Dietary and microbiome factors determine longevity in Caenorhabditis elegans

B. subtilis fed worms live longer and die because of different reasons than E. coli fed worms

Feeding the gram positive B. subtilis to C. elegans instead of the gram negative standard laboratory diet E. coli considerably increases worm longevity [14,17,19,21]. To understand the nature of this life span increase we first examined whether it is B. subtilis strain-specific. We fed worms three different WT strains of B. subtilis (PY79, 3610, and 168) and compared their longevity to worms fed the standard E. coli OP50. Worms fed any of the B. subtilis strains lived longer (43%-58%) than E. coli fed worms (Figure 1A), thus feeding B. subtilis to worms extends their life span independently of the B. subtilis strain used.

Figure 1. B. subtilis fed worms live longer and die because of different reasons than E. coli fed worms. (A) Life span curves for adult worms maintained on E. coli (OP50) or on different wild type strains of B. subtilis: PY79, 3610, and 168. (B) Age-related mortality for worms fed B. subtilis or E. coli (see also Figure S2 for life span and death frequency over time data). (C) Life span curves for adult worms maintained on E. coli, B. subtilis, or the sporeless B. subtilis mutant (1S143). (D) Life span curves for adult worms maintained on live E. coli, UV-killed E. coli, live B. subtilis, or UV-killed B. subtilis. (E) Life span curves for adult worms maintained on E. coli; on B. subtilis; or on E. coli as late L4 stage and then switched to B. subtilis. (A-E) y-axis indicates percentage of worms that are alive. x-axis indicates day of adulthood. See also Table S1.

To investigate the overall health of B. subtilis fed worms, we compared C. elegans gut autofluorescence accumulation and muscle decline during aging for B. subtilis and E. coli fed worms. Age-related fluorescent breakdown products accumulate in the gut during aging leading to increasing levels of autofluorescence with age [19]. B. subtilis fed worms displayed less gut autofluorescence late in life than E. coli fed worms (Figure S1A). UNC-54 is the major myosin heavy chain expressed in C. elegans and is required for C. elegans locomotion [22]. We used a unc-54::mCherry reporter C. elegans strain [19] to measure muscle decline and observed a lower decline of unc-54 expression in B. subtilis fed worms than in E. coli fed worms during aging (Figure S1B). These data indicate that the overall health of B. subtilis fed worms declines at a slower rate during aging than that of E. coli fed worms.

Previous work indicated that factors associated with the E. coli and the B. subtilis diets make worms die because of different reasons [19]. To further investigate the underlying mechanism, we performed demographic longevity studies in which we subjected 500 individuals to each diet and examined their mortality rates and death frequency over time (Figure 1B; Figure S2). We observed a lowering of the initial mortality rate but not a major impact on the rate of aging when worms were fed B. subtilis as compared to E. coli. This suggests that feeding worms E. coli would add additional risk factors to the worms at all ages or that feeding worms B. subtilis would eliminate risk factors at all ages.

B. subtilis are able to sporulate while E. coli are not. Spores are not digestible and upon germination might produce factors that affect worm life span. We tested this possibility by using a B. subtilis strain that is unable to sporulate. Worms fed this spore-less B. subtilis diet lived 46% longer than E. coli fed worms (Figure 1C). Although B. subtilis spore presence increased worm longevity by an additional 10% (Figure 1C), the data show that the life span difference between E. coli and B. subtilis fed worms is not solely due to the presence of B. subtilis spores.

Since life span studies are usually performed with live bacteria, one possibility is that worms that feed on E. coli are more prone to infection than worms that feed on B. subtilis. Bacteria that are not properly processed before being digested can enter the worm gut and proliferate [23]. E. coli proliferation has been shown to be harmful to C. elegans [24]. Therefore, excessive bacterial gut proliferation could be the cause of the life span shortening when worms are fed E. coli instead of B. subtilis. We compared the longevity of worms fed either live E. coli or live B. subtilis versus non-infectious UV killed E. coli or UV killed B. subtilis. Worms fed live or UV killed B. subtilis had very similar life spans (Figure 1D). However, as previously reported [19,25], worms fed UV killed E. coli lived approximately 20% longer than worms fed live E. coli (Figure 1D), indicating that E. coli infection is a contributing factor for the shortening of the life span when worms are fed E. coli. However, E. coli infection does not explain the overall life span increase observed when worms are fed B. subtilis instead of E. coli as worms that feed on B. subtilis (live or UV killed) live longer than UV killed E. coli fed worms.

