Genome-wide screen identifies Escherichia coli TCA-cycle-related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia-inducible transcription factor ArcA
Valter D. Longo
Andrus Gerontology Center, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA
Search for more papers by this authorValter D. Longo
Andrus Gerontology Center, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA
Search for more papers by this authorSummary
Single-gene mutants with extended lifespan have been described in several model organisms. We performed a genome-wide screen for long-lived mutants in Escherichia coli, which revealed strains lacking tricarboxylic acid (TCA)-cycle-related genes that exhibit longer stationary-phase survival and increased resistance to heat stress compared to wild-type. Extended lifespan in the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with the reduced production of superoxide and increased stress resistance. On the other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is associated with reduced oxygen consumption and requires the acetate-producing enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription factor ArcA, acting independently of acetate metabolism, is also required for maximum lifespan extension in the lipA and lpdA mutants, indicating that these mutations promote entry into a mode normally associated with a low-oxygen environment. Because analogous changes from respiration to fermentation have been observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans strains, such metabolic alterations may represent an evolutionarily conserved strategy to extend lifespan.
Supporting Information
Fig. S1 Schematic representation of the absorbance-based, genome-wide screen for extended stationary phase survival in E. coli.
Fig. S2 Related to Fig. 1. (A–C) CFU titers of experiments shown in Fig. 1A–C. (D) Growth curves as CFU counts of wt and the long-lived strains. (E) Survival of 10-ml cultures of wt and the lipA strain in 125-ml flasks. (F) Survival of the shown strains.
Fig. S3 Related to Fig. 4. (A) Effect of the overexpression of SodB (pHS1-7) on the survival of wt (pBR322 empty vector). (B) Time-dependent frequency of rifampicin-resistant mutants in wt and the sdhA strain. (C) Growth curves as CFU counts of the shown strains. (D) Stationary phase survival of all strains included in the mutation frequency experiment.
Fig. S4 Related to Fig. 6. (A) Extracellular acetate concentration at late stationary phase of the shown mutants. () Effect of sodium chloride or sodium acetate added to 6 mM at the time-point designated by the arrow on the survival of the lipA mutant. (C, D) Stationary phase survival of the shown mutants at pH 7.5. (E) Stationary phase survival of the shown mutants at pH 9. (F) Extracellular acetate concentration of the shown mutants over time. (G, H) Expression of pta and ackA in wt and the lipA strain over time.
Fig. S5 (A) Rate of oxygen consumption of the shown mutants at early stationary phase. (B) ATP levels of the shown strains, measured using firefly luciferase. (C) Survival of the wt and pta strains.
Table S1. Stationary phase regrowth ratios of strains of the KEIO collection (see supplementary experimental procedures for details).
Table S2. Stationary-phase pH and CPII density of wt and longlived mutants.
Table S3. List of strains used in this study.
Data S1. Supplementary materials and methods.
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References
- Abdel-Hamid AM, Attwood MM, Guest JR (2001) Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli. Microbiology 147, 1483–1498.
- 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.
- Baev MV, Baev D, Radek AJ, Campbell JW (2006) Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays. Appl. Microbiol. Biotechnol. 71, 310–316.
- Benov L, Fridovich I (1996) The rate of adaptive mutagenesis in Escherichia coli is enhanced by oxygen (superoxide). Mutat. Res. 357, 231–236.
- Burtner CR, Murakami CJ, Kennedy BK, Kaeberlein M (2009) A molecular mechanism of chronological aging in yeast. Cell Cycle 8, 1256–1270.
- Carlioz A, Touati D (1986) Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 5, 623–630.
- Chang EC, Kosman DJ (1989) Intracellular Mn (II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress. J. Biol. Chem. 264, 12172–12178.
- Chen D, Thomas EL, Kapahi P (2009) HIF-1 modulates dietary restriction-mediated lifespan extension via IRE-1 in Caenorhabditis elegans. PLoS Genet. 5, e1000486.
- Clark DP (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol. Rev. 5, 223–234.
- Conter A, Gangneux C, Suzanne M, Gutierrez C (2001) Survival of Escherichia coli during long-term starvation: effects of aeration, NaCl, and the rpoS and osmC gene products. Res. Microbiol. 152, 17–26.
- Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U S A 97, 6640–6645.
- Desnues B, Cuny C, Gregori G, Dukan S, Aguilaniu H, Nystrom T (2003) Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells. EMBO Rep. 4, 400–404.
