Escherichia coli K-12 substr. MG1655 Enzyme: serine/threonine kinase HipA

Gene: hipA Accession Numbers: EG10443 (EcoCyc), b1507, ECK1500

Regulation Summary Diagram: ?

Regulation summary diagram for hipA

Component of: HipAB toxin/antitoxin complex and DNA-binding transcriptional repressor (extended summary available)

Alternative forms of serine/threonine kinase HipA: phosphorylated serine/threonine kinase HipA

HipA is a serine/threonine kinase that mediates the phenomenon of persistence in E. coli. A small fraction of cells within a population are dormant persister cells; these cells are phenotypic variants that are not killed by antibiotics, leading to multidrug tolerance (MDT). The HipAB system can be categorized as a toxin/antitoxin module.

HipA expression was shown to activate ppGpp synthesis by RelA, which then leads to inhibition of macromolecular synthesis, triggering growth arrest, and enabling resistance to β-lactam antibiotics [Bokinsky13]. ppGpp synthesis may be triggered by an increased concentration of uncharged tRNAGlu that is caused by HipA inhibition of GluRS activity [Germain13]. Autophosphorylation of HipA may be the crucial initial event that allows persister cells to revert to a growth phenotype [Schumacher12].

HipA is toxic in the absence of its binding partner HipB, a transcriptional repressor [Black91, Black94, Rotem10]. Expression of HipA leads to growth arrest due to a shutdown of macromolecular synthesis, but does not cause cell death [Korch06]. The HipA-mediated phenotypic switch has been investigated at the single cell level [Balaban04], and a mathematical model has been developed [Lou08]. Above a certain threshold, the level of HipA in the cell determines the typical duration of growth arrest. The threshold level is determined by the level of HipB [Rotem10].

Modeling suggests that phenotypic variation in persister levels within a population can be due to different levels of noise in the regulation of the gene circuit [Koh12].

HipA is a member of the PI 3/4-kinase superfamily, capable of autophosphorylation at Ser150. This activity depends on two aspartates, one in the active site at Asp309, the other binding the Mg2+ cofactor at Asp332. This phosphorylation activity is required both for HipA toxicity and for antibiotic resistance conferred by overexpression of HipA [Correia06]. One target of the kinase activity was suggested to be EF-Tu, which is inactivated by phosphorylation at Thr382 [Schumacher09]. However, it was later shown that the in vivo target of HipA is glutamyl-tRNA synthetase (GluRS). HipA phosphorylates the conserved Ser239 residue near the active site of GluRS and inhibits its aminoacylation activity [Germain13].

Unlike in other kinases, the autophosphorylated Ser150 residue is not located in a solvent-accessible loop, but in the catalytic core P-loop of the protein. Crystal structures revealed that this P loop exists in an "in-out" conformational equilibrium, thereby allowing access to the residue for intermolecular autophosphorylation. Phosphorylation of Ser150 appears to stabilize the "out" state and thereby inactivates HipA [Schumacher12].

Although HipA does not itself bind the hip regulatory region, it plays an indirect role in the regulation of hipBA transcription via its binding to HipB [Black91, Black94]. Binding to HipB may lock HipA into a catalytically inactive conformation [Schumacher09].

A variety of crystal structures of HipA, including in the apo form, in complex with ATP or ADP, and in a complex with DNA and HipB have been solved [Schumacher09, Evdokimov09, Schumacher12].

The hipA7 allele that was initially isolated [Moyed83] is a gain-of-function mutant that contains two amino acid changes [Korch03], one of which likely results in weakened interaction between HipA and HipB and thus resulting in higher HipA kinase activity [Schumacher09].

A hipBA deletion strain shows a decrease in stationary phase persister cells after treatment with antibiotics [Keren04] and a decrease in biofilm formation even in the absence of antibiotics [Zhao13a]. Expression of wild-type hipA in excess of hipB results in the shutdown of macromolecular synthesis and a high frequency of persister formation [Korch06]. Overexpression of gltX suppresses HipA toxicity [Germain13]. Ectopic expression of HipA leads to expression of the relBEF toxin/antitoxin module [Kasari13]. HipA enhances repression of the relA promoter by HipB [Lin13].

hipA is most highly expressed during stationary phase [Kawano09].