To examine whether E. coli fed worms live shorter than B. subtilis fed worms because of E. coli-specific pathogenic factors, we fed worms E. coli only during development (L1 to late L4) and then changed their diet to B. subtilis for the remainder of their lives. These worms lived 30% shorter than worms that were exclusively fed on B. subtilis (Figure 1E) suggesting that E. coli fed worms are exposed to negative effects by E. coli, which shorten C. elegans longevity.

B. subtilis do not induce dietary restriction to C. elegans

Dietary restriction has been shown to extend C. elegans life span [26,27]. If the B. subtilis diet induced a state of dietary restriction to the worms, then it would explain the longevity difference with respect to E. coli fed worms. We determined that the caloric content of B. subtilis and E. coli is very similar (Table S2). We also showed that these two types of bacteria have very similar water content and that the major calorie contribution is conferred by protein and carbohydrate.

It has been shown that C. elegans do not display noticeable feeding behavior modifications when fed B. subtilis and that these bacteria are efficiently ingested and digested by C. elegans (Laaberki and Dworkin, 2008b). Independently, we also tested whether B. subtilis cells might not be well digested by C. elegans, thus not allowing worms to get the same amount of nourishment as worms that feed on E. coli. First, we examined markers of worm metabolic activity. Egg production and developmental growth are high-energy demanding metabolic processes and thus good indicators of the metabolic status of the worm. Interventions that compromise C. elegans metabolism such as dietary restriction lead to reduced brood size and/or delayed development [25,28–30]. We monitored the daily and the overall brood size of B. subtilis and E. coli fed worms but did not observe any differences between the two feeding conditions (Figure 2A). We compared the percentage of WT L1 larvae that developed to adults after 2.7 days of feeding E. coli or two different strains of B. subtilis and, similar to previously published results [17,29] we did not see any differences (Figure 2B). We also compared the percentage of L1 larvae that developed to adults when worms were fed E. coli or B. subtilis for a number of C. elegans mutants known to develop slower. These mutant strains included the daf-2 insulin-like growth factor 1 receptor; the daf-16 forkhead box O (FOXO) homologue; the isp-1 and nuo-6 mitochondrial electron transport chain subunit components; the eat-2 nicotinic acetylcholine receptor subunit, which mimics dietary restriction; and the coQ-deficient clk-1. With the exception of clk-1 worms, we did not see differences in the percentage of L1 larvae that developed to adults when these mutant worms were fed either diet (Figure 2B).

Figure 2. B. subtilis fed worms are not dietary restricted. (A) Daily average number of progeny ± S.E.M per worm and diet (n = 5 for each group). Inset bars represent the total average progeny ± S.E.M per worm on E. coli and B. subtilis. y-axis shows progeny number. x-axis shows age of adult worms. No statistical differences for each diet and group were observed (p > 0.05, Student’s t test). (B) Percentage of L1 larvae that became adults when fed E. coli or B. subtilis, wild type (PY79) and spore-less mutant (1S143), after 2.7 days (wild type worms), 2.7 days (daf-16 worms), 2.9 days (eat-2 worms), 3.6 days (daf-2 worms), 3.2 days (clk-1 worms), 5.0 days (nuo-6 worms), 5.0 days (isp-1 worms). y-axis shows percentage of L1 larvae that reached adulthood. x-axis shows E. coli or B. subtilis fed worms. n = 44-160 worms per group. Bars indicate the mean value ± S.E.M. No statistical differences for each diet and group were observed (p > 0.05, Student’s t test) except for clk-1 worms. (C) pha-4::mCherry fluorescent marker expression for 2 day old adults exposed to plenty of food (Control) or dietary restricted (sDR). y-axis shows levels of fluorescent expression in arbitrary units. x-axis shows E. coli or B. subtilis fed worms. Bars indicate mean fluorescent marker expression ± S.E.M. n = 10-20 for each group (**p < 0.01, Student’s t test).

We also used a transgenic pha-4::mCherry transcriptional reporter strain [19], which can indicate the nutritional state of worms as it displays elevated fluorescence expression upon dietary restriction conditions (Figure S3). PHA-4 is a Foxa transcription factor that has been shown to play an important role in the C. elegans starvation response [31] as well as to mediate diet-restriction-induced longevity in C. elegans [32]. We found that ad libitum E. coli and B. subtilis fed worms displayed the same pha-4::mCherry expression levels (Figure 2C). We also found that E. coli or B. subtilis fed worms that were subjected to dietary restriction increased pha-4::mCherry expression. Importantly, the increase in pha-4::mCherry expression was similar regardless of the diet type (Figure 2C).