- Dukan S, Nystrom T (1998) Bacterial senescence: stasis results in increased and differential oxidation of cytoplasmic proteins leading to developmental induction of the heat shock regulon. Genes Dev. 12, 3431–3441.
- Eisenstark A, Miller C, Jones J, Leven S (1992) Escherichia coli genes involved in cell survival during dormancy: role of oxidative stress. Biochem. Biophys. Res. Commun. 188, 1054–1059.
- Ericsson M, Hanstorp D, Hagberg P, Enger J, Nystrom T (2000) Sorting out bacterial viability with optical tweezers. J. Bacteriol. 182, 5551–5555.
- Fabrizio P, Longo VD (2003) The chronological life span of Saccharomyces cerevisiae. Aging Cell 2, 73–81.
- Fabrizio P, Liou LL, Moy VN, Diaspro A, Valentine JS, Gralla EB, Longo VD (2003) SOD2 functions downstream of Sch9 to extend longevity in yeast. Genetics 163, 35–46.
- Farrell MJ, Finkel SE (2003) The growth advantage in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment. J. Bacteriol. 185, 7044–7052.
- Finkel SE (2006) Long-term survival during stationary phase: evolution and the GASP phenotype. Nat. Rev. Microbiol. 4, 113–120.
- Fontaine F, Stewart EJ, Lindner AB, Taddei F (2008) Mutations in two global regulators lower individual mortality in Escherichia coli. Mol. Microbiol. 67, 2–14.
- Garibyan L, Huang T, Kim M, Wolff E, Nguyen A, Nguyen T, Diep A, Hu K, Iverson A, Yang H, Miller JH (2003) Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome. DNA Repair (Amst.) 2, 593–608.
- Georgellis D, Kwon O, Lin EC (2001) Quinones as the redox signal for the arc two-component system of bacteria. Science 292, 2314–2316.
- Groat RG, Schultz JE, Zychlinsky E, Bockman A, Matin A (1986) Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation survival. J. Bacteriol. 168, 486–493.
- Hahm DH, Pan J, Rhee JS (1994) Characterization and evaluation of a pta (phosphotransacetylase) negative mutant of Escherichia coli HB101 as production host of foreign lipase. Appl. Microbiol. Biotechnol. 42, 100–107.
- Halpern YS, Even-Shoshan A, Artman M (1964) Effect of glucose on the utilization of succinate and the activity of tricarboxylic acid-cycle enzymes in Escherichia Coli. Biochim. Biophys. Acta 93, 228–236.
- Hassan HM, Fridovich I (1977) Regulation of the synthesis of superoxide dismutase in Escherichia coli. Induction by methyl viologen. J. Biol. Chem. 252, 7667–7672.
- Hengge-Aronis R (2002) Recent insights into the general stress response regulatory network in Escherichia coli. J. Mol. Microbiol. Biotechnol. 4, 341–346.
- Iuchi S, Lin EC (1991) Adaptation of Escherichia coli to respiratory conditions: regulation of gene expression. Cell 66, 5–7.
- Jones RW, Garland PB (1977) Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem. J. 164, 199–211.
- 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, 117–123.
- Koland JG, Miller MJ, Gennis RB (1984) Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase. Biochemistry 23, 445–453.
- Kumari S, Tishel R, Eisenbach M, Wolfe AJ (1995) Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli. J. Bacteriol. 177, 2878–2886.
- Kumari S, Beatty CM, Browning DF, Busby SJ, Simel EJ, Hovel-Miner G, Wolfe AJ (2000) Regulation of acetyl coenzyme A synthetase in Escherichia coli. J. Bacteriol. 182, 4173–4179.
- Kwon O, Georgellis D, Lin EC (2003) Rotational on-off switching of a hybrid membrane sensor kinase Tar-ArcB in Escherichia coli. J. Biol. Chem. 278, 13192–13195.
- Levanon SS, San KY, Bennett GN (2005) Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses. Biotechnol. Bioeng. 89, 556–564.
- Li M, Ho PY, Yao S, Shimizu K (2006) Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments. J. Biotechnol. 122, 254–266.
- Longo VD, Finch CE (2003) Evolutionary medicine: from dwarf model systems to healthy centenarians? Science 299, 1342–1346.
- Lynch AS, Lin EC (1996) Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters. J. Bacteriol. 178, 6238–6249.
