Escherichia coli K-12 substr. MG1655 Enzyme: guanosine 3'-diphosphate 5'-triphosphate 3'-diphosphatase [multifunctional]

Gene: spoT Accession Numbers: EG10966 (EcoCyc), b3650, ECK3640

Regulation Summary Diagram: ?

Regulation summary diagram for spoT

SpoT is a key enzyme involved in the stringent response in Escherichia coli. It is a bifunctional enzyme with both hydrolase and synthetase activities [Hernandez91]. The ppGpp hydrolase activity of SpoT has a major physiological role in ppGpp degradation [Somerville79a] and is inhibited under conditions of physiological stress. Its amino acid sequence has been shown to be extensively related to that of RelA [Metzger89]. RelA is involved in the E. coli stringent response triggered by amino acid starvation, activating (p)ppGpp synthesis via a ribosomal mechanism [Wendrich02]. SpoT provides the hydrolase activity for this RelA-dependent synthetase activity.

The physiological role of the RelA-independent, spoT-encoded synthetase activity is less clear. This activity is also referred to as ppGpp synthetase II or PSII, whereas the RelA synthetase activity is referred to as PSI [Hernandez91, Gentry96]. PSII activity was demonstrated in relA null mutants and it was shown that relA/spoT null mutants did not synthesize ppGpp [Xiao91]. The SpoT PSII activity has been proposed to have a role in maintaining the basal level of ppGpp during exponential growth. PSII is relatively inactive during amino acid starvation, is activated by other sources of stress, and is active during exponential growth. In contrast, the RelA-encoded PSI activity is activated by amino acid starvation and has little activity during exponential growth [Murray96].

Although incompletely understood, the regulation of ppGpp levels by the spoT-encoded hydrolase and PSII synthetase activities involves sensing sources of nutrient stress other than amino acid starvation. These include a limitation in fatty acid synthesis [Battesti06], phosphate starvation (which elevates (p)ppGpp levels by a mechanism involving IraP, RssB and RpoS) [Bougdour07], and iron limitation [Vinella05]. SpoT PSII activity may be necessary for increasing the ppGpp levels in these conditions, although how it detects them is still under investigation. In the case of fatty acid starvation, there is in vivo and in vitro evidence for a model in which acyl carrier protein specifically interacts with SpoT and may trigger a SpoT conformational change leading to (p)ppGpp accumulation. The interaction between ACP and SpoT was demonstrated in co-purification experiments and in a bacterial two-hybrid system [Battesti06].

Another interaction has been reported between SpoT and the 50S ribosomal subunit-associated GTPase, CgtAE (ObgE). The two proteins were shown to co-purify. The possible involvement of CgtAE in the control of SpoT function, or an effect of SpoT on CgtAE activity were suggested [Wout04].

In addition to the ppGpp hydrolase and synthetase activity of SpoT, SpoT also has pppGpp hydrolase activity [Heinemeyer78] and possibly pppGpp synthetase activity. The putative pppGpp synthetase activity is based the observation that in ΔrelAgpp mutants starved for glucose pppGpp accumulates, which suggests phosphoryl group transfer to GTP as well as GDP, although the phsophoryl donor is not known (cited as unpublished data in Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96]).

SpoT has been studied both mutationally and biochemically. Deletion mutants of spoT were analyzed for their ability to complement chromosomal mutations defective in synthetase or hydrolase activities. This allowed mapping of ppGpp hydrolase activity to the first 203 amino acids and the PSII activity to an overlapping region comprising amino acids 67-374. This suggested separate, but closely linked catalytic sites in the amino acid sequence [Gentry96]. A site-directed mutagenesis study suggested that Asp293 is required for ppGpp synthetase activity [Fujita02]. Proteolysis studies of partially purified, recombinant SpoT identified two major domains (Met1-Phe373 and Glu374-Asn702) each containing two minor sub-domains. In vivo expression studies suggested that ppGpp synthetic activity was associated with the N-terminal domain [Fujita02a].

In comparison, proteolysis studies done on the bifunctional Rel/Spo homolog from Streptococcus equisimilis localized hydrolase and synthase activities to the N-terminal half of the protein, whereas the C-terminal half appeared to have a regulatory function [Mechold02]. The crystal structure of a fragment containing the catalytic region of this S. equisimilis protein was determined at 2.1 Å resolution [Hogg04].

