Characterization of Siderophore-Host Interactions

Emma Worden-Sapper

Thesis Defense Date April 8, 2019

Thesis Advisor: Min Han, MCDB

Honors Council Representative: Christy Fillman, MCDB

Other Committee Member: Stephanie Renfrow, EBIO

Abstract The role of the gut microbiome in influencing human health has received increasing attention in the past decade, with many of the interactions between symbiont and host being elucidated. A recent study found that the siderophore enterobactin, secreted by E. coli to scavenge iron from its environment, is an essential metabolite for Caenorhabditis elegans (C. elegans) development. The benefit of enterobactin, which appears to be conserved in mammals, carries potential for treating iron deficiency disorder. This thesis focuses on characterizing the nature of the interaction of C. elegans with siderophores other than enterobactin, to assess the uniqueness of the benefit enterobactin conveys to the host. By performing a series of assays with C. elegans, I provide evidence that yersiniabactin, ornibactin, and bacillibactin do not promote C. elegans growth, implying that these siderophores do not convey a benefit similar to enterobactin. 1. Introduction The increasing attention towards the health impacts of human microbial residents (the microbiome) has produced many surprising contradictions to the previous notions of the nature of host-microbe interactions. The microbiome has been found to impact mental health (Dinan and Cryan 2017; Hsiao et al. 2013; Sharon et al. 2014), the development of the immune system (Brestoff and Artis 2013; Hooper, Littman, and Macpherson 2012; Ellermann and Arthur 2017), and metabolism (Sharon et al. 2014; Ellermann and Arthur 2017) among many other physiological functions. One of these previous notions, that bacterial residents of the gastrointestinal (GI) tract “steal” iron from their hosts (Ellermann and Arthur 2017), has recently been challenged (Qi and Han 2018), suggesting a new interaction between host and resident that may carry implications for anemia treatments (Eschner 2018). Iron, as a metabolite that is essential for many biological processes, generates a conflict between host and resident, with both essentially fighting for its uptake (Ellermann and Arthur 2017). To this end, most bacteria secrete siderophores, small molecules which bind ferric (III) iron. After their reuptake by the cell—or by a different cell that is able to pirate the siderophore—the ferric iron is released from the siderophore and reduced to soluble ferrous (II) iron (Miethke and Marahiel 2007; Ellermann and Arthur 2017; Andrews, Robinson, and Rodríguez-Quiñones 2003). Siderophores have been thought to be key measures of bacterial virulence (Miethke and Marahiel 2007; Visser et al. 2004; Cassat and Skaar 2013). This paradigm is supported by the response of the mammalian host, which is to produce and secrete a protein (lipocalin-2) that binds and sequesters the E. coli produced siderophore enterobactin (Ellermann and Arthur 2017). The model organism Caenorhabditis elegans (C. elegans), a roundworm with a well-documented microbiome (Berg et al. 2016; Zhang et al. 2017; Shapira 2017), has provided a useful platform for testing this interplay between host and microbial resident. (Qi and Han 2018) recently revealed an interaction between C. elegans and one of its GI tract residents, E. coli, that challenged this canonical idea of siderophores as agents of virulence. Qi previously found that heat killed E. coli does not provide C. elegans with key nutrients required for normal growth (Qi, Kniazeva, and Han 2017), but that growth was rescued when a small amount of live, wild type E. coli was provided to the worms as a supplement. Thus, the live bacteria provide the worms with essential metabolites. However, replacing the live wild type E. coli with a live mutant unable to synthesize enterobactin yielded severely attenuated worm growth, implying that one of the key nutrients E. coli provides to its C. elegans host is enterobactin. Both worm growth and iron levels were rescued by the addition of enterobactin to this assay. This result carries great significance for the treatment of iron deficiency if it is conserved in mammals. Anemia affects roughly a quarter of the world’s population, especially the elderly (Tettamanti et al. 2010), children, pregnant women, and the populations of poorer countries (de Benoist B et al., eds. 2008; Pena-Rosas, Rogers, and Stevens 2015). Enterobactin, if it has the same effect in humans as in worms, could be used as a more effective treatment for this condition. The current treatment, iron supplements, poses several health threats, such as damage to the liver, heart, and other tissues that respond poorly to oxidative stress, due to the high reactivity of iron (Fisher and Naughton 2004; Wessling-Resnick 2017). There is still much unknown about the interaction between host and microbial residents, however, which must be elucidated prior to the use of treatments such as enterobactin. One of these unknowns is if siderophores other than enterobactin have the same beneficial effect on the host. This result would shed light on the role enterobacteria play in the gut as symbionts, and would potentially offer further explanation of the mediation of iron homeostasis in the host, an important clarification to make before introducing enterobactin to the host as a treatment for iron deficiency. If there is an interplay between beneficial siderophores, the host, and the symbionts in the GI tract, it would not be ideal to potentially interrupt this homeostasis with an abundance of a particular siderophore. Ruling out other siderophores would also allow for increased focus on enterobactin as the principal drug target. In this work, I addressed the question: Are siderophores other than enterobactin necessary or beneficial for C. elegans growth and/or iron levels? I tested C. elegans due to its previous use in the work in which the beneficial effect of enterobactin was discovered (Qi and Han 2018), making it the most likely organism to use other siderophores. In addition, C. elegans has a well- documented gut microbiome (Berg et al. 2016; Zhang et al. 2017; Shapira 2017). Knowing the types of bacteria likely to be present in the guts of wild-type worms narrows down the list of potentially beneficent siderophores, since if the worm does not live with a certain bacteria as a canonical symbiont, it is not likely to have evolved a way to use its siderophore. With this model, I performed the same assay as described by Qi (Qi and Han 2018) to test four siderophores, yersiniabactin, ornibactin, salmochelin, and bacillibactin. Of these, salmochelin requires further investigation, and the rest did not provide a benefit to the host.