HipA: "high persistence factor A" [Moyed83]

Reviews: [Yamaguchi11, Lewis08, Jayaraman08, Lewis07, Lewis05a]

Citations: [Bruel12, Keren12, Lewis12]

Locations: cytosol

Map Position: [1,588,878 <- 1,590,200] (34.25 centisomes, 123°)
Length: 1323 bp / 440 aa

Molecular Weight of Polypeptide: 49.276 kD (from nucleotide sequence), 50.0 kD (experimental) [Moyed86 ]

Unification Links: ASAP:ABE-0005022 , DIP:DIP-9898N , EchoBASE:EB0438 , EcoGene:EG10443 , EcoliWiki:b1507 , Mint:MINT-1292260 , OU-Microarray:b1507 , PortEco:hipA , PR:PRO_000022889 , Pride:P23874 , Protein Model Portal:P23874 , RefSeq:NP_416024 , RegulonDB:EG10443 , SMR:P23874 , String:511145.b1507 , UniProt:P23874

Relationship Links: InterPro:IN-FAMILY:IPR012893 , InterPro:IN-FAMILY:IPR012894 , InterPro:IN-FAMILY:IPR017508 , PDB:RELATED-TO:3DNV , PDB:Structure:2WIU , PDB:Structure:3DNT , PDB:Structure:3DNU , PDB:Structure:3FBR , PDB:Structure:3HZI , PDB:Structure:3TPB , PDB:Structure:3TPD , PDB:Structure:3TPE , PDB:Structure:3TPT , PDB:Structure:3TPV , Pfam:IN-FAMILY:PF07804 , Pfam:IN-FAMILY:PF07805 , Pfam:IN-FAMILY:PF13657

In Paralogous Gene Group: 156 (3 members)

Reactions known to consume the compound:

Not in pathways:
serine/threonine kinase HipA + ATP → phosphorylated serine/threonine kinase HipA + ADP

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for hipA

GO Terms:

Biological Process: GO:0022611 - dormancy process Inferred from experiment [Korch06]
GO:0036289 - peptidyl-serine autophosphorylation Inferred from experiment [Correia06]
GO:0043086 - negative regulation of catalytic activity Inferred from experiment [Germain13]
GO:0044010 - single-species biofilm formation Inferred from experiment [Zhao13a]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11a]
GO:0046677 - response to antibiotic Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Schumacher09]
GO:0004674 - protein serine/threonine kinase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Correia06]
GO:0005515 - protein binding Inferred from experiment [Black94, Schumacher09]
GO:0005524 - ATP binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Schumacher09]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003677 - DNA binding Inferred by computational analysis [UniProtGOA11a]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: information transfer protein related posttranslational modification
information transfer protein related translation
regulation type of regulation posttranscriptional covalent modification, demodification, maturation

Essentiality data for hipA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Last-Curated ? 31-Oct-2013 by Keseler I , SRI International

Enzymatic reaction of: serine/threonine kinase

EC Number: 2.7.11.-

serine/threonine kinase HipA + ATP <=> phosphorylated serine/threonine kinase HipA + ADP

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Cofactors or Prosthetic Groups: Mg2+ [Correia06]

Subunit of: HipAB toxin/antitoxin complex and DNA-binding transcriptional repressor

Synonyms: HipBA

Subunit composition of HipAB toxin/antitoxin complex and DNA-binding transcriptional repressor = [(HipB)2][HipA]2
         HipB antitoxin and DNA-binding transcriptional repressor = (HipB)2
         serine/threonine kinase HipA = HipA (extended summary available)

The transcriptional repressor HipB, for "High persistence," is negatively autoregulated and controls the transcription of a critical persistence factor [Kawano09, Lewis05a, Keren04, Black91, Korch03, Balaban04]. hipB foms an operon with hipA, and the products of this operon are classified as a toxin (HipA)-antitoxin (HipB) system. This HipAB system is involved in the high persistence, which is the capacity of the bacteria to survive prolonged exposure to antibiotics [Balaban04, Inouye06], and Kawano et al. also showed that this system is important for survival during the long-term stationary phase [Kawano09, Lewis05a, Keren04].

The crystal structure of the HipB-HipA-DNA complex has been solved at 2.68 Å resolution [Schumacher09]. The complex is tetrameric and is comprised of a HipB homodimer that interacts with DNA, sandwiched by a monomer of HipA on each side [Schumacher09]. In the HipB-HipA complex, each monomer of HipA interacts with two monomers of HipB [Schumacher09, Evdokimov09]. HipB is an antitoxin that counteracts and neutralizes the HipA toxin, and HipA is toxic in the absence of its binding partner HipB [Schumacher09, Black94, Black91]. Although HipA does not bind the Hip regulatory region, it plays an indirect role via its binding to HipB.

