Open Access Highly Accessed Research article

Excessive folate synthesis limits lifespan in the C. elegans: E. coli aging model

Bhupinder Virk1, Gonçalo Correia123, David P Dixon12, Inna Feyst1, Jie Jia1, Nikolin Oberleitner1, Zoe Briggs1, Emily Hodge1, Robert Edwards12, John Ward4, David Gems5 and David Weinkove125*

Author Affiliations

1 School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK

2 Biophysical Sciences Institute, Durham University, South Road, Durham, DH1 3LE, UK

3 Faculty of Science, University of Lisbon, Campo Grande, 1749-016, Lisbon, Portugal

4 Department of Structural and Molecular Biology, ISMB, University College London, London WC1E 6BT, UK

5 Institute of Healthy Ageing and Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK

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BMC Biology 2012, 10:67  doi:10.1186/1741-7007-10-67

Published: 31 July 2012

Additional files

Additional file 1:

Summary of all lifespan experiments conducted in this study. File showing individual experiments, number of animals scored as dead, number censored and, where relevant, percent increase in lifespan between mutant and control, and P-values from Log-Rank and Wilcoxon tests of the Kaplan-Meier survival model.

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Additional file 2:

Spontaneous E. coli mutant rather than RNAi plasmid extends C. elegans lifespan. A) A new RNAi strain containing the ugt-27 plasmid has no effect on lifespan. Survival of rrf-3 worms (20°C) on the control HT115(DE3) strain with the L4440 plasmid, (control, n = 61), the original ugt-27 strain (mutant, n = 98) and a new strain consisting of HT115(DE3) transformed with the ugt-27 plasmid (new, n = 50). Difference between mutant and control, 29.3% (P = < 0.0001).

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Additional file 3:

PABA supplementation has no toxic effect. A) PABA supplementation reverses the lifespan extension on the mutant bacteria (aroD + PABA, n = 85, aroD, n = 124), but has no effect on the control bacteria (control + PABA, n = 112, control, n = 84). B) Addition of PABA has no effect on ubiG bacteria (ubiG + PABA, n = 90, ubiG, n = 76) or ubiG- + rescue plasmid (ubiG:pAHG + PABA, n = 116, ubiG:pAHG, n = 118). C) PHB supplementation had no effect of lifespan of C. elegans maintained on either the mutant (aroD, n = 161, aroD + 25 μM PHB, n = 129, aroD + 250 μM, n = 105) or control bacteria (control, n = 173, control + 25 μM PHB, n = 121, control + 250 μM PHB, n = 130). See Additional file 1 for a full listing of all lifespan data in this study.

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Additional file 4:

Relevant traces from the HPLC/MS analysis from E. coli. A) Traces from HT115(DE3) and HT115(DE3)aroD of m/z = -730.244 corresponding to the negative ion of formylTHFGlu3. B) Traces from formylTHFGlu3 show that this species becomes undetectable in OP50 with increasing concentrations of SMX. C) Traces from C. elegans lysates corresponding to the negative ion of 5-methylTHFGlu5, with 0, 0.1 and 1 μg/ml SMX.

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Additional file 5:

Effect of media supplementation with folate on C. elegans lifespan on aroD and ubiG mutants. (A) glp-4(bn2) animals were raised on the aroD mutant until L4 (15°C) and then transferred (25°C) to the aroD mutant (n = 100), control (n = 126), aroD + 1 × folate (n = 101), aroD + 2 × folate (n = 149). aroD vs aroD + 1 × folate, P = < 0.0001; aroD + 1 × folate vs aroD + 2 × folate, P = 0.13 (Log Rank), P = 0.055 (Wilcoxon). (B) glp-4 worms were raised on E. coli OP50 (15°C) and transferred (25°C) at L4 to ubiG (n = 145), ubiG + pAHG (ubiG+ rescue plasmid), GD1 + 1 × folate (n = 145), GD1+ 2 × folate (n = 146). (C) Traces from the HPLC/MS analysis of the wild type (HT115(DE3)) and aroD mutant (HT115(DE3)aroD) bacteria. Peaks shown are m/z = 730.25 and 548.67 corresponding to formylTHFGlu3 and the methotrexateGlu6 spiked standard respectively with increasing folic acid supplementation (1 × folate is equal to 294 μM folic acid).

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Additional file 6:

PABA reverses the lifespan increase caused by 16 μg/ml and 128 μg/ml SMX. Lifespan curves showing Control (n = 102), 16 μg/ml SMX (n = 176), 128 μg/ml SMX (n = 229), 16 μg/ml SMX + 250 μM PABA (n = 160), 128 μg/ml SMX + 250 μM PABA (n = 217).

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Additional file 7:

Lawn density of plates treated with various concentrations of SMX. Relative bacterial content of lawns from the mean values from 10 plates per conditions (see Methods). Error bars are ± standard deviations. Student's t-test values (Control vs 8 μg/ml SMX: P = 0.02, Control vs 16 μg/ml: P = 0.04, Control vs 64 μg/ml: P = 0.12, Control vs 128 μg/ml: P = 0.04).

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