2. Methods and Materials 2.1 C. elegans maintenance, strains, and preparation for plating C. elegans were grown on full bacterial lawns of OP50 (wild-type E. coli), on NGM (nematode growth medium) plates (20 mL petri dishes containing 10 mL NGM) at 20o C. Two different strains of C. elegans were used in this work, N2 (wild type) and an XA 6901 pftn-2::gfp reporter strain. This strain fluoresces with high iron levels (Qi and Han 2018), so it was used in order to analyze iron in the worm. While the reporter strain does not reach the same volume as the N2 strain (Fig. S2), the relative volumes are conserved. The N2 worms were used for the initial volumetric assays (Fig. 1, 2), and the reporter strain was used for the assay involving ampicillin (Fig. 3) and the assay performed over three days with full lawns of B. subtilis and Ent A- (Fig. S4), due to availability. The worms were plated at the L1 (juvenile) stage, roughly 300 worms per plate, roughly 5 plates per group. The worms were synchronized to L1 via bleaching of well-fed gravid adults the day before plating. 2.2 Characterization and maintenance of bacteria Four types of E. coli were used in this work: OP50, K12, Ent A-, and Ent F-, the former two being wild-type, the latter two being members of the Keio collection (Baba et al. 2006), a library of single knockouts of K12 genes. Ent A- and Ent F- are unable to synthesize enterobactin (Qi and Han 2018). Five types of B. subtilis were used in this work: wild-type B. subtilis, BKK 31990 ΔdhbC, BKK 31980 ΔdhbE, BKK 31970 ΔdhbB, and BKK 31960 ΔdhbF. The mutants are all knockouts for genes that encode enzymes that catalyze reactions in the bacillibactin biosynthesis pathway, and are thus unable to synthesize bacillibactin (Pi and Helmann 2018; May, Wendrich, and Marahiel 2001; Caspi 2008). BKK 31990 ΔdhbC is a knockout of ΔdhbC, BKK 31980 ΔdhbE is a knockout of ΔdhbE, BKK 31970 ΔdhbB is a knockout of ΔdhbB, and BKK 31960 ΔdhbF is a knockout of ΔdhbF. The bacterial mutants were streaked onto LB (lumina broth) plates with kanamycin, and the wild-type strains were streaked onto regular LB plates. 2.3 Preparation of bacteria Full lawns of bacteria were spotted onto NGM plates from an overnight culture in LB at 37o C. Roughly 0.5 mL of culture was used per plate. Small food spots for the assays with heat killed bacteria were prepared similarly in LB. The mutants were incubated in LB treated with 0.05 mg/mL kanamycin, while the wild type strains were incubated in untreated LB. Optical density was measured with a wavelength of 600 nm. In the case of the assays testing bacillibactin and yersiniabactin (Fig. 1B, 1C), the ODs were not synchronized. In the case of the assay testing ornibactin and salmochelin (Fig. 1D), the ODs of these food spots were synchronized to roughly 1, while in the case of the assay with ampicillin and the B. subtilis mutants (Fig. 3) the ODs were synchronized to roughly 0.7. The bacteria was heat killed as previously described by Qi (Qi and Han 2018; Qi, Kniazeva, and Han 2017). Overnight cultures of OP50 in LB were concentrated tenfold, then incubated in a 75o C water bath for 90 minutes. Preparation of heat-killed B. subtilis was originally performed in the same way, but after this procedure failed to sufficiently kill the bacteria, 87o C for 90 minutes was used instead for heat killing, based off of a procedure outlined to kill B. subtilis spores (Coleman et al. 2007). 150 μL of heat killed bacteria was then spotted onto an NGM plate. 2.4 Preparation and quantification of siderophores The siderophores were dissolved in DMSO, ethanol, or water at a concentration of 1 mg/mL. In the case of the preliminary volumetric assay (Fig. 1), they were spotted directly on top of the heat-killed OP50. The amount of each used was based on molarity. For the enterobactin (and the mock, DMSO), 5 μL of 1 mg/mL solution was used, or 7.47E-9 mol enterobactin. The same number of moles of the other siderophores was used (Fig. S1), so 6.59 μL bacillibactin 1 mg/mL solution, 3.60 μL yersiniabactin 1 mg/mL solution, 7.42 μL salmochelin 1 mg/mL solution, and 5.29 μL ornibactin 1 mg/mL solution were spotted onto their respective plates. For the full lawn assay with Ent A- and B. subtilis (Fig. S4), 5 μL enterobactin or DMSO was spotted just outside of the lawn, and the worms were plated directly on top of the siderophore or mock. 2.5 Incubation of experimental plates All plates were incubated at 20o C. All plates were scored after 3 days of incubation except the initial volumetric assays of bacillibactin and yersiniabactin (Fig 1B, 1C), which were scored after 4 days, and the volumetric and iron level assay of B. subtilis and Ent A- with a siderophore supplement (Fig S4), which was scored after 1, 2, and 3 days of incubation. 2.6 Ampicillin soaked plates The final assay (Fig. 3) involved plating a small amount of B. subtilis and a large amount of heat-killed B. subtilis to determine the effect of bacillibactin on C. elegans. However, I was not able to analyze B. subtilis plates that were uncontaminated by extensive growth of the bacteria across the culturing plates used. The success of this assay relies on the worm consuming enough bacteria to obtain metabolites, but not enough to live on as food. After several trials involving the progressive lowering of the optical density of the live bacteria, all unsuccessful, I resorted to soaking the plates with a 0.01 mg/mL ampicillin solution prior to plating any bacteria, heat killed or live. This concentration was determined by a dilution assay (Fig. S3). While not ideal for this trial, which involves live bacteria, this tactic did seem to impede the growth of B. subtilis. The average volume and iron level of the worms fed K12 was significantly greater than that of the worms fed Ent A- (Fig. 3B, 3C). This difference is expected when worms have access to a small amount of live food and a large amount of dead food, indicating that the level of antibiotic was not high enough to kill all live bacteria on the plate.