- Maklashina E, Berthold DA, Cecchini G (1998) Anaerobic expression of Escherichia coli succinate dehydrogenase: functional replacement of fumarate reductase in the respiratory chain during anaerobic growth. J. Bacteriol. 180, 5989–5996.
- Mehta R, Steinkraus KA, Sutphin GL, Ramos FJ, Shamieh LS, Huh A, Davis C, Chandler-Brown D, Kaeberlein M (2009) Proteasomal regulation of the hypoxic response modulates aging in C. elegans. Science 324, 1196–1198.
- Messner KR, Imlay JA (2002) Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase. J. Biol. Chem. 277, 42563–42571.
- Miller RA (2009) Cell stress and aging: new emphasis on multiplex resistance mechanisms. J. Gerontol. A Biol. Sci. Med. Sci. 64, 179–182.
- Miller JR, Busby RW, Jordan SW, Cheek J, Henshaw TF, Ashley GW, Broderick JB, Cronan JE Jr, Marletta MA (2000) Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. Biochemistry 39, 15166–15178.
- Nystrom T, Larsson C, Gustafsson L (1996) Bacterial defense against aging: role of the Escherichia coli ArcA regulator in gene expression, readjusted energy flux and survival during stasis. EMBO J. 15, 3219–3228.
- Partridge L, Barton NH (1993) Optimality, mutation and the evolution of ageing. Nature 362, 305–311.
- Phue JN, Noronha SB, Hattacharyya R, Wolfe AJ, Shiloach J (2005) Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. Biotechnol. Bioeng. 90, 805–820.
- Powers RW III, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S (2006) Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20, 174–184.
- Pruss BM, Nelms JM, Park C, Wolfe AJ (1994) Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids. J. Bacteriol. 176, 2143–2150.
- Rea S, Johnson TE (2003) A metabolic model for life span determination in Caenorhabditis elegans. Dev. Cell 5, 197–203.
- Salmon KA, Hung SP, Steffen NR, Krupp R, Baldi P, Hatfield GW, Gunsalus RP (2005) Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA. J. Biol. Chem. 280, 15084–15096.
- Semenza GL (2000) HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J. Appl. Physiol. 88, 1474–1480.
- Shen C, Nettleton D, Jiang M, Kim SK, Powell-Coffman JA (2005) Roles of the HIF-1 hypoxia-inducible factor during hypoxia response in Caenorhabditis elegans. J. Biol. Chem. 280, 20580–20588.
- Smith MW, Neidhardt FC (1983a) 2-Oxoacid dehydrogenase complexes of Escherichia coli: cellular amounts and patterns of synthesis. J. Bacteriol. 156, 81–88.
- Smith MW, Neidhardt FC (1983b) Proteins induced by aerobiosis in Escherichia coli. J. Bacteriol. 154, 344–350.
- Stewart EJ, Madden R, Paul G, Taddei F (2005) Aging and death in an organism that reproduces by morphologically symmetric division. PLoS Biol. 3, e45.
- Touati D (1983) Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12. J. Bacteriol. 155, 1078–1087.
- Vanden Boom TJ, Reed KE, Cronan JE Jr (1991) Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system. J. Bacteriol. 173, 6411–6420.
- Vemuri GN, Eiteman MA, Altman E (2006) Increased recombinant protein production in Escherichia coli strains with overexpressed water-forming NADH oxidase and a deleted ArcA regulatory protein. Biotechnol. Bioeng. 94, 538–542.
- Visick JE, Cai H, Clarke S (1998) The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses. J. Bacteriol. 180, 2623–2629.
- Vulic M, Kolter R (2002) Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli. J. Bacteriol. 184, 2898–2905.
- Wei M, Fabrizio P, Madia F, Hu J, Ge H, Li LM, Longo VD (2009) Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension. PLoS Genet. 5, e1000467.
- Wolfe AJ (2005) The acetate switch. Microbiol. Mol. Biol. Rev. 69, 12–50.
-
Yang YT,
Bennett GN,
San KY (1999) Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli.
Biotechnol. Bioeng.
65, 291–297.
10.1002/(SICI)1097-0290(19991105)65:3<291::AID-BIT6>3.0.CO;2-F CAS PubMed Web of Science® Google Scholar
- Yu L, Yu CA (1980) Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase. Biochim. Biophys. Acta 593, 24–38.
- Zhang Y, Shao Z, Zhai Z, Shen C, Powell-Coffman JA (2009) The HIF-1 hypoxia-inducible factor modulates lifespan in C. elegans. PLoS ONE 4, e6348.
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