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PhoB DNA-binding transcriptional dual regulator

Synonyms: PhoB-Phosphorylated, PhoB
PhoB is a dual transcription regulator that activates expression of the Pho regulon in response to environmental Pi. The Pho regulon includes operons and genes whose products are involved in phosphorus uptake and metabolism [3, 19, 20] Expression of the periplasmic binding proteins for peptide transport, OppA and DppA, is repressed by PhoB [21]
PhoB is a response regulator and belongs to the two-component system PhoR/PhoB. Under phosphate limitation conditions the inner membrane sensor kinase PhoR autophosphorylates. Subsequent transfer of the phosphate group to PhoB results in activation of PhoB [22] When phosphate is in excess, autophosphorylation of PhoR is inhibited and PhoB-P is dephosphorylated. This negative regulation requires in addition to PhoR an intact Pst system and PhoU [23] In the absence of PhoR, cross-regulation of PhoB by CreC or acetyl phosphate results in phosphorylation of PhoB in response to carbon sources [24, 25]
PhoB consists of two functional domains, the N-terminal receiver domain, which is phosphorylated, and the C-terminal output domain, which binds to DNA and interacts with the σ70-subunit of RNA polymerase to activate transcription [26, 27] The crystal structure of a subcomplex, which includes the σ4 domain of the σ70 factor fused with the RNAP β-subunit flap tip helix, shows that the σ4 domain is recruited to the pho box promoters by PhoB and reorients the σ4 domain with respect to its binding site [28] The output domain belongs to the winged helix-turn-helix family of transcription factors [29] Its activity is silenced by the receiver domain, and phosphorylation relieves inhibition [30] The 3D structure has been solved for the receiver domain [31]and two constitutively active mutants of this domain [32] as well as that for the output domain [33] Aspartic acid 196 (D196) and arginine 219 (R219), located in the C-terminal DNA-binding domain minor groove, are necessary for the binding process for the PhoB transcriptional regulator [34] Based on biochemical analysis, a positive cooperative binding mechanism of PhoBDBD (DNA-binding domain) to its cognate DNA sequence and a decisive contribution of dimerization to the complex stability were demonstrated [35]
PhoB binds to the Pho box, which has been described as two direct 11-bp repeats consisting of successive 7-bp direct repeats followed by an A/T-rich region of 4 bp, situated 10 bp upstream of the -10 region [10, 16, 36] The pho promoters contain functional -10 sequences but lack the consensus -35 sequence [15]
The 3D structures of the receiver domain in the absence and presence of the phosphoryl analog beryllium fluoride [31, 37] and of two constitutively active mutants of this domain [32] have been solved.
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Transcription factor      
TF conformation(s):
Name Conformation Type TF-Effector Interaction Type Apo/Holo Conformation Evidence (Confirmed, Strong, Weak) References
PhoB Non-Functional   Apo [BPP], [IPI] [1]
PhoB-Phosphorylated Functional Covalent Holo [BPP], [IPI] [1]
Evolutionary Family: OmpR
Sensing class: External-Two-component systems
Connectivity class: Local Regulator
Gene name: phoB
  Genome position: 417142-417831
  Length: 690 bp / 229 aa
Operon name: phoBR
TU(s) encoding the TF:
Transcription unit        Promoter

