sites whereas OmpR-P cooperatively binds to other sites only after the upstream F1 and C1 sites are occupied by OmpR-P (21Head C.G. Tardy A. Kenney L.J. J. Mol. Biol. 1998; 281: 857-870Crossref PubMed Scopus (122) Google Scholar, 27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar). The ompB operon of E. coli is comprised of two genes, ompR and envZ, which are co-transcribed as a polycistronic mRNA from a promoter located 5′ to theompR gene (30Hall M.N. Silhavy T.J. J. Mol. Biol. 1981; 151: 1-15Crossref PubMed Scopus (159) Google Scholar). It has been shown that significantly fewer EnvZ molecules are produced compared with OmpR molecules in a cell, and it has been estimated that there are about 1000 molecules of OmpR whereas there are only 10 molecules of EnvZ per cell (5Pratt L. Silhavy T.J. Hoch J. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology Press, Washington, D. C.1995: 105-107Google Scholar). In this report, we reexamined the cellular OmpR and EnvZ levels under different growth conditions by quantitative Western blot analysis. We also determined the dissociation constant of OmpR and EnvZ by using His10-OmpR and Ni-NTA-agarose. These findings provide new insights into the mechanism by which the EnvZ/OmpR system regulates the osmosensory pathway in E. coli. DISCUSSIONIn the present study, OmpR and EnvZ levels in E. colicells grown in L-broth at mid-log phase were determined to be 3500 and 100 molecules per cell, respectively. OmpR and EnvZ levels were almost the same from cells grown in L-broth medium or in a high osmolarity medium (NB + 20% sucrose). However, when cells were grown in a low osmolarity medium (NB), both OmpR and EnvZ levels were about 60% of those in the high osmolarity medium. The mechanism for the differential expression of the ompB locus under different osmolarities remains to be elucidated. The autoregulation of theompB locus has been reported, where the ompBexpression is regulated by OmpR, the first product of theompB operon (35Liljestrom P. Laamanen I. Palva E.T J. Mol. Biol. 1988; 201: 663-673Crossref PubMed Scopus (46) Google Scholar). It should be noted that under all the different growth conditions used, the ratio of OmpR to EnvZ was almost constant, and OmpR molecules existed in 30–35-fold excess over EnvZ monomers or in 60–70-fold excess compared with EnvZ dimers. It has been reported that translation of OmpR mRNA is ten times more efficient than that of EnvZ using lacZ as a reporter inSalmonella typhimurium (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar). Because bothenvZ and ompR genes are transcribed as a single polycistronic mRNA (30Hall M.N. Silhavy T.J. J. Mol. Biol. 1981; 151: 1-15Crossref PubMed Scopus (159) Google Scholar), the expression of the two genes are differentially regulated at the level of translation initiation (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar).The apparent K d value of EnvZc and His10-OmpR interaction was 1.20 ± 0.17 μm under the conditions used. Assuming the cell volume is 10−15 liters (37Wanner B.L. Neidhardt F.C. Escherichia coli and Salmonella. 1. American Society of Microbiology, Washington, D. C.1996: 1359Google Scholar), the cellular concentrations of OmpR and EnvZ were calculated to be 6 and 0.18 μm, respectively. Therefore on the basis of the K d value thus obtained, ∼85% of EnvZ molecules in a cell are estimated to be occupied with OmpR. It is important to note that as OmpR molecules exist in concentrations 60–70-fold higher than EnvZ dimers, OmpR-P molecules formed on EnvZ dimers are always competed with free OmpR molecules in the cytoplasmic fraction. It has been demonstrated that OmpR-P has a much higher affinity for the ompF andompC promoter regions than OmpR, so that once the OmpR-P molecules released into the cytoplasm are bound to the ompFand ompC promoter regions they are readily sequestered from the EnvZ phosphatase activity (38Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 908-913PubMed Google Scholar).Previous reports (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar) have proposed that at low osmolarity, OmpR-P binds to the C1 site and cooperatively to F1, F2, and somewhat loosely to F3 sites resulting only in ompF