A relA spoT mutant was found to form biofilms with reduced catalase activity and elevated levels of hydroxyl radicals; the mutant biofilms are more sensitive to the antibiotics ofloxacin and tobramycin than wild type [Nguyen11].

Gene Citations: [Kalman92]

Locations [Comment 1]: cytosol

Map Position: [3,820,423 -> 3,822,531] (82.34 centisomes, 296°)
Length: 2109 bp / 702 aa

Molecular Weight of Polypeptide: 79.342 kD (from nucleotide sequence), 80.0 kD (experimental) [An79 ]

pI: 8.98

Isozyme Sequence Similarity:
GDP pyrophosphokinase / GTP pyrophosphokinase: YES

Unification Links: ASAP:ABE-0011935 , CGSC:156 , DIP:DIP-29378N , EchoBASE:EB0959 , EcoGene:EG10966 , EcoliWiki:b3650 , Mint:MINT-1219858 , ModBase:P0AG24 , OU-Microarray:b3650 , PortEco:spoT , PR:PRO_000023977 , Pride:P0AG24 , Protein Model Portal:P0AG24 , RefSeq:NP_418107 , RegulonDB:EG10966 , SMR:P0AG24 , String:511145.b3650 , UniProt:P0AG24

Relationship Links: InterPro:IN-FAMILY:IPR002912 , InterPro:IN-FAMILY:IPR003607 , InterPro:IN-FAMILY:IPR004095 , InterPro:IN-FAMILY:IPR004811 , InterPro:IN-FAMILY:IPR006674 , InterPro:IN-FAMILY:IPR007685 , InterPro:IN-FAMILY:IPR012675 , InterPro:IN-FAMILY:IPR012676 , Panther:IN-FAMILY:PTHR21262 , Panther:IN-FAMILY:PTHR21262:SF1 , Pfam:IN-FAMILY:PF01966 , Pfam:IN-FAMILY:PF02824 , Pfam:IN-FAMILY:PF04607 , Prosite:IN-FAMILY:PS51671 , Smart:IN-FAMILY:SM00471 , Smart:IN-FAMILY:SM00954

In Paralogous Gene Group: 457 (2 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for spoT

GO Terms:

Biological Process: GO:0015949 - nucleobase-containing small molecule interconversion Inferred from experiment [Fiil77]
GO:0015969 - guanosine tetraphosphate metabolic process Inferred from experiment Inferred by computational analysis [GOA01, Laffler74]
GO:0042594 - response to starvation Inferred from experiment [Laffler74]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01]
GO:0015970 - guanosine tetraphosphate biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Battesti09, Gully06, Butland05]
GO:0008893 - guanosine-3',5'-bis(diphosphate) 3'-diphosphatase activity Inferred from experiment Inferred by computational analysis [GOA01a, Heinemeyer78]
GO:0008728 - GTP diphosphokinase activity Inferred by computational analysis [GOA01a]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11]
GO:0016597 - amino acid binding Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11]

MultiFun Terms: cell processes adaptations starvation
metabolism central intermediary metabolism nucleotide and nucleoside conversions
regulation type of regulation posttranscriptional proteases, cleavage of compounds

Essentiality data for spoT knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 2]

Last-Curated ? 08-Mar-2010 by Fulcher C , SRI International

Enzymatic reaction of: guanosine 3'-diphosphate 5'-triphosphate 3'-diphosphatase

Synonyms: pppGppase, pentaphosphate guanosine-3'-pyrophosphohydrolase, pppGpp 3'-pyrophosphohydrolase

EC Number:

pppGpp + H2O <=> GTP + diphosphate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown. [Heinemeyer78]

Alternative Substrates for pppGpp: ppGpp [Heinemeyer78 ]

In Pathways: ppGpp biosynthesis

In addition to ppGpp hydrolase activity, SpoT can also hydrolyze pppGpp [Heinemeyer78].

Enzymatic reaction of: GDP diphosphokinase

Synonyms: guanosine 3',5'-polyphosphate synthase, guanosine 5'-diphosphate 3'-diphosphate synthetase, ppGpp synthetase

ATP + GDP <=> AMP + ppGpp

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: ppGpp biosynthesis

The spoT gene also encodes a ppGpp synthetase II activity (also referred to as PSII) [Hernandez91]. The activity is unstable and has not been well characterized. This synthetase activity is similar to the relA-encoded ppGpp synthetase I (PSI) activity, which uses GDP and ATP to synthesize ppGpp. The spoT-dependent synthesis of ppGpp (PSII activity) has been analyzed in a ΔrelA strain of E. coli [Murray96].