2.7 Microscopy The initial volumetric assay of bacillibactin (Fig. 1B) was photographed with an AF Micro Nikkor 60 mm Nikon camera and a WormLab Illuminator Base. The rest of the plates were observed with a Leica MZ16F microscope. Samples for Nomarski were prepared by washing plates with M9, then adding a small amount (~0.3 μL) of NaN3 to the suspended worms (to paralyze the worms), which were then mounted on slides. Nomarski microscopy was performed with a Zeiss Axioplan 2 microscope. 2.8 Analysis of C. elegans volume and fluorescence The surface areas of the worms in the initial volumetric assay with bacillibactin (Fig. 1B) were analyzed with WormLab software, while the volumes of the rest of the samples were analyzed with WormSizer (Image J plugin) software. Fluorescence was analyzed with Image J. 2.9 Statistical Analysis All statistical analyses are a student’s t-test, two-winged, assuming unequal variance. A p-value of less than 0.05 was considered significant. 3. Results This work was proposed and funded by principle investigator Min Han. I performed the trials and analyzed the data. The first assay was described by Dr. Qi of the Han lab, but the later assays were conceived of after discussion with Han and other lab members. I assayed four of the many potentially beneficial siderophores: yersiniabacin, salmochelin, ornibactin, and bacillibactin (Fig. S1). These four were chosen based on their likelihood of mimicking enterobactin’s beneficial activity. Yersiniabactin and salmochelin are both produced by E. coli (Searle et al. 2015). In testing these, I aimed to determine if other E. coli generated siderophores supply the same benefit to the host. Ornibactin is produced by bacteria of the Burkholderia genus (Deng et al. 2017; Visser et al. 2004), which is a common resident of C. elegans (Berg et al. 2016; Zhang et al. 2017), indicating a possible evolutionary link. Bacillibactin is produced by Bacillus subtilis. While Bacillus is not a canonical resident of C. elegans, bacillibactin has a very similar structure to enterobactin (Fig. S1), and C. elegans can use Bacillus as a food source (Gómez-Orte et al. 2017), (Fig. 2B). Of these four siderophores, only salmochelin and bacillibactin showed a potentially beneficial effect. To test bacillibactin further, I used the bacteria that produces it, Bacillus subtilis. This test, however, revealed a negative for bacillibactin as well. 3.1 Only salmochelin and bacillibactin showed potential benefit to the host The initial assay performed was as described by Qi (Qi and Han 2018), replacing enterobactin with test siderophores on the experimental plates (Fig. 1A). Normal growth was expected on plates supplemented with a small amount of K12 (wild-type E. coli), and on plates supplemented with a small amount of Ent A- (enterobactin-deficient E. coli) and enterobactin. Attenuated growth was expected on plates supplemented with a small amount of Ent A- and DMSO. Any siderophore that increased worm volume compared to the mock (DMSO) would be tested further. The worms supplemented with ornibactin and yersiniabactin exhibited no significant difference in volume from the mock worms (Fig. 1C, 1D), indicating that these siderophores were not able to mimic enterobactin in C. elegans. The worms supplemented with bacillibactin, however, did have a significantly larger volume than the mock worms, although they were still significantly smaller than the worms supplemented with enterobactin (Fig. 1B). The worms supplemented with salmochelin were significantly larger than the mock worms (Fig. 1D), but this difference was less significant than that observed in the worms supplemented with bacillibactin, so I did not pursue it for further investigation in this project. Figure 1: Volumetric assay of four test siderophores