Three independent lines of evidence including the similar caloric content for B. subtilis and E. coli; the equal progeny number and developmental time to reach adulthood for worms fed B. subtilis or E. coli ; and the pha-4::mCherry nutritional biomarker response for worms fed either diet indicate that B. subtilis fed worms are not under the effects of dietary restriction and thus their longevity increase compared to E. coli fed worms must be due to some other reason.

Worms fed B. subtilis instead of E. coli live longer mainly because this food source does not contain coenzyme Q

CoQ-deficient clk-1 larvae were unable to develop when fed B. subtilis (Figure 2B). We confirmed this result using another clk-1 allele (Figure S4). B. subtilis fed clk-1 L1 larvae developed into L2 stage and arrested. After approximately one week, most L2 larvae continued their development to adulthood but worms were sterile. We maintained clk-1 larvae on B. subtilis for up to 30 days but these still failed to develop into fertile adults. However, if arrested larvae were transferred to E. coli plates, development resumed and larvae became fertile adults. Conversely, fertile E. coli fed clk-1 young adults became sterile 2 days upon transfer to B. subtilis plates.

CoQ is a crucial mitochondrial electron transport chain (ETC) carrier [33]. C. elegans synthesize their own coQ (Q₉) and the demethoxyubiquinone hydroxylase CLK-1 is a necessary enzyme in this process. Thus, clk-1 mutants are Q₉ deficient [34]. If worms completely lack coQ, then they become sterile and larvae are not able to complete development [35,36]. Although clk-1 mutant worms cannot synthesize Q₉, they uptake coQ (Q₈) from the E. coli diet, which explains why under standard laboratory conditions clk-1 worms can develop and function [36,37]. Unlike E. coli, B. subtilis do not synthesize coQ [38]. Therefore, feeding coQ-less B. subtilis to coQ-deficient clk-1 worms leads to developmental arrest and adult sterility. These phenotypes are comparable to those observed when clk-1 worms are fed coQ-deficient E. coli [35–37,39]. Worms fed coQ-deficient E. coli mutant strains live up to 60% longer than coQ-producing E. coli fed worms [8]. CoQ-less B. subtilis fed worms also live much longer than coQ-producing E. coli fed worms [17] (Figure 1). Therefore, we wanted to investigate whether the extended longevity of B. subtilis versus E. coli fed worms was due to their lack of dietary coQ.

We compared the life span of B. subtilis fed worms with the life span of B. subtilis fed worms supplemented with coQ. CoQ is highly hydrophobic, thus supplementation of synthetic coQ to the aqueous C. elegans media makes it difficult for worms to incorporate this compound [10]. To ensure an effective level of coQ supplementation as well as to supplement with the same coQ species worms receive when they feed on the standard E. coli diet, we developed a coQ supplementation method using non-nutritious E. coli extracts that retained coQ activity (see Experimental Procedures). CoQ-deficient clk-1 L1 larvae reared on the coQ-less B. subtilis diet were able to develop when the E. coli extract was present, demonstrating that the E. coli extract retained coQ activity. The rate of clk-1 L1 to adult development was proportional to the concentration of E. coli extract used (Figure 3A) and the adult sterility when clk-1 worms were fed B. subtilis was reversed when the E. coli extract was added (Table S3). Importantly, the E. coli extract was not nutritious as clk-1 L1 larvae that are solely fed with it arrested at L1 stage (Figure 3A).

Figure 3. B. subtilis fed worms supplemented with coQ-active E. coli extract have shortened longevity. (A) Development of synchronized clk-1(qm30) L1 larvae after 4.7 days feeding on E. coli, E. coli extract at different concentrations, or B. subtilis with or without supplementation of E. coli extract at different concentrations. y-axis shows percentage of individuals that reached each developmental stage. x-axis shows developmental stages. n= 51-160 worms per group (B) Life span curves for adult worms maintained on E. coli or B. subtilis with or without supplementation of coQ-active E. coli (OP50) extract at different concentrations. (C) Life span curves for adult worms maintained on E. coli or B. subtilis with or without supplementation of coQ-deficient E. coli (GD1) extract at different concentrations. (B and C) y-axis indicates percentage of worms that are alive. x-axis indicates day of adulthood. See also Table S4.