expression, whereas at high osmolarity, the concentration of OmpR-P increases allowing C2, C3, and F4 sites (the repressor site for the ompF expression) to be also occupied by OmpR-P, resulting in the induction ofompC expression and repression of ompFexpression. In this fashion, the reciprocal expression ofompF and ompC is achieved depending upon the medium osmolarity. According to the footprinting experiment, F1, F2, and a significant fraction of the F3 site were protected when the OmpR-P concentrations were between 108 and 135 nm whereas all C1, C2, and C3 sites became protected only when the OmpR-P concentration reached 540 nm (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar), assuming that the OmpR added was fully phosphorylated. Assuming that these results may be applied in vivo, at low osmolarity the phosphorylation of only 3.5% (120 nm or 70 OmpR-P molecules/cell) of total OmpR molecules in a cell (2024 OmpR molecules per cell) would be enough to activate the expression of ompF, whereas at high osmolarity the phosphorylation of about 10% (580 nm or 350 OmpR-P molecules/cell) of total OmpR molecules in a cell (3500 molecules per cell) would be sufficient to promote the expression ofompC and to repress the expression of ompF. Clearly the 60 EnvZ monomers or 30 EnvZ dimers per cell at low osmolarity and 100 EnvZ monomers or 50 EnvZ dimers per cell at high osmolarity are sufficient to fulfill the effective regulation ofompF and ompC. On the basis of OmpR amounts it has been shown that the OmpR-P level at high osmolarity was about 4-fold higher than that at low osmolarity (39Forst S. Delgado J. Rampersaud A. Inouye M. J. Bacteriol. 1990; 172: 3473-3477Crossref PubMed Google Scholar). Considering that the cellular OmpR level is increased by more than 70% at high osmolarity than that at low osmolarity, the actual OmpR-P level could be increased more than 7-fold at high osmolarity in comparison with the level of OmpR-P at low osmolarity. In addition, the cell volume increases at low osmolarity whereas it decreases at high osmolarity resulting in even more dramatic changes in the OmpR-P concentration. This sharp change of the OmpR-P concentration appears to account well for the expression patterns of ompF and ompC at high and low osmolarity. However, the regulation of ompF andompC in the cell may be more complex, as there are cross-talks among the His-Asp phosphorelay systems (40Wanner B.L. J. Bacteriol. 1992; 174: 2053-2058Crossref PubMed Google Scholar), and other genes besides ompF and ompC are known to be regulated by OmpR-P (41Verhoef C. Lugtenberg B. van Boxtel R. de Graaff P. Verheij H. Mol. Gen. Genet. 1979; 169: 137-146Crossref PubMed Scopus (44) Google Scholar, 42Shin S. Park C. J. Bacteriol. 1995; 177: 4696-4702Crossref PubMed Google Scholar, 43Mao W. Siegele D.A. Mol. Microbiol. 1998; 27: 415-424Crossref PubMed Scopus (12) Google Scholar).A similar quantitative Western blot analysis has been carried out with the proteins involved in chemotaxis signal transduction. The cellular contents of the total MCPs (methyl-accepting chemotaxis protein), CheA, CheY, and CheW were estimated to be 6000 molecules each per cell inE. coli (44Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 45Kuo S.C. Koshland D.E., Jr. J. Bacteriol. 1987; 169: 1307-1314Crossref PubMed Google Scholar). However it is important to know that CheA belongs to Class II histidine kinases, whereas EnvZ belongs to Class I, the major signal transducing kinases in E. coli as 29 of 30 histidine kinases in E. coli belong to Class I (46Mizuno T. DNA Res. 1997; 4: 161-168Crossref PubMed Scopus (298) Google Scholar). Class II kinases are involved in regulating flagella rotation whereas Class I kinases regulate transcription of specific genes (47Yamaguchi S. Aizawa S. Kihara M. Isomura M. Jones C.J. Macnab R.M. J. Bacteriol. 