Nematode Culture Conditions Brenner, 1974 Brenner S. The genetics of Caenorhabditis elegans. d -glucose (0.25, 1%). For the carbenicillin treatment of bacteria, 80 μl of 500 mM carbenicillin stock solution was added to the plates after 48 hr of bacterial growth as described ( Garigan et al., 2002 Garigan D.

Hsu A.L.

Fraser A.G.

Kamath R.S.

Ahringer J.

Kenyon C. Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Lenaerts et al., 2008 Lenaerts I.

Walker G.A.

Van Hoorebeke L.

Gems D.

Vanfleteren J.R. Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes. Kaeberlein et al., 2006 Kaeberlein T.L.

Smith E.D.

Tsuchiya M.

Welton K.L.

Thomas J.H.

Fields S.

Kennedy B.K.

Kaeberlein M. Lifespan extension in Caenorhabditis elegans by complete removal of food. Nematode growth media was prepared as described (). Where indicated molten agar was supplemented with phenformin (1.5, 3, 4.5 mM), metformin (25, 50, 100 mM), carbenicillin (120 μM), trimethoprim (TMP 0.1, 0.2, 0.5, 1 μg/ml),-glucose (0.25, 1%). For the carbenicillin treatment of bacteria, 80 μl of 500 mM carbenicillin stock solution was added to the plates after 48 hr of bacterial growth as described (). For the UV treatment of bacteria, 80 μl of E. coli OP50 suspension was seeded onto an NGM plate and left to grow at 20°C overnight. Plates were then irradiated for 30 min on a UV Stratalinker 2400 (Stratagene) containing bulbs irradiating at 254 nm. Plates were further irradiated for 5 min on the day of transfer. Axenic plates were prepared as previously described (). Briefly, axenic solid media is composed of 3% peptone, 3% yeast extract and 0.05% hemoglobin (diluted in 0.1 M KOH). Hemoglobin is added to the autoclaved molten agar with constant stirring. Plates are kept at 4°C and in the dark. Bacterial deprivation is performed with NGM plates in the absence of E. coli, as previously described (). Plates at pH = 6.0-6.5 were used and the pH was confirmed using pH indicator strips (note that metformin effects on E. coli are pH-sensitive; see Figures S2 G and S2H). All chemicals were purchased from Sigma-Aldrich.

Lifespan and Mortality Analysis Lifespan measurements were performed as follows, unless indicated otherwise in Supplementary Tables. Axenic worm eggs were obtained using alkaline hypochlorite treatment of gravid adult hermaphrodites. These were then placed onto plates containing the test bacterial strain and maintained at 20°C for 2 generations. Lifespan measurements were initiated by transfer of L4-stage worms (day 0) to plates containing bacteria grown in the presence or absence of treatment for 96 hr. Worms were transferred to fresh plates every 4 days until day 12, to plates containing bacteria grown for 96 hr at 20°C. To prevent progeny development plates were supplemented with 5-fluoro-2′-deoxyuridine (FUdR, 15 μM) on the day prior to use, unless indicated otherwise. For the survival assays involving metformin-pretreatment of E. coli, 4-day old E. coli grown on control or metformin-treated plates were transferred using a 24 mm cell culture scraper (TPP®, Switzerland) to NGM plates supplemented with carbenicillin and FUdR. Worms growing on E. coli OP50 for 2 generations were transferred at the L4 stage to start the lifespan. Worms were transferred every 2 days to fresh plates containing metformin-pretreated or control E. coli. For axenic, bacterial deprivation and UV-treated bacteria lifespans, bleached eggs were placed onto UV-irradiated bacterial plates. This allows worm development and prevents bacterial contamination. At the L4 stage, worms were transferred to each of the treatment/condition plates containing FUdR (15 μM) and transferred every 2 days to new fresh plates. For RNAi experiments, eggs were added to plates seeded with the bacteria expressing double-stranded RNA to sams-1. Lifespans were initiated by the transfer of L4 stage worms to new fresh plates containing FUdR (10 μM). Worms were transferred to new fresh plates every 2 days until days 12 and then every 5 days. Worms that showed severe vulva protrusion or bagging were censored. Survival was monitored at regular time points and worms scored as dead if they did not show any movement when prodded with a platinum wire. Statistical significance of effects on lifespan was estimated using the log rank test, performed using JMP, Version 7 (SAS Institute). The mortality plots were defined as log(−log(1−n/event(x)/n.risk(x)/time(x)−time(x-1))) over time (x). Smoothing was applied using a sliding window approach.

RNA-Mediated Interference Clones Kamath et al., 2001 Kamath R.S.

Martinez-Campos M.

Zipperlen P.

Fraser A.G.

Ahringer J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. RNAi by feeding was performed as previously described (). RNAi E. coli feeding clones were derived from the Ahringer RNAi Library, kindly provided by Steven Nurrish.