Figure 1. A: The experimental setup was as previously described by Qi (Qi and Han 2018). N2 worms were used for this assay. B-D: The average volume of all worms fed test siderophores was significantly lower than those fed enterobactin, but worms fed bacillibactin and salmochelin were on average larger than those fed DMSO (mock). The first two trials (B, C) were incubated for four days, while the last trial (D) was incubated for three days.

3.2 C. elegans is able to use bacillus subtilis as a food source To further analyze bacillibactin, I began using Bacillus subtilis (B. subtilis), the bacteria that produces bacillibactin, in a functional assay; similar to that where E. coli was used to analyze the role of enterobactin. As a preliminary test, I plated worms with full bacterial lawns of OP50 (E. coli), B. subtilis, or one of four different B. subtilis mutants unable to synthesize bacillibactin (Fig. 2A). This was done in part to ensure that worms could grow and develop on full lawns of this bacteria, since the later assay with heat-killed bacteria depended on the worms being able to use B. subtilis or one of its mutants as food. There was no significant difference between the volumes of worms fed B. subtilis and worms fed B. subtilis mutants, but all of these groups were significantly lower in volume than the worms fed OP50 (Fig. 2B). All worms reached the L4 stage, however, indicating that C. elegans is able to use B. subtilis as a food source. Figure 2: Full lawn assay of B. subtilis and B. subtilis mutants

Figure 2. A: The plates used for this assay varied only in the identity of the live bacteria used, which was present in a quantity sufficient to keep the worms well fed. N2 worms were used for this assay. B: While all the worms reached the L4 (adult) stage, the average volume of the worms fed OP50 was significantly higher than that of the groups fed B. subtilis. These plates were incubated for three days.