We supplemented B. subtilis fed worms with non-nutritious coQ-active E. coli extract. These worms had a shorter life span than B. subtilis fed worms. This life span shortening effect is dose-dependent upon the concentration of coQ-active E. coli extract (Figure 3B). Importantly, supplementation of B. subtilis fed worms with coQ-deficient E. coli extract led only to a minor life span decrease compared to worms fed exclusively on B. subtilis (Figure 3C). E. coli extracts likely contain other components besides coQ that are detrimental to the worm such as lipopolysaccharides (LPS) (Maier et al., 2010), folic acid (Virk et al., 2012), etc. Therefore, the minor life span decrease observed when B. subtilis fed worms are supplemented with coQ-deficient E. coli extract is likely due to the presence of these other E. coli components. The fact that coQ-active E. coli extracts significantly decreased the life span extension of B. subtilis fed worms whereas coQ-deficient E. coli extracts only led to a slight life span decrease indicates that the contribution of other factors besides coQ from the E. coli extract to the life span shortening of B. subtilis fed worms is smaller, thus suggesting that B. subtilis fed worms live longer mainly because this diet does not provide supplemental coQ to the worms.

E. coli fed worms to which coQ-active E. coli extracts were added lived the same as E. coli fed worms (Figure 3B). Moreover, E. coli fed worms to which coQ-less extracts from either mutant E. coli or B. subtilis were added lived the same as E. coli fed worms (Figure 3C; Figure S5).

Dietary coQ from E. coli acts as an antioxidant and shortens C. elegans life span

Besides being an essential ETC acceptor/donor of electrons, coQ is also a potent antioxidant [40]. C. elegans and E. coli, but not B. subtilis, synthesize their own coQ. Thus, worms that feed on E. coli are subjected to persistent dietary coQ supplementation and this may act as a lifelong antioxidant treatment for the worms affecting negatively their life span. To begin testing this possibility, we maintained WT L1 larvae on E. coli or B. subtilis seeded plates to which we had added a small concentration of the oxidant paraquat (PQ). If dietary coQ supplementation by E. coli indeed acts as an antioxidant, then it should counteract the effect of the oxidant. E. coli fed L1 larvae maintained on PQ containing plates suffered a minor developmental delay. However, B. subtilis fed L1 larvae maintained on PQ containing plates exhibited a severe developmental delay (Figure 4A-C). We performed the same experiment using coQ-deficient clk-1 worms. clk-1 L1 larvae maintained on E. coli seeded plates that contained PQ displayed a severe developmental delay compared with control E. coli fed clk-1 L1 larvae (Figure 4D-F). Although clk-1 L1 larvae are not able to complete development when maintained on B. subtilis, the arrested development phenotype was more severe when plates contained PQ (Figure 4D-F). After 14 days, the B. subtilis fed clk-1 L1 larvae had transitioned into sterile adults, whereas all the B. subtilis fed clk-1 larvae maintained on PQ plates continued arrested at L1 stage. The development in the presence of the PQ was also severely affected when WT larvae were fed a coQ-deficient E. coli diet (Figure S6). Our results show that worms with more coQ content such as E. coli fed WT worms, are able to better offset the oxidative effect of PQ and thus counteract the developmental delay induced by PQ.

Figure 4. Treatment with PQ severely affects the development of worms feeding on coQ-less B. subtilis and development of coQ-deficient clk-1 mutant worms. (A-C) Development of synchronized wild type N2 L1 larvae after 2, 3 and 4 days feeding on E. coli or B. subtilis with or without PQ treatment (0.1 mM). (A-C) Development of synchronized clk-1(qm30) L1 larvae after 3, 4 and 5 days feeding on E. coli or B. subtilis with or without PQ treatment (0.1 mM). (A-F) y-axis shows percentage of individuals that reached each developmental stage after the indicated time. x-axis shows developmental stages. Bars indicate the mean value ± S.D. n = 118-196 worms per group.

If E. coli fed worms were indeed subjected to the persistent antioxidant effect induced by coQ supplementation, then they should be in a less oxidized cellular state than B. subtilis fed worms. We examined the overall cellular reactive oxygen species (ROS) levels of worms and observed that B. subtilis fed worms had cellular ROS levels 40% higher than E. coli fed worms (Figure 5A). E. coli fed worms maintained on PQ-containing plates also had increased cellular ROS levels and these were similar to those observed for B. subtilis fed worms. E. coli or B. subtilis diet plates that contained the chemical antioxidant N-acetyl-cysteine (NAC) led to worms with approximately 40% lower cellular ROS levels than worms fed solely E. coli or B. subtilis, respectively (Figure 5A). Worms maintained on B. subtilis seeded plates that contained the oxidant PQ displayed higher ROS levels than B. subtilis fed worms or E. coli fed worms that were maintained on PQ plates (Figure 5A). E. coli fed clk-1 worms as well as coQ-deficient E. coli fed WT worms also displayed higher cellular ROS levels than E. coli fed WT worms (Figure S7). Next, we used cellular lysates obtained from worms fed either E. coli or B. subtilis and compared their amounts of malondialdehyde (MDA), a commonly used marker of lipid peroxidation [41]. B. subtilis fed worms as well as PQ-treated E. coli fed controls had 1.7 and 2.2 fold higher MDA levels than E. coli fed worms, respectively (Figure 5B). The ROS and the MDA data indicate that worms feeding on the coQ-containing E. coli diet are in a less oxidized cellular state than worms feeding on the coQ-less B. subtilis diet.