HipB contains a helix-turn-helix motif near the N terminus and is a Cro-like DNA-binding protein. It binds to four operator sites with the conserved inverted repeat sequence motif TATCCN8GGATA, and its binding occurs cooperatively and almost simultaneously on the same face of the DNA helix. HipB forms dimers in solution [Black94, Schumacher09].

Reviews: [Yamaguchi11, Jayaraman08]

Relationship Links: PDB:Structure:3DNV , PDB:Structure:3DNW

GO Terms:

Biological Process: GO:0045892 - negative regulation of transcription, DNA-templated Inferred from experiment [Black94]
Molecular Function: GO:0043565 - sequence-specific DNA binding Inferred from experiment [Black94]

DNA binding site length: 20 base-pairs

Symmetry: Inverted Repeat

Consensus DNA Binding Sequence: tTATCCgctatAGCGGATAa

Regulated Transcription Units (2 total): ?


Transcription-unit diagram

Transcription-unit diagram

Sequence Features

Protein sequence of serine/threonine kinase HipA with features indicated

Feature Class Location Common Name Attached Group Citations Comment State
Mutagenesis-Variant 22    
[Korch03, UniProt11]
UniProt: Loss of toxicity, does not confer high persistence. Loss of toxicity, high persistence and cold sensitivity; in hipA7; when associated with A-291.
Sequence-Conflict 40    
[Black91, UniProt10]
UniProt: (in Ref. 1; AAA56878);
Mutagenesis-Variant 88    
[Korch03, UniProt11]
UniProt: Loss of toxicity, still confers high persistence.
Mutagenesis-Variant 150    
[Correia06, UniProt11]
UniProt: No phosphorylation; cells grow normally.
Phosphorylation-Modification 150 Phosphorylation site  
[Schumacher12, Correia06]
Nucleotide-Phosphate-Binding-Region 152 -> 157   ATP
[Schumacher12, UniProt15]
UniProt: ATP.
Amino-Acid-Sites-That-Bind 181    
[Schumacher12, UniProt15]
UniProt: ATP.
Sequence-Conflict 214 -> 215    
[Black91, UniProt10]
UniProt: (in Ref. 1; AAA56878);
Nucleotide-Phosphate-Binding-Region 234 -> 236   ATP
[Schumacher12, UniProt15]
UniProt: ATP.
Sequence-Conflict 274    
[Black91, UniProt10]
UniProt: (in Ref. 1; AAA56878);
Mutagenesis-Variant 291    
[Korch03, UniProt11]
UniProt: Retains toxicity and high persistence but not cold-sensitive. Loss of toxicity, high persistence and cold sensitivity; in hipA7; when associated with S-22.
Mutagenesis-Variant 309    
[Correia06, UniProt11]
UniProt: Loss of autophosphorylation; cells grow normally; protein can accumulate to high levels in E.coli.
Active-Site 309 kinase active site  
This aspartate is critical for kinase activity [Correia06].
Nucleotide-Phosphate-Binding-Region 311 -> 314   ATP
[Schumacher12, UniProt15]
UniProt: ATP.
Nucleotide-Phosphate-Binding-Region 331 -> 332   ATP
[Schumacher12, UniProt15]
UniProt: ATP.
Mutagenesis-Variant 332    
[Correia06, UniProt11]
UniProt: Loss of autophosphorylation; cells grow normally.
Metal-Binding-Site 332 Magnesium binding site Mg2+
DNA-Binding-Region 379 -> 382    

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


1/22/1998 (pkarp) Merged genes G7995/hipA and EG10443/hipA
Markus Krummenacker on Mon Oct 20, 1997:
The location of this gene on the E. Coli chromosome has not yet been determined.


Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Balaban04: Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S (2004). "Bacterial persistence as a phenotypic switch." Science 305(5690);1622-5. PMID: 15308767

Black91: Black DS, Kelly AJ, Mardis MJ, Moyed HS (1991). "Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis." J Bacteriol 173(18);5732-9. PMID: 1715862

Black94: Black DS, Irwin B, Moyed HS (1994). "Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis." J Bacteriol 1994;176(13);4081-91. PMID: 8021189

Bokinsky13: Bokinsky G, Baidoo EE, Akella S, Burd H, Weaver D, Alonso-Gutierrez J, Garcia-Martin H, Lee TS, Keasling JD (2013). "HipA-triggered growth arrest and β-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis." J Bacteriol 195(14);3173-82. PMID: 23667235

Bruel12: Bruel N, Castanie-Cornet MP, Cirinesi AM, Koningstein G, Georgopoulos C, Luirink J, Genevaux P (2012). "Hsp33 controls elongation factor-Tu stability and allows Escherichia coli growth in the absence of the major DnaK and trigger factor chaperones." J Biol Chem 287(53);44435-46. PMID: 23148222