Generation of Bacterial Strains OP50R and OP50-tmpR E. coli OP50 was grown on NGM plates containing an initial metformin concentration of 50 mM. Positive clones were transferred to fresh plates containing increasing concentrations of metformin up to 300 mM. To favor selection of metformin-resistant bacteria, the plates were incubated at 25°C. When it was ensured that bacterial growth was not compromised, the growth temperature was shifted to 37°C. For the generation of E. coli OP50 TMP-resistant strain, a fragment of 349 bp comprising the trimethoprim (TMP) resistant cassette was PCR-amplified from plasmid pEAK16_GFP (kindly provided by Brian Seed) using primers 5′-CCAGCAACGCAAGCTAGAGTT and 5′-GTCCTCCTTACCAGAAATTTATCC and cloned into pGEM-T Easy Vector System (Promega). Resulting plasmid (pNV6) was transformed into E. coli OP50 competent cells by standard procedure. Transformants were plated in LB (Luria-Bertani) agar containing ampicillin (100 mg/ml) and trimethoprim (50 μg/ml) and incubated at 37°C for 12–14 hr. Positive clones were selected and the presence of plasmid pNV6 was verified by plasmid extraction and sequencing.

Genome Sequencing of Bacterial Strains OP50 and OP50R Li and Durbin, 2009 Li H.

Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Koboldt et al., 2012 Koboldt D.C.

Zhang Q.

Larson D.E.

Shen D.

McLellan M.D.

Lin L.

Miller C.A.

Mardis E.R.

Ding L.

Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. An Illumina MiSeq machine was used to generate 150bp paired-end reads from the genomes of the OP50 parental (5.86 million reads) and the resistant (4.06 million reads) strains. Fastq files were aligned to the Escherichia coli B strain REL606 genome using the Burrows-Wheeler Aligner (BWA) () and the median coverage for the parental strain was 325x and 189x for the resistant strain. VarScan () was used to identify SNP’s and indels. There were eight SNP’s with variant frequency above 90% in the resistant strain but not present (i.e., variant frequency less than 1%) in the parental or reference genome. Two of these SNP’s result in single amino acid changes in the genes argG and glyA (which is a serine hydroxymethyltransferase involved in folate-mediated one-carbon metabolism).

Fecundity, Size, and Development Time Measurements Synchronized L1 larvae were obtained by bleaching of gravid adults and left overnight in M9 solution. Larvae were then raised on control and respective drug treatments until reaching the L4 stage. For developmental assays the proportion of worms that had reached the L4 stage was determined at 12 hr intervals. Worm length was measured at the L4 stage using a Leica RXA2 compound microscope and the image analysis application Volocity (Improvision, UK). For brood size and fecundity timing assays, starting with L4 worms, animals were transferred daily to fresh new plates during the reproductive period and progeny numbers counted. All data present the average of at least 3 independent trials.

GST-4::GFP Fluorescence Quantitation CL2166 dvIs19 [pAF15(gst-4::GFP::NLS)] and GA1112 aak-2(ok524); dvIs19 [pAF15 (gst-4::GFP::NLS)] animals were raised from synchronized L1 larvae in control, 50 mM metformin and 4.5 mM phenformin plates. L4 worms were picked and placed in a drop of 0.06% levamisole and observed under a 10x objective. Quantification of GFP expression from the transgene was carried out using a Leica DMRXA2 microscope using a GFP filter cube (excitation: 470/40 nm; emission: 525/50 nm), an Orca C10600 digital camera (Hamamatsu) and Volocity image analysis software (Improvision, UK). GFP intensity was measured as the pixel density in the entire cross sectional area of each worm from which the background pixel density was subtracted (90 worms per condition).

Bacterial Growth Measurements Liquid bacterial growth was performed in microtiter plates containing the respective bacterial strain (previously grown overnight in LB and diluted 1,000-fold) and drugs in 200 μl of LB at pH = 7.0. The absorbance (OD 600 nm) was measured every 5 min for an 18 hr incubation period with regular shaking at 37°C using a Tecan Infinite M2000 microplate reader and Magellan V6.5 software. Data analysis was performed on 3 or more replicate trials for each condition. Values for bar graphs are taken from OD 600 values at 18 hr of growth. Each bacterial strain was previously grown overnight in LB at 37°C (200 rpm). For colony forming unit counts, bacteria growing in control or treatments plates was scraped from the plate, resuspended in M9 and normalized to an OD 600 = 0.1. Each sample was diluted to 1x10−5 and 5 μl streaked on an LB agar plate followed by incubation at 37°C for 16 hr. The number of colonies formed was scored for each condition.

Bacterial Respiration Lenaerts et al., 2008 Lenaerts I.

Walker G.A.

Van Hoorebeke L.

Gems D.

Vanfleteren J.R. Dietary restriction of Caenorhabditis elegans by axenic culture reflects nutritional requirement for constituents provided by metabolically active microbes. 2 O assuming 406 nmol O 2 /ml at 37°C. Bacteria grown on plates ±50 mM metformin were resuspended in LB medium (OD 600 ∼0.25–0.35), and oxygen consumption was measured in the presence of exogenously added 50 mM metformin or H 2 O carrier. Oxygen concentration was plotted as a function of time, and the slope of the linear part of the graph was used to estimate the oxygen consumption rate. Bacterial respiration was measured in a Clark-type oxygen electrode (Rank Brothers, Cambridge, UK) in a 1 ml stirred chamber set at 37°C (). The electrode was calibrated using air-saturated HO assuming 406 nmol O/ml at 37°C. Bacteria grown on plates ±50 mM metformin were resuspended in LB medium (OD∼0.25–0.35), and oxygen consumption was measured in the presence of exogenously added 50 mM metformin or HO carrier. Oxygen concentration was plotted as a function of time, and the slope of the linear part of the graph was used to estimate the oxygen consumption rate.