Regulated gene(s) adiC, amn, argP, asr, cra, cusA, cusB, cusC, cusF, cusR, cusS, eda, feaR, gadW, gadX, mipA, ompF, phnC, phnD, phnE_1, phnE_2, phnF, phnG, phnH, phnI, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, phoA, phoB, phoE, phoH, phoQ, phoR, phoU, pitB, prpR, psiE, psiF, pstA, pstB, pstC, pstS, sbcC, sbcD, tktB, ugpA, ugpB, ugpC, ugpE, ugpQ, waaH, ydfH, yedX, yegH, yhjC
Multifun term(s) of regulated gene(s) MultiFun Term (List of genes associated to the multifun term)
phosphorous metabolism (20)
membrane (13)
Transcription related (11)
activator (8)
repressor (7)
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Regulated operon(s) adiC, amn, argP, asr, cra, cusCFBA, cusRS, edd-eda, feaR, gadAXW, mipA, ompF, phnCDE_1E_2FGHIJKLMNOP, phoA-psiF, phoBR, phoE, phoH, phoPQ, pitB, prpR, psiE, pstSCAB-phoU, sbcDC, talA-tktB, ugpBAECQ, waaH, ydfH, yedX, yegH, yhjC
First gene in the operon(s) adiC, amn, argP, asr, cra, cusC, cusR, eda, feaR, gadX, gadX, mipA, ompF, phnC, phoA, phoB, phoE, phoH, phoQ, phoU, pitB, prpR, psiE, pstS, pstS, pstS, sbcD, tktB, ugpB, ugpB, waaH, ydfH, yedX, yegH, yhjC
Simple and complex regulons ArgP,PhoB
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Simple and complex regulatory phrases Regulatory phrase (List of promoters regulated by the phrase)