Correia06: Correia FF, D'Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, Koonin EV, Lewis K (2006). "Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli." J Bacteriol 188(24);8360-7. PMID: 17041039

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Evdokimov09: Evdokimov A, Voznesensky I, Fennell K, Anderson M, Smith JF, Fisher DA (2009). "New kinase regulation mechanism found in HipBA: a bacterial persistence switch." Acta Crystallogr D Biol Crystallogr 65(Pt 8);875-9. PMID: 19622872

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

Germain13: Germain E, Castro-Roa D, Zenkin N, Gerdes K (2013). "Molecular mechanism of bacterial persistence by HipA." Mol Cell 52(2);248-54. PMID: 24095282

Inouye06: Inouye M (2006). "The discovery of mRNA interferases: implication in bacterial physiology and application to biotechnology." J Cell Physiol 209(3);670-6. PMID: 17001682

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Jayaraman08: Jayaraman R (2008). "Bacterial persistence: some new insights into an old phenomenon." J Biosci 33(5);795-805. PMID: 19179767

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kasari13: Kasari V, Mets T, Tenson T, Kaldalu N (2013). "Transcriptional cross-activation between toxin-antitoxin systems of Escherichia coli." BMC Microbiol 13(1);45. PMID: 23432955

Kawano09: Kawano H, Hirokawa Y, Mori H (2009). "Long-term survival of Escherichia coli lacking the HipBA toxin-antitoxin system during prolonged cultivation." Biosci Biotechnol Biochem 73(1);117-23. PMID: 19129642

Keren04: Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004). "Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli." J Bacteriol 186(24);8172-80. PMID: 15576765

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Koh12: Koh RS, Dunlop MJ (2012). "Modeling suggests that gene circuit architecture controls phenotypic variability in a bacterial persistence network." BMC Syst Biol 6;47. PMID: 22607777

Korch03: Korch SB, Henderson TA, Hill TM (2003). "Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis." Mol Microbiol 50(4);1199-213. PMID: 14622409

Korch06: Korch SB, Hill TM (2006). "Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation." J Bacteriol 188(11);3826-36. PMID: 16707675

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Lewis08: Lewis K (2008). "Multidrug tolerance of biofilms and persister cells." Curr Top Microbiol Immunol 322;107-31. PMID: 18453274

Lewis12: Lewis K (2012). "Persister cells: molecular mechanisms related to antibiotic tolerance." Handb Exp Pharmacol (211);121-33. PMID: 23090599

Lin13: Lin CY, Awano N, Masuda H, Park JH, Inouye M (2013). "Transcriptional Repressor HipB Regulates the Multiple Promoters in Escherichia coli." J Mol Microbiol Biotechnol 23(6);440-447. PMID: 24089053

Lou08: Lou C, Li Z, Ouyang Q (2008). "A molecular model for persister in E. coli." J Theor Biol 255(2);205-9. PMID: 18721814

Moyed83: Moyed HS, Bertrand KP (1983). "hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis." J Bacteriol 155(2);768-75. PMID: 6348026

Moyed86: Moyed HS, Broderick SH (1986). "Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis." J Bacteriol 166(2);399-403. PMID: 3516974

Rotem10: Rotem E, Loinger A, Ronin I, Levin-Reisman I, Gabay C, Shoresh N, Biham O, Balaban NQ (2010). "Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence." Proc Natl Acad Sci U S A 107(28);12541-6. PMID: 20616060

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Schumacher12: Schumacher MA, Min J, Link TM, Guan Z, Xu W, Ahn YH, Soderblom EJ, Kurie JM, Evdokimov A, Moseley MA, Lewis K, Brennan RG (2012). "Role of unusual P loop ejection and autophosphorylation in HipA-mediated persistence and multidrug tolerance." Cell Rep 2(3);518-25. PMID: 22999936

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-11 released on 2011-11-22 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Yamaguchi11: Yamaguchi Y, Park JH, Inouye M (2011). "Toxin-antitoxin systems in bacteria and archaea." Annu Rev Genet 45;61-79. PMID: 22060041

Zhao13a: Zhao J, Wang Q, Li M, Heijstra BD, Wang S, Liang Q, Qi Q (2013). "Escherichia coli toxin gene hipA affects biofilm formation and DNA release." Microbiology 159(Pt 3);633-40. PMID: 23329678

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 19.0 on Fri Oct 9, 2015, biocyc14.