Figure 5. B. subtilis fed worms live longer and are in a higher oxidation state than E. coli fed worms. (A) B. subtilis fed worms have higher ROS levels than E. coli fed worms. Treatment with the antioxidant NAC (10 mM) decreases ROS levels of worms in both diets. Treatment with the pro-oxidant PQ (0.1 mM) increases the ROS levels of worms in both diets. Bars indicate the relative mean fluorescent marker expression ± S.E.M difference to the E. coli fed worms control. n = 10-20 for each group (**p < 0.01, *p < 0.05, Student’s t test). Statistical significance for B. subtilis fed worms treated with NAC and for B. subtilis fed worms treated with PQ is established with respect to B. subtilis fed worms. y-axis indicates relative fluorescence from DCFDA. x-axis indicates diet and treatment of worms. (B) B. subtilis fed worms have higher MDA levels than E. coli fed worms. n = 3-4 for each group (*p < 0.05, Bonferroni). y-axis indicates μg of MDA per mg of total protein. x-axis indicates diet and treatment of worms. (C) Life span curves for adult worms maintained on E. coli or B. subtilis with or without NAC treatment. (D) Life span curves for adult worms maintained on E. coli or B. subtilis with or without PQ treatment. Worms were subjected to PQ treatment since L1, or since adulthood (a.d.). (C-D) y-axis indicates percentage of worms that are alive. x-axis indicates day of adulthood. See also Table S5.

If the life span difference between E. coli and B. subtilis fed worms is due to the less oxidized state induced by the persistent coQ antioxidant effect of the E. coli diet, then treating B. subtilis fed worms with an antioxidant should lead to worms with lower cellular ROS levels and shorter life spans. Treatment of B. subtilis fed worms with the antioxidant NAC leads to worms with lower ROS levels than B. subtilis fed worms (Figure 5A). NAC treatment had no life span effect on E. coli fed worms. However, worms maintained on B. subtilis seeded plates that also contained NAC lived shorter than control B. subtilis fed worms (Figure 5C). This life span decrease is partial as NAC-treated B. subtilis fed worms still lived 27% longer than E. coli fed worms. The partial life span reduction effect might be due to other beneficial effects induced by the B. subtilis diet such as the lack of LPS.

Treating E. coli fed worms with a low concentration of PQ increases cellular ROS levels (Figure 5A) and, as already reported [42,43], worms live longer than untreated E. coli fed worms. B. subtilis fed worms and PQ-treated E. coli fed worms exhibit similar cellular ROS levels (Figure 5A) and live considerably longer than E. coli fed worms (Figure 1A; Figure 5A). If the mechanism by which B. subtilis fed worms live longer than E. coli fed worms is independent of the mechanism by which PQ-treated E. coli fed worms live longer than E. coli fed worms, then treating B. subtilis fed worms with PQ should lead to a further longevity increase. We tested this possibility by measuring the life span of E. coli and B. subtilis fed worms when these were subjected to PQ treatment. PQ-treated B. subtilis fed worms lived only 8.4% longer than PQ-treated E. coli fed worms. Moreover, starting the PQ treatment after worms reached adulthood led to a minimal 6.4% life span difference between E. coli and B. subtilis fed worms (Figure 5D). The partial life span increase observed between PQ-treated B. subtilis and PQ-treated E. coli fed worms might be due to other beneficial effects induced by the B. subtilis diet such as the lack of LPS. Interestingly, coQ-deficient E. coli fed worms also failed to further increase their longevity upon PQ treatment (Figure S8). These results indicate that the mechanism by which B. subtilis, coQ-deficient E. coli, and PQ-treated E. coli fed worms live longer than E. coli fed worms is likely the same.