Transcription factor binding sites (TFBSs) arrangements       

  Functional conformation Function Promoter Sigma factor Central Rel-Pos Distance to first Gene Genes Sequence LeftPos RightPos Evidence (Confirmed, Strong, Weak) References
  PhoB-Phosphorylated activator adiCp7 Sigma32 nd nd adiC nd nd [GEA] [2]
  PhoB-Phosphorylated activator amnp1 nd -33.0 -63.0 amn
2054989 2055007 [AIBSCS], [GEA] [3]
  PhoB-Phosphorylated activator argPp nd -27.0 -50.0 argP
3059694 3059712 [GEA], [HIBSCS] [4]
  PhoB-Phosphorylated activator asrp Sigma38 -30.0 -79.0 asr
1671288 1671306 [BCE], [GEA], [HIBSCS] [5]
  PhoB-Phosphorylated activator cusCp Sigma70 -54.0 -81.0 cusC, cusF, cusB, cusA
595510 595528 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator cusRp Sigma70 -57.0 -76.0 cusR, cusS
595510 595528 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated repressor edap3 nd -53.0 -81.0 eda
1932827 1932847 [BPP], [GEA], [HIBSCS] [7]
  PhoB-Phosphorylated repressor feaRp2 Sigma70 -17.0 -43.0 feaR
1447317 1447335 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated repressor fruRp8 Sigma32 nd nd cra nd nd [GEA] [2]
  PhoB-Phosphorylated activator gadXp Sigma38 nd nd gadX, gadW nd nd [GEA] [2]
  PhoB-Phosphorylated activator mipAp nd -146.5 -180.5 mipA
1866643 1866662 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator ompFp Sigma38 nd nd ompF nd nd [GEA] [2]
  PhoB-Phosphorylated activator phnCp Sigma70 nd nd phnC, phnD, phnE_1, phnE_2, phnF, phnG, phnH, phnI, phnJ, phnK, phnL, phnM, phnN, phnO, phnP nd nd [BPP], [GEA], [HIBSCS] [2], [8], [9]
  PhoB-Phosphorylated activator phoAp Sigma70 -31.0 -71.0 phoA, psiF
401667 401685 [BCE], [GEA], [SM] [2], [10]
  PhoB-Phosphorylated activator phoBp Sigma70 -31.0 -72.0 phoB, phoR
417061 417079 [BPP], [GEA], [HIBSCS] [2], [6], [10]
  PhoB-Phosphorylated activator phoEp Sigma70 -113.0 -170.5 phoE
260261 260280 [BCE], [GEA], [SM] [2], [11]
  PhoB-Phosphorylated activator phoEp Sigma70 -88.0 -146.0 phoE
260237 260255 [BCE], [GEA], [SM] [2], [11]
  PhoB-Phosphorylated activator phoEp Sigma70 -32.0 -90.0 phoE
260181 260199 [BCE], [GEA], [SM] [2], [11]
  PhoB-Phosphorylated activator phoHp1 Sigma70 -31.0 -158.0 phoH
1084825 1084843 [BPP], [GEA], [HIBSCS] [2], [12]
  PhoB-Phosphorylated activator phoQp5 Sigma24 nd nd phoQ nd nd [GEA] [2]
  PhoB-Phosphorylated activator phoUp Sigma70 nd nd phoU nd nd [GEA] [2]
  PhoB-Phosphorylated repressor pitBp Sigma70 nd nd pitB nd nd [IMP] [13]
  PhoB-Phosphorylated repressor prpRp Sigma70 -24.0 -52.0 prpR
348486 348504 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator psiEp Sigma70 -53.0 -78.0 psiE
4240238 4240256 [BPP], [GEA], [HIBSCS] [14]
  PhoB-Phosphorylated activator psiEp Sigma70 -31.0 -56.0 psiE
4240260 4240278 [BPP], [GEA], [HIBSCS] [14]
  PhoB-Phosphorylated activator pstSp Sigma70 -54.0 -97.0 pstS, pstC, pstA, pstB, phoU
3911613 3911631 [BCE], [GEA], [HIBSCS], [SM] [2], [15], [16]
  PhoB-Phosphorylated activator pstSp Sigma70 -32.0 -75.0 pstS, pstC, pstA, pstB, phoU
3911591 3911609 [BCE], [GEA], [HIBSCS], [SM] [2], [15], [16], [17]
  PhoB-Phosphorylated repressor sbcDp Sigma70 -93.0 -118.0 sbcD, sbcC
417061 417079 [BPP], [GEA], [HIBSCS] [2], [6], [10]
  PhoB-Phosphorylated activator tktBp Sigma38 nd nd tktB nd nd [GEA] [2]
  PhoB-Phosphorylated activator ugpBp1 Sigma70 -52.0 -105.0 ugpB, ugpA, ugpE, ugpC, ugpQ
3592421 3592439 [BPP], [GEA], [HIBSCS] [2], [18]
  PhoB-Phosphorylated activator ugpBp1 Sigma70 -30.0 -83.0 ugpB, ugpA, ugpE, ugpC, ugpQ
3592399 3592417 [BPP], [GEA], [HIBSCS] [2], [18]
  PhoB-Phosphorylated repressor ugpBp2 Sigma70 -26.0 -127.0 ugpB, ugpA, ugpE, ugpC, ugpQ
3592443 3592461 [BPP], [HIBSCS] [18]
  PhoB-Phosphorylated repressor ugpBp2 Sigma70 -4.0 -105.0 ugpB, ugpA, ugpE, ugpC, ugpQ
3592421 3592439 [BPP], [GEA], [HIBSCS] [2], [18]
  PhoB-Phosphorylated activator ydfHp nd -20.0 -46.0 ydfH
1628297 1628315 [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator yedXp Sigma70 nd nd yedX nd nd [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator yegHp nd nd nd yegH nd nd [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator yhjCp Sigma54 nd nd yhjC nd nd [BPP], [GEA], [HIBSCS] [6]
  PhoB-Phosphorylated activator yibDp1 Sigma70 -113.0 -181.0 waaH
3790249 3790275 [AIBSCS], [GEA] [3]
  PhoB-Phosphorylated activator yibDp1 Sigma70 -59.5 -127.5 waaH
3790199 3790218 [AIBSCS], [GEA] [3]

Alignment and PSSM for PhoB TFBSs    

Aligned TFBS of PhoB   

Position weight matrix (PWM).   
A	8	10	4	3	4	5	6	17	1	17	13	12	7	5	0	0	2	9	20	3	13	8
C	8	5	13	0	4	5	12	0	7	2	2	2	0	12	2	0	5	12	2	12	6	2
G	3	1	1	1	12	1	2	3	3	0	0	2	3	3	0	20	1	2	0	2	2	1
T	5	8	6	20	4	13	4	4	13	5	9	8	14	4	22	4	16	1	2	7	3	13