1986; 168: 1172-1179Crossref PubMed Google Scholar). The present report is the first to accurately estimate the cellular contents of a Class I kinase and its cognate response regulator, suggesting that the Class I kinase system is substantially different from the Class II kinase system in their regulatory mechanisms.The interaction between EnvZ and OmpR-P is not discussed in the present study. A recent report concerning this issue raised a question about the role of EnvZ phosphatase activity in osmoregulation (48Mattison K. Kenney L. J. Biol. Chem. 2002; 277: 11143-11148Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We have reexamined the OmpR-P interaction with EnvZc, and these results will be presented elsewhere. The characterization of the OmpR-P-EnvZ interaction provides important insights into the bi-functional roles of EnvZ in the osmoregulation of ompC and ompF. In the present study, OmpR and EnvZ levels in E. colicells grown in L-broth at mid-log phase were determined to be 3500 and 100 molecules per cell, respectively. OmpR and EnvZ levels were almost the same from cells grown in L-broth medium or in a high osmolarity medium (NB + 20% sucrose). However, when cells were grown in a low osmolarity medium (NB), both OmpR and EnvZ levels were about 60% of those in the high osmolarity medium. The mechanism for the differential expression of the ompB locus under different osmolarities remains to be elucidated. The autoregulation of theompB locus has been reported, where the ompBexpression is regulated by OmpR, the first product of theompB operon (35Liljestrom P. Laamanen I. Palva E.T J. Mol. Biol. 1988; 201: 663-673Crossref PubMed Scopus (46) Google Scholar). It should be noted that under all the different growth conditions used, the ratio of OmpR to EnvZ was almost constant, and OmpR molecules existed in 30–35-fold excess over EnvZ monomers or in 60–70-fold excess compared with EnvZ dimers. It has been reported that translation of OmpR mRNA is ten times more efficient than that of EnvZ using lacZ as a reporter inSalmonella typhimurium (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar). Because bothenvZ and ompR genes are transcribed as a single polycistronic mRNA (30Hall M.N. Silhavy T.J. J. Mol. Biol. 1981; 151: 1-15Crossref PubMed Scopus (159) Google Scholar), the expression of the two genes are differentially regulated at the level of translation initiation (36Berman M.L. Jackson D.E. J. Bacteriol. 1984; 159: 750-756Crossref PubMed Google Scholar). The apparent K d value of EnvZc and His10-OmpR interaction was 1.20 ± 0.17 μm under the conditions used. Assuming the cell volume is 10−15 liters (37Wanner B.L. Neidhardt F.C. Escherichia coli and Salmonella. 1. American Society of Microbiology, Washington, D. C.1996: 1359Google Scholar), the cellular concentrations of OmpR and EnvZ were calculated to be 6 and 0.18 μm, respectively. Therefore on the basis of the K d value thus obtained, ∼85% of EnvZ molecules in a cell are estimated to be occupied with OmpR. It is important to note that as OmpR molecules exist in concentrations 60–70-fold higher than EnvZ dimers, OmpR-P molecules formed on EnvZ dimers are always competed with free OmpR molecules in the cytoplasmic fraction. It has been demonstrated that OmpR-P has a much higher affinity for the ompF andompC promoter regions than OmpR, so that once the OmpR-P molecules released into the cytoplasm are bound to the ompFand ompC promoter regions they are readily sequestered from the EnvZ phosphatase activity (38Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 908-913PubMed Google Scholar). Previous reports (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar) have proposed that at low osmolarity, OmpR-P binds to the C1 site and cooperatively to F1, F2, and somewhat loosely to F3 sites resulting only in ompF expression, whereas at high osmolarity, the concentration of OmpR-P increases allowing C2, C3, and F4 sites (the repressor site for the ompF expression) to be also occupied by OmpR-P, resulting in the induction ofompC expression and repression of ompFexpression. In this fashion, the reciprocal expression ofompF and ompC is achieved depending upon the medium osmolarity. According to the footprinting experiment, F1, F2, and a significant fraction of the F3 site were protected when the OmpR-P concentrations were between 108 and 135 nm whereas all C1, C2, and C3 sites became protected only when the OmpR-P concentration reached 540 nm (27Bergstrom L.C. Qin L. Harlocker S.L. Egger L.A. Inouye M. Genes Cells. 