Mutations that lead to life span alterations when worms are fed E. coli do not always lead to proportional life span changes when worms are fed B. subtilis

Most C. elegans aging research is done using E. coli as the standard control diet. Since the longevity of E. coli fed worms is affected by dietary factors such as the persistent exposure to dietary coQ, we assayed whether genetic interventions that alter longevity by affecting C. elegans aging, immunity, and stress response lead to similar results when worms are fed E. coli or B. subtilis. We used 14 C. elegans mutant strains including the insulin/IGF-1-like signaling pathway receptor daf-2 [44]; two alleles of the daf-16 forkhead box O (FOXO) transcription factor homologue, daf-16(mu86) and daf-16(mgDf50), which regulate insulin/IGF-1-mediated signaling activity [45]; and the double mutant daf-2;daf-16. We also used the stress response transcription factor mutant hsf-1 [46]; and the hypoxia-induced factor mutant hif-1 [42]. The mutant strains we used that affect the innate immune response were the mitogen-activated protein kinase (MAPK) pmk-1 [47]; the member of the transforming growth factor beta (TGFβ) superfamily dbl-1 [48]; the serine/threonine kinase member of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases jnk-1 [49]; and the Toll-like receptor (TLR) tol-1 [50]. We also used several mutant strains that are known to increase worm longevity including the nicotinic acetylcholine receptor subunit eat-2, which mimics dietary restriction [51]; the Notch family receptor glp-1 [52]; and the mitochondrial electron transport chain complex I and III subunits nuo-6 and isp-1 [43].

WT worms fed B. subtilis lived 45% longer than E. coli fed WT worms. Using this result as a reference point, we examined the life span difference between the 14 mutants when worms were fed either of the two diets. We observed that in the case of dbl-1, jnk-1, isp-1, nuo-6, and daf-2 mutants the life span difference when worms were fed either diet was similar (Figure 6A and Table S7). However, in the case of the glp-1, eat-2, hif-1, pmk-1, hsf-1, daf-16, daf-2; daf-16, and tol-1 mutants the life span difference with respect to WT worms when worms were fed E. coli or B. subtilis was not proportionally similar. These mutants could be divided in 4 categories according to their life span differences with respect to WT controls. The two daf-16 alleles, daf-2;daf-16, pmk-1, and hsf-1 mutant worms lived proportionally shorter than WT worms when fed B. subtilis than when fed E. coli (Figure 6). glp-1 and eat-2 mutant worms lived proportionally longer than WT worms when fed E. coli than when fed B. subtilis (Figure 6). hif-1 worms displayed a slightly shorter life span than WT worms when fed B. subtilis, yet they displayed a slightly longer life span than WT worms when fed E. coli. Interestingly, tol-1 mutant worms showed a strong differential longevity response with respect to WT worms depending on the diet. These worms lived 28% longer than WT worms when fed B. subtilis, yet they lived slightly shorter than WT worms when fed E. coli. Taken together, the data show that different C. elegans signaling pathways involved in aging, immunity and stress response can be more or less relevant for worm survival depending on dietary and microbiome factors.

Figure 6. Genetic interventions that affect the life span of worms fed E. coli do not always lead to similar effects in worms fed B. subtilis. (A) Median life span ± S.E.M. of 14 mutant strains as well as WT worms when worms are fed E. coli or B. subtilis. Perpendicular dotted lines are reference lines representing the median life span of WT worms on each diet. The diagonal dotted line represents a perfect proportionality between median life span for mutant worms upon feeding B. subtilis relative to feeding E. coli, based on the WT median life span results. See also Table S6. (B) Bars represent the percentage of life span increase or decrease for mutant worms fed E. coli or B. subtilis relative to WT worms fed the same type of diet. Indicated is the mutant and allele used in each life span experiment.

Reagents

NAC (Sigma) was prepared as 500 mM stock solution in distilled water. PQ (Sigma) was prepared as 1 M stock solution in distilled water. FUDR (Sigma) was prepared as 30 mM stock solution in distilled water. H₂DCFDA (Life Technologies) was prepared as 10 mg/ml stock solution in DMSO and stored at -20 ^oC. MDA standard was purchased from Sigma.

C. elegans maintenance

Worms were maintained and handled as previously described [2]. Unless otherwise stated, all the reagents used in C. elegans experiments were added to the molten NGM agar and cast into culture plates. Hardened agar was seeded with the proper bacterial culture. All C. elegans experiments were conducted at 20 °C.

C. elegans development assay

C. elegans embryos were isolated following standard bleaching protocol using a 1:2 solution of 5 M NaOH:5% NaOCl [2]. Embryos were allowed to hatch overnight at 20 °C in M9 buffer. Arrested L1 larvae were transferred to NGM plates seeded with the proper bacterial culture. Worms were examined over time and the percentage of adults and larvae at different stages was determined.

C. elegans progeny determination

Total progeny number was determined as previously published [58] by counting the daily as well as the cumulative number of progeny from 5 single worms.

Dietary restriction conditions

We subjected adult worms to solid dietary restriction (sDR) as previously described [59] with slight modifications. sDR conditions were achieved by seeding plates with 10⁸ CFU/ml (strong sDR) or with 10⁹ CFU/ml (mild sDR).