Enzymatic reaction of: guanosine-3',5'-bis(diphosphate) 3'-diphosphatase

Synonyms: ppGppase, guanosine 3',5'-bis(diphosphate) 3'-pyrophosphatase, guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase, ppGpp 3'-pyrophosphohydrolase

EC Number:

ppGpp + H2O <=> GDP + diphosphate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown. [Heinemeyer78]

Alternative Substrates for ppGpp: pppGpp [Heinemeyer78 ]

In Pathways: ppGpp biosynthesis

This spoT-encoded ppGpp hydrolase activity is the major route of cellular ppGpp degradation (Cashel et. al. (1996) "The Stringent Response", chapter 92, in [Neidhardt96]).

Uncharged tRNAPhe from a yeast source was shown to inhibit this enzyme reaction [Richter80].

Cofactors or Prosthetic Groups [Comment 3]: Mn2+ [Heinemeyer78]

Activators (Unknown Mechanism): ATP [Heinemeyer77]

Inhibitors (Unknown Mechanism): pGpp [Heinemeyer77] , o-phenanthroline [Xiao91] , tetracycline [Heinemeyer78a] , levallorphan [Heinemeyer77] , Li+ [Heinemeyer78] , picolinate [Xiao91]

Kinetic Parameters:

Km (μM)

T(opt): 37 °C [Heinemeyer78]

pH(opt): 7.5-8 [Heinemeyer78]

Sequence Features

Protein sequence of guanosine 3'-diphosphate 5'-triphosphate 3'-diphosphatase [multifunctional] with features indicated

Feature Class Location Citations Comment
Conserved-Region 45 -> 144
UniProt: HD;
Mutagenesis-Variant 73
[Boehm09, UniProt11]
UniProt: Obviates hydrolysis of ppGpp, moderately decreases biofilm formation, loss of biofilm induction in response to translation inhibitors.
Mutagenesis-Variant 259
[Boehm09, UniProt11]
UniProt: Obviates synthesis of ppGpp, strongly increases biofilm formation.
Mutagenesis-Variant 293
[Fujita02, UniProt11]
UniProt: Unable to restore (p)ppGpp synthesis nor complement a relA/spoT double disruption.
Mutagenesis-Variant 294
UniProt: Synthesizes (p)ppGpp, complements a relA/spoT double disruption.
Conserved-Region 628 -> 702
UniProt: ACT.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram


10/20/97 Gene b3650 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10966; confirmed by SwissProt match.


An79: An G, Justesen J, Watson RJ, Friesen JD (1979). "Cloning the spoT gene of Escherichia coli: identification of the spoT gene product." J Bacteriol 137(3);1100-10. PMID: 374338

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

Battesti06: Battesti A, Bouveret E (2006). "Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism." Mol Microbiol 62(4);1048-63. PMID: 17078815

Battesti09: Battesti A, Bouveret E (2009). "Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction." J Bacteriol 191(2);616-24. PMID: 18996989

Boehm09: Boehm A, Steiner S, Zaehringer F, Casanova A, Hamburger F, Ritz D, Keck W, Ackermann M, Schirmer T, Jenal U (2009). "Second messenger signaling governs Escherichia coli biofilm induction upon ribosomal stress." Mol Microbiol 72(6);1500-16. PMID: 19460094

Bougdour07: Bougdour A, Gottesman S (2007). "ppGpp regulation of RpoS degradation via anti-adaptor protein IraP." Proc Natl Acad Sci U S A 104(31);12896-901. PMID: 17640895

Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043

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

Fiil77: Fiil NP, Willumsen BM, Friesen JD, von Meyenburg K (1977). "Interaction of alleles of the relA, relC and spoT genes in Escherichia coli: analysis of the interconversion of GTP, ppGpp and pppGpp." Mol Gen Genet 150(1);87-101. PMID: 319345

Fujita02: Fujita C, Maeda M, Fujii T, Iwamoto R, Ikehara K (2002). "Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein." Biosci Biotechnol Biochem 66(12);2735-8. PMID: 12596879

Fujita02a: Fujita C, Nishimura A, Iwamoto R, Ikehara K (2002). "Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthetic activities on Escherichia coli SpoT domains." Biosci Biotechnol Biochem 66(7);1515-23. PMID: 12224635

Gentry96: Gentry DR, Cashel M (1996). "Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation." Mol Microbiol 19(6);1373-84. PMID: 8730877