1998; 3: 777-788Crossref PubMed Scopus (32) Google Scholar), assuming that the OmpR added was fully phosphorylated. Assuming that these results may be applied in vivo, at low osmolarity the phosphorylation of only 3.5% (120 nm or 70 OmpR-P molecules/cell) of total OmpR molecules in a cell (2024 OmpR molecules per cell) would be enough to activate the expression of ompF, whereas at high osmolarity the phosphorylation of about 10% (580 nm or 350 OmpR-P molecules/cell) of total OmpR molecules in a cell (3500 molecules per cell) would be sufficient to promote the expression ofompC and to repress the expression of ompF. Clearly the 60 EnvZ monomers or 30 EnvZ dimers per cell at low osmolarity and 100 EnvZ monomers or 50 EnvZ dimers per cell at high osmolarity are sufficient to fulfill the effective regulation ofompF and ompC. On the basis of OmpR amounts it has been shown that the OmpR-P level at high osmolarity was about 4-fold higher than that at low osmolarity (39Forst S. Delgado J. Rampersaud A. Inouye M. J. Bacteriol. 1990; 172: 3473-3477Crossref PubMed Google Scholar). Considering that the cellular OmpR level is increased by more than 70% at high osmolarity than that at low osmolarity, the actual OmpR-P level could be increased more than 7-fold at high osmolarity in comparison with the level of OmpR-P at low osmolarity. In addition, the cell volume increases at low osmolarity whereas it decreases at high osmolarity resulting in even more dramatic changes in the OmpR-P concentration. This sharp change of the OmpR-P concentration appears to account well for the expression patterns of ompF and ompC at high and low osmolarity. However, the regulation of ompF andompC in the cell may be more complex, as there are cross-talks among the His-Asp phosphorelay systems (40Wanner B.L. J. Bacteriol. 1992; 174: 2053-2058Crossref PubMed Google Scholar), and other genes besides ompF and ompC are known to be regulated by OmpR-P (41Verhoef C. Lugtenberg B. van Boxtel R. de Graaff P. Verheij H. Mol. Gen. Genet. 1979; 169: 137-146Crossref PubMed Scopus (44) Google Scholar, 42Shin S. Park C. J. Bacteriol. 1995; 177: 4696-4702Crossref PubMed Google Scholar, 43Mao W. Siegele D.A. Mol. Microbiol. 1998; 27: 415-424Crossref PubMed Scopus (12) Google Scholar). A similar quantitative Western blot analysis has been carried out with the proteins involved in chemotaxis signal transduction. The cellular contents of the total MCPs (methyl-accepting chemotaxis protein), CheA, CheY, and CheW were estimated to be 6000 molecules each per cell inE. coli (44Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 45Kuo S.C. Koshland D.E., Jr. J. Bacteriol. 1987; 169: 1307-1314Crossref PubMed Google Scholar). However it is important to know that CheA belongs to Class II histidine kinases, whereas EnvZ belongs to Class I, the major signal transducing kinases in E. coli as 29 of 30 histidine kinases in E. coli belong to Class I (46Mizuno T. DNA Res. 1997; 4: 161-168Crossref PubMed Scopus (298) Google Scholar). Class II kinases are involved in regulating flagella rotation whereas Class I kinases regulate transcription of specific genes (47Yamaguchi S. Aizawa S. Kihara M. Isomura M. Jones C.J. Macnab R.M. J. Bacteriol. 1986; 168: 1172-1179Crossref PubMed Google Scholar). The present report is the first to accurately estimate the cellular contents of a Class I kinase and its cognate response regulator, suggesting that the Class I kinase system is substantially different from the Class II kinase system in their regulatory mechanisms. The interaction between EnvZ and OmpR-P is not discussed in the present study. A recent report concerning this issue raised a question about the role of EnvZ phosphatase activity in osmoregulation (48Mattison K. Kenney L. J. Biol. Chem. 2002; 277: 11143-11148Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We have reexamined the OmpR-P interaction with EnvZc, and these results will be presented elsewhere. The characterization of the OmpR-P-EnvZ interaction provides important insights into the bi-functional roles of EnvZ in the osmoregulation of ompC and ompF. We thank Ling Qin for supplying His10-OmpR and Yan Zhu for providing EnvZc. We are grateful to Ling Qin, Yan Zhu and Takeshi Yoshida for their critical reading of the manuscript.