C. elegans lifespan assay

Synchronized L4 larvae were transferred to bacterial seeded NGM plates containing FUDR (15 μM). 200 μl of the corresponding bacterial culture were used to seed plates. When a specific bacterium was used for a life span experiment, worms were fed this type of bacteria during development and adulthood. Life span assays with UV-killed E. coli or B. subtilis were done by UV treating bacteria as described [24]. Plates were scored for dead worms every other day. Worms that did not respond to prodding with a platinum wire were considered dead. Age refers to days following adulthood. Individuals were excluded from the analysis when their gonad was extruded, or when they desiccated by crawling onto the edge of the housing plate.

C. elegans fluorescence microscopy

A Zeiss Axioplan microscope equipped with Zeiss AxioVision 4.6 software and a Leica DM6000B microscope equipped with Metamorph 7.6.2.0 software were used for quantitative fluorescence microscopy. Worm gut autofluorescence was imaged using a 650 nm bandpass filter. Images were captured with 20X lens and analyzed using ImageJ. To analyze fluorescence, the same area size was selected from worm to worm. This area included the anterior part of the intestine. Gut autofluorescence time courses were done using 10-15 worms per age. All pictures were taken on the same day with the same microscope settings. unc-54::mCherry and pha-4::mCherry fluorescence worms were photographed using 10x or 20x lenses and images were analyzed using ImageJ. All the mCherry fluorescence experiments were done using at least 15 worms per treatment and/or per age. For a given experiment, all pictures were taken on the same day with the same microscope settings. To analyze unc-54::mCherry and pha-4::mCherry fluorescence, the same area size was selected from worm to worm. In the case of unc-54::mCherry worms this area included the head of the worm until the end of the pharynx. In the case pha-4::mCherry worms this area included the first two pairs of intestinal cells.

ROS determination

2:1 parts of M9 buffer:H₂DCFDA stock solution were mixed and divided into 0.5 ml tubes. 2-day-old worms of each sample were placed in each tube and incubated for 20 minutes at room temperature with moderate shaking (protected from the light). Worms were then centrifuged at 2000 rpm and washed twice with M9. Worms were pipetted onto agar pads (2% agarose in water) that were prepared on glass slides and covered with a coverslip. Individual worms were photographed using the GFP channel at 10X magnification. Fluorescence intensity was analyzed with ImageJ. To analyze fluorescence, the same area size was selected from worm to worm. This area included the head and the first two pairs of intestinal cells.

Bacterial extract preparations

40 ml of fresh overnight E. coli or B. subtilis cultures were distributed into conical tubes and incubated at 70^o C in a water bath for 30 minutes. 0.4 ml of WEC lysis buffer was added to each of the 40 ml samples and these were incubated at 4^o C for 30 minutes. Samples were then subjected to 4 cycles of liquid nitrogen followed by 30^o C water bath and centrifuged (4000 rpm, 15 minutes, 4^o C). After centrifugation, 28 ml of supernatant were discarded from each 40 ml tube. The pellet extract was then resuspended in the remaining 12 ml of supernatant. Dilutions (dH₂O) of the extract (1:2, 1:5, 1:10, 1:20) were done to prepare the extract at different concentrations. Prior to using an extract, we verified that there were not bacteria alive by inoculating two LB plates with 200 μl of extract and incubating them overnight at 37^o C. E. coli (GD1) and B. subtilis bacterial cultures grew at a concentration of fewer CFU/ml than E. coli (OP50). Thus, bacterial extracts were concentrated to achieve CFU/ml equivalent to those obtained for E. coli (OP50). 75 μl of extracts were dispensed on top of the bacterial lawn of worm plates every other day for the first 20 days of worm adulthood as well as during development.

Bacterial nutritional composition

To determine the water content, approximately 5 g of pelleted bacteria were placed in conical tubes and samples were freeze-dried. The water content was determined from the loss of weight of the samples before and after lyophilization.

Protein composition was determined by the Kjeldalh method (AOAC International, 2005a). Bacterial pellets were freeze-dried and 0.2-0.3 g of each sample were used in the procedure. Samples were transferred into a Kjeldahl digestion flask containing 10 g of catalyst (96% K₂SO₄ / 4% CuSO₄^.5H₂O) and 20 ml of H₂SO₄ and subjected to 2 hours of digestion in a Kjeldalh digestion stand. Once the samples were cooled down at room temperature, the minerals were transferred to a distillation apparatus with 400 ml of distilled water, phenolphthalein as indicator, and NaOH (30%). The ammonia gas liberated from the solution was transferred from the digestion flask into the receiving flask, which contained an excess of H₂SO₄ (25 ml, 0.05385 M). The excess of H₂SO₄ was titrated by a NaOH solution (0.1093 M) using a mixed indicator endpoint (0.1 g/100 ml bromocresol green and 0.1 g/100 ml methyl red in 95% ethanol). The nitrogen content was determined and protein content was calculated using the conversion factor 6.25.