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

Gully06: Gully D, Bouveret E (2006). "A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli." Proteomics 6(1);282-93. PMID: 16294310

Heinemeyer77: Heinemeyer EA, Richter D (1977). "In vitro degradation of guanosine tetraphosphate (ppGpp) by an enzyme associated with the ribosomal fraction from Escherichia coli." FEBS Lett 1977;84(2);357-61. PMID: 340264

Heinemeyer78: Heinemeyer EA, Richter D (1978). "Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli." Biochemistry 1978;17(25);5368-72. PMID: 365225

Heinemeyer78a: Heinemeyer EA, Geis M, Richter D (1978). "Degradation of guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT gene product of Escherichia coli." Eur J Biochem 1978;89(1);125-31. PMID: 359325

Hernandez91: Hernandez VJ, Bremer H (1991). "Escherichia coli ppGpp synthetase II activity requires spoT." J Biol Chem 1991;266(9);5991-9. PMID: 2005135

Hogg04: Hogg T, Mechold U, Malke H, Cashel M, Hilgenfeld R (2004). "Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]." Cell 117(1);57-68. PMID: 15066282

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Kalman92: Kalman M, Murphy H, Cashel M (1992). "The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12." Gene 110(1);95-9. PMID: 1544582

Laffler74: Laffler T, Gallant J (1974). "spoT, a new genetic locus involved in the stringent response in E. coli." Cell, Vol. 1, No. 1, 27-30.

Mechold02: Mechold U, Murphy H, Brown L, Cashel M (2002). "Intramolecular regulation of the opposing (p)ppGpp catalytic activities of Rel(Seq), the Rel/Spo enzyme from Streptococcus equisimilis." J Bacteriol 184(11);2878-88. PMID: 12003927

Metzger89: Metzger S, Sarubbi E, Glaser G, Cashel M (1989). "Protein sequences encoded by the relA and the spoT genes of Escherichia coli are interrelated." J Biol Chem 1989;264(16);9122-5. PMID: 2542299

Murray96: Murray KD, Bremer H (1996). "Control of spoT-dependent ppGpp synthesis and degradation in Escherichia coli." J Mol Biol 1996;259(1);41-57. PMID: 8648647

Neidhardt96: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low Jr KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE "Escherichia coli and Salmonella, Cellular and Molecular Biology, Second Edition." American Society for Microbiology, Washington, D.C., 1996.

Nguyen11: Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, McKay G, Siehnel R, Schafhauser J, Wang Y, Britigan BE, Singh PK (2011). "Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria." Science 334(6058);982-6. PMID: 22096200

Richter80: Richter D (1980). "Uncharged tRNA inhibits guanosine 3',5'-bis (diphosphate) 3'-pyrophosphohydrolase [ppGppase], the spoT gene product, from Escherichia coli." Mol Gen Genet 178(2);325-7. PMID: 6156378

Somerville79a: Somerville CR, Ahmed A (1979). "Mutants of Escherichia coli defective in the degradation of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp)." Mol Gen Genet 169(3);315-23. PMID: 372753

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

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

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

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

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Vinella05: Vinella D, Albrecht C, Cashel M, D'Ari R (2005). "Iron limitation induces SpoT-dependent accumulation of ppGpp in Escherichia coli." Mol Microbiol 56(4);958-70. PMID: 15853883

Wendrich02: Wendrich TM, Blaha G, Wilson DN, Marahiel MA, Nierhaus KH (2002). "Dissection of the mechanism for the stringent factor RelA." Mol Cell 10(4);779-88. PMID: 12419222

Wout04: Wout P, Pu K, Sullivan SM, Reese V, Zhou S, Lin B, Maddock JR (2004). "The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase." J Bacteriol 186(16);5249-57. PMID: 15292126

Xiao91: Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, Cashel M (1991). "Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations." J Biol Chem 1991;266(9);5980-90. PMID: 2005134

Other References Related to Gene Regulation

Gentry93: Gentry D, Bengra C, Ikehara K, Cashel M (1993). "Guanylate kinase of Escherichia coli K-12." J Biol Chem 1993;268(19);14316-21. PMID: 8390989

Lemke11: Lemke JJ, Sanchez-Vazquez P, Burgos HL, Hedberg G, Ross W, Gourse RL (2011). "Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA." Proc Natl Acad Sci U S A 108(14);5712-7. PMID: 21402902

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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