PWM logo   


Evolutionary conservation of regulatory elements    
     Note: Evolutionary conservation of regulatory interactions and promoters is limited to gammaproteobacteria.
TF-target gene evolutionary conservation
Promoter-target gene evolutionary conservation


 [BPP] Binding of purified proteins

 [IPI] Inferred from physical interaction

 [GEA] Gene expression analysis

 [AIBSCS] Automated inference based on similarity to consensus sequences

 [HIBSCS] Human inference based on similarity to consensus sequences

 [BCE] Binding of cellular extracts

 [SM] Site mutation

 [IMP] Inferred from mutant phenotype


 [1] Yamamoto K., Hirao K., Oshima T., Aiba H., Utsumi R., Ishihama A., 2005, Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli., J Biol Chem. 280(2):1448-56

 [2] Marzan LW., Hasan CM., Shimizu K., 2013, Effect of acidic condition on the metabolic regulation of Escherichia coli and its phoB mutant., Arch Microbiol. 195(3):161-71

 [3] Baek JH., Lee SY., 2006, Novel gene members in the Pho regulon of Escherichia coli., FEMS Microbiol Lett. 264(1):104-9

 [4] Han JS., Park JY., Lee YS., Thony B., Hwang DS., 1999, PhoB-dependent transcriptional activation of the iciA gene during starvation for phosphate in Escherichia coli., Mol Gen Genet. 262(3):448-52

 [5] Suziedeliene E., Suziedelis K., Garbenciete V., Normark S., 1999, The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon., J Bacteriol. 181(7):2084-93

 [6] Yang C., Huang TW., Wen SY., Chang CY., Tsai SF., Wu WF., Chang CH., 2012, Genome-wide PhoB binding and gene expression profiles reveal the hierarchical gene regulatory network of phosphate starvation in Escherichia coli., PLoS One. 7(10):e47314

 [7] Murray EL., Conway T., 2005, Multiple regulators control expression of the Entner-Doudoroff aldolase (Eda) of Escherichia coli., J Bacteriol. 187(3):991-1000

 [8] Makino K., Kim SK., Shinagawa H., Amemura M., Nakata A., 1991, Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12., J Bacteriol. 173(8):2665-12

 [9] Wanner BL., Boline JA., 1990, Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli., J Bacteriol. 172(3):1186-96

 [10] Makino K., Shinagawa H., Amemura M., Nakata A., 1986, Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12., J Mol Biol. 190(1):37-44

 [11] Tommassen J., Koster M., Overduin P., 1987, Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein., J Mol Biol. 198(4):633-41

 [12] Kim SK., Makino K., Amemura M., Shinagawa H., Nakata A., 1993, Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli., J Bacteriol. 175(5):1316-24

 [13] Harris RM., Webb DC., Howitt SM., Cox GB., 2001, Characterization of PitA and PitB from Escherichia coli., J Bacteriol. 183(17):5008-14

 [14] Kim SK., Kimura S., Shinagawa H., Nakata A., Lee KS., Wanner BL., Makino K., 2000, Dual transcriptional regulation of the Escherichia coli phosphate-starvation-inducible psiE gene of the phosphate regulon by PhoB and the cyclic AMP (cAMP)-cAMP receptor protein complex., J Bacteriol. 182(19):5596-9

 [15] Kimura S., Makino K., Shinagawa H., Amemura M., Nakata A., 1989, Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene., Mol Gen Genet. 215(3):374-80

 [16] Makino K., Shinagawa H., Amemura M., Kimura S., Nakata A., Ishihama A., 1988, Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro., J Mol Biol. 203(1):85-95

 [17] Otsuka J., Watanabe H., Mori KT., 1996, Evolution of transcriptional regulation system through promiscuous coupling of regulatory proteins with operons; suggestion from protein sequence similarities in Escherichia coli., J Theor Biol. 178(2):183-204

 [18] Kasahara M., Makino K., Amemura M., Nakata A., Shinagawa H., 1991, Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters., J Bacteriol. 173(2):549-58

 [19] Wanner BL., 1993, Gene regulation by phosphate in enteric bacteria., J Cell Biochem. 51(1):47-54