Fat composition was determined by the Soxhlet method (AOAC International, 2005b). Crude fat content was determined by extracting the fat from the sample using diethyl ether as solvent, and then by weighing the fat that was recovered. Bacterial pellets were freeze-dried and 0.8-1.2 g of each sample were used in the procedure. Samples were introduced in an extraction thimble, which was placed in a Soxhlet apparatus. Approximately 300 ml of diethyl ether were added. The Soxhlet apparatus was placed in a water bath (40^o C) prior to the extraction. The solvent completed 20 cycles of extraction, then it was removed and the fat extract was placed in a desiccation stove (100 ^o C) to remove the ether and to allow samples to dry completely. The fat content was calculated from the difference in weights before and after the procedure.

To determine the ash composition, bacterial pellets were freeze-dried and 1.5 g of each sample were placed in incineration melting pots (previously heated at 910^o C for 15 min), allowed to cool in a desiccator, and weighed as soon as they reached room temperature. Samples were placed in incineration melting pots in a uniform layer and placed at the entrance of the oven with the door open. Incineration was kept at 910^o C until total combustion of the samples. Incineration melting pots were removed from the oven, allowed to cool in a desiccator and weighed as soon as they reached room temperature. Ash content was calculated from the weight difference before and after incineration.

Carbohydrate composition was determined by the difference between the total weight of the dehydrated sample minus protein, fat and ashes.

Determination of MDA by dinitrophenylhydrazine assay

MDA determination was based on HPLC measurement after derivatization with 2,4-dinitrophenylhydrazine (DNPH) as described [60]. 500 µl of trichloroacetic acid was added to 400 µl of worm homogenate to remove proteins. After 15 min at 4º C the homogenate was centrifuged (10000 g for 10 min at 4º C) and the supernatant was collected and mixed with 100 µl of 5 mM DNPH in 2M HCl (prepared daily) and incubated for 1 hour at room temperature. The mixture was extracted three times with 400 µl of chloroform. The organic phase was collected and molecular sieves were added to the organic phase for 30 min. The molecular sieves were removed and the organic phase concentrated to dryness under nitrogen steam, and resuspended in 80 µl of acetic acid:acetonitrile (62:38, v/v), sonicated twice for 5 s, centrifuged (10000 g, 5 min) and injected in the HPLC system. A calibration curve with concentrations ranging from 0.25 to 100 µg/ml of MDA standard was performed. Protein content in worm homogenate was determined by the Bradford method [61] after digestion of the homogenate.

HPLC-DAD-ESI/MS analysis

We used a Hewlett-Packard 1100 chromatograph (Agilent Technologies) with a quaternary pump and a diode array detector (DAD) coupled to an HP Chem Station (rev. A.05.04) data-processing station. The column was a Waters Spherisorb S3 ODS-2 C8, 3 µm (4.6 x 150 mm) and the solvents were: (A) 0.2% acetic acid, and (B) acetonitrile. The elution gradient established was: isocratic 38% of B for 10 min, from 38% to 75% of B in 10 min, from 75% to 76% of B in 4 min, from 76% to 77% in 11 min, from 77% to 80% B in 5 min and wash and re-equilibrate the column. The flow rate established was 0.6 ml/min. Double online detection was carried out in the DAD using 310 nm, 360 nm and 380 nm as preferred wavelengths and in a mass spectrometer connected to HPLC system via the DAD cell outlet. Ms detection was performed in an API 3200 Qtrap (Applied Biosystems) equipped with an APCI source and a triple quadrupole-ion trap mass analyzer, which was controlled by the Analyst 5.1 software. The analytes were ionized in negative ion mode. The APCI temperature was set at 400 ºC, the nebulizer gas was 40 psi. Nitrogen served as the curtain was 10 psi and collision gas (medium). Data acquisition was performed in Enhanced Ms analysis mode, which produces single Ms type spectra. Method settings were: declustering potential (DP), -20 V; entrance potential (EP), -10 V; and collision energy (CE), -10V. In order to obtain the fragmentation pattern of the parent ion, enhanced product ion (EPI) mode was also applied using the following settings: declustering potential (DP), -40 V; entrance potential (EP), -6 V and collision energy (CE), -10V. MDA was quantified from their chromatographic peak recorded at 310 nm compared with calibration curve of the standard.