Sample Preparation for Metabolite Analysis Bacteria: 4-day old bacterial lawns growing on control and metformin plates were washed from the plates using M9. The bacteria was then centrifuged at 4°C, 4,000 rpm for 20 min. The supernatant was discarded and the bacterial pellet kept at −80°C until further analysis. Worms: Approximately 600 synchronized 4-day adult worms per biological replicate from both control and metformin plates were collected. Worms were washed three times using M9 to remove bacteria and frozen at −70°C until further analysis. Test conditions for metabolite analysis were similar to those for lifespan assays at day 4. At least 3 biological replicates from each type of bacteria/worm strain and drug treatment were collected for each measurement. Buffer containing 20 mM ammonium acetate, 0.1% ascorbic acid, 0.1% citric acid and 100 mM DTT at pH 7.0 was added to cells. Suspensions were sonicated for 10 s using a hand-held sonicator at 40% amplitude for bacteria and 60% for C. elegans. Protein was removed by precipitation by addition of 2 sample volume of acetonitrile, mixing for two minutes and centrifugation for 15 min at 12,000 g and 4°C. Supernatants were transferred to fresh tubes, lyophilized and stored at −80°C prior to analysis.

Folate Analysis in Bacteria and C. elegans by LC-MS/MS Garratt et al., 2005 Garratt L.C.

Ortori C.A.

Tucker G.A.

Sablitzky F.

Bennett M.J.

Barrett D.A. Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry. Lyophilized samples were resuspended in 50 μl water (milli-Q) and centrifuged for 5 min at 12,000 g at 4°C. Supernatants were transferred to glass sample vials for LC-MS/MS analysis. Metabolites were resolved by reversed-phase chromatography (Luna C18 column; 150 mm x 2.0 mm; 5 μm bead size; Phenomenex, UK) using a 2795XE high performance liquid chromatography unit with solvent divert valve (Waters Corporation, UK). The HPLC was coupled to a MicroMass Quattro triple quadrupole tandem mass spectrometer (Waters Corporation, UK) operating in negative-ion mode using the following settings: capillary 3.54 kV, source temperature 150°C, desolvation temperature 350°C, cone gas flow rate 25 L/h and desolvation gas flow rate 950 L/h. Folates were measured by multiple reaction monitoring (MRM) with optimized cone voltage and collision energy for precursor and product ions as previously described ().

Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine Burren et al., 2006 Burren K.A.

Mills K.

Copp A.J.

Greene N.D. Quantitative analysis of s-adenosylmethionine and s-adenosylhomocysteine in neurulation-stage mouse embryos by liquid chromatography tandem mass spectrometry. Quantification of SAMe and SAH was performed as described previously ().

Metabolomic Principal Component Analysis Xia and Wishart, 2011 Xia J.

Wishart D.S. Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst. Raw LC-MS/MS spectral data from 3 biological replicates of each condition was uploaded into MetaboAnalyst (). Missing values were replaced by default by low signal intensity values (below detection limit). Row-wise normalization was achieved by sample normalization prior to analysis. Column-wise normalization was achieved using Pareto scaling in order to produce a typical “bell curve” shape of transformed data. To avoid propensity to data overfitting, PCA analysis was used to create the 2D analysis plot. The dendrogram plot was obtained through clustering of the data using the Ward method.

Western Blotting Eggs were placed in 0, 25, 50 and 100 mM metformin plates and allowed to develop in the presence of drug until reaching 1-day adults. Additionally, L4 wild-type were placed on 0, 1.5, 3 and 4.5 mM phenformin plates for 2 days or in 50 mM metformin for 8 and 16 days. The worms were then washed 3 times using M9 to remove the bacteria and frozen at −70°C. Phospho-safe lysis buffer (Pierce) was supplemented with protease inhibitors (Roche). The lysates were homogenized using a Bioruptor (Cosmo Bio Co., Ltd., Tokyo, Japan) in 2 ml microcentrifuge tubes and then centrifuged at 16,000 g for 30 min at 4°C. The supernatants were then assayed for protein concentration using the Bradford assay. 30 mg of protein extracts were run on SDS-PAGE gel and transferred onto nitrocellulose membrane. Phosphorylation of AAK-2 subunit (pAMPKα) was detected using a rabbit antibody specific to pAMPKα (cell signaling) at a 1:1,000 dilution. As a loading control, β-actin protein was used with mouse anti-actin at a 1:5,000 dilution (Santa-Cruz Biotechnology). Blots were developed using the SuperSignal West Pico chemiluminescent substrate (Perbio Sciences). Films were scanned and the density of each band or the entire lane was quantified by densitometry using ImageQuant TL (GE Healthcare Europe GmbH).