 [20] VanBogelen RA., Olson ER., Wanner BL., Neidhardt FC., 1996, Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli., J Bacteriol. 178(15):4344-66

 [21] Smith MW., Payne JW., 1992, Expression of periplasmic binding proteins for peptide transport is subject to negative regulation by phosphate limitation in Escherichia coli., FEMS Microbiol Lett. 79(1-3):183-90

 [22] Makino K., Shinagawa H., Amemura M., Kawamoto T., Yamada M., Nakata A., 1989, Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins., J Mol Biol. 210(3):551-9

 [23] Wanner BL., 1996, Signal transduction in the control of phosphate-regulated genes of Escherichia coli., Kidney Int. 49(4):964-7

 [24] Wanner BL., Wilmes-Riesenberg MR., 1992, Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli., J Bacteriol. 174(7):2124-30

 [25] Amemura M., Makino K., Shinagawa H., Nakata A., 1990, Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2., J Bacteriol. 172(11):6300-7

 [26] Makino K., Amemura M., Kawamoto T., Kimura S., Shinagawa H., Nakata A., Suzuki M., 1996, DNA binding of PhoB and its interaction with RNA polymerase., J Mol Biol. 259(1):15-26

 [27] Makino K., Amemura M., Kim SK., Nakata A., Shinagawa H., 1993, Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli., Genes Dev. 7(1):149-60

 [28] Blanco AG., Canals A., Bernues J., Sola M., Coll M., 2011, The structure of a transcription activation subcomplex reveals how ¿¿(70) is recruited to PhoB promoters., EMBO J. 30(18):3776-85

 [29] Martinez-Hackert E., Stock AM., 1997, Structural relationships in the OmpR family of winged-helix transcription factors., J Mol Biol. 269(3):301-12

 [30] Ellison DW., McCleary WR., 2000, The unphosphorylated receiver domain of PhoB silences the activity of its output domain., J Bacteriol. 182(23):6592-7

 [31] Sola M., Gomis-Ruth FX., Serrano L., Gonzalez A., Coll M., 1999, Three-dimensional crystal structure of the transcription factor PhoB receiver domain., J Mol Biol. 285(2):675-87

 [32] Arribas-Bosacoma R., Kim SK., Ferrer-Orta C., Blanco AG., Pereira PJ., Gomis-Ruth FX., Wanner BL., Coll M., Sola M., 2007, The X-ray crystal structures of two constitutively active mutants of the Escherichia coli PhoB receiver domain give insights into activation., J Mol Biol. 366(2):626-41

 [33] Martinez-Hackert E., Stock AM., 1997, The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor., Structure. 5(1):109-24

 [34] Ritzefeld M., Wollschlager K., Niemann G., Anselmetti D., Sewald N., 2011, Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study., Mol Biosyst. 7(11):3132-42

 [35] Ritzefeld M., Walhorn V., Kleineberg C., Bieker A., Kock K., Herrmann C., Anselmetti D., Sewald N., 2013, Cooperative binding of PhoB(DBD) to its cognate DNA sequence-a combined application of single-molecule and ensemble methods., Biochemistry. 52(46):8177-86

 [36] Blanco AG., Sola M., Gomis-Ruth FX., Coll M., 2002, Tandem DNA recognition by PhoB, a two-component signal transduction transcriptional activator., Structure. 10(5):701-13

 [37] Bachhawat P., Swapna GV., Montelione GT., Stock AM., 2005, Mechanism of activation for transcription factor PhoB suggested by different modes of dimerization in the inactive and active states., Structure. 13(9):1353-63

 [38] Okamura H., Hanaoka S., Nagadoi A., Makino K., Nishimura Y., 2000, Structural comparison of the PhoB and OmpR DNA-binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box., J Mol Biol. 295(5):1225-36

 [39] Mack TR., Gao R., Stock AM., 2009, Probing the roles of the two different dimers mediated by the receiver domain of the response regulator PhoB., J Mol Biol. 389(2):349-64