3.3 C. elegans growth was the same on both bacillus subtilis and bacillus subtilis mutants Since worms plated on full lawns of bacteria were not arrested in growth, I repeated the same volumetric assay as described earlier, but with experimental variations among the type of live bacteria added to the plate instead of the type of siderophore (Fig. 3A). Since worms fed a small amount of wild-type E. coli and heat-killed E. coli are not arrested in growth, while worms fed a small amount of E. coli unable to synthesize enterobactin and heat-killed E. coli are arrested (Qi and Han 2018), I reasoned that if C. elegans can utilize bacillibactin, this same pattern should be observed with B. subtilis as well. Figure 3: Volumetric and iron level assay of worms fed B. subtilis

Figure 3. A: The experimental setup of this assay was similar to the previous assay (Figure 1A), but with a different spot of live bacteria in each experimental group rather than a test siderophore. These plates were also soaked with 1 mL of 0.01 mg/mL ampicillin solution prior to addition of bacteria. Incubation time was three days, and a gfp reporter strain was used for this assay. B, C: The worms fed a small spot of live K12 had a significantly larger average volume and iron level than that of the worms fed Ent A-. D: There was no significant difference between the average volumes of the worms fed wild type B. subtilis and those fed the mutants. E: There was no significant difference between the iron levels of the bacillus fed worms, except for a slight difference between B. subtilis and one mutant. Worms were taken from two plates per group for Nomarski analysis of fluorescence. There was no significant difference in worm volume between B. subtilis and its corresponding mutants (Fig. 3D). I analyzed the iron levels of the same worms, and found only a slight difference between B. subtilis and one of its mutants (Fig. 3E), which could likely be explained by small sample size. 3.4 Summary of results The trials performed in this work are summarized in table 1. Table 1—Summary of Siderophore Assay Results Description of assay Result Conclusion Initial volumetric assay of N2 Attenuated worm growth on Ornibactin and yersiniabactin with test siderophores plates with ornibactin and are not essential for C. bacillibactin, yersiniabactin, yersiniabactin; slight growth elegans growth; bacillibactin salmochelin, and ornibactin on plates with salmochelin and salmochelin inconclusive. (Fig. 1A). and bacillibactin (Fig 1B-D). Volumetric assay of N2 on Worms all reached the L4 Worms can use B. subtilis as full lawns of B. subtilis and stage, but worms fed OP50 food. bacillibactin deficient B. (E. coli) were larger than subtilis mutants (Fig 2A). worms fed B. subtilis (Fig 2B). Volumetric assay of B. B. subtilis spread over the Inconclusive. subtilis and bacillibactin plates, making them deficient B. subtilis mutants impossible to score (data not with a small amount of live shown). bacteria and heat killed bacteria (Fig 3A)—no antibiotic. Volumetric assay of B. No difference in volume or Worms do not require subtilis and bacillibactin iron level between worms fed bacillibactin. deficient B. subtilis mutants a small amount of live B. with a small amount of live subtilis and worms fed a bacteria and heat killed small amount of live B. bacteria (Fig 3A)—plates subtilis mutant (Fig 3D, 3E). soaked with 0.01 mg/mL ampicillin. Iron level assay of worms fed Not enough worms per slide Inconclusive. only heat-killed bacteria and to produce meaningful results a siderophore supplement or (data not shown). mock. Volumetric and iron level Not enough worms per slide Needs to be repeated before assay of worms fed a full to produce meaningful results speculation can be made on lawn of live bacteria and a on day 1; on days 2 and 3 the result. siderophore supplement or results were inconclusive mock (Fig. S4A). (Fig. S4B-S4I).

4. Discussion These results provide evidence that ornibactin, yersiniabactin, and bacillibactin cannot mimic the effects of enterobactin. The preliminary screens of yersiniabactin and ornibactin revealed no difference between worms fed these siderophores and those fed a mock (Fig. 1C, 1D). The worms fed bacillibactin had a significantly higher average volume than those fed the mock, but a significantly lower volume than those fed enterobactin (Fig. 1B). Further testing of bacillibactin, using a bacteria that produces this siderophore, B. subtilis, revealed no significant difference between worms fed a small amount of B. subtilis and worms fed a small amount of a B. subtilis mutant unable to synthesize bacillibactin (Fig. 3D, 3E), indicating that the lack of this metabolite had no effect on the worms. These results are in favor of the hypothesis that no siderophore on this list can perform the function of enterobactin. Drug design with enterobactin is somewhat simplified by this result. Other beneficial siderophores represent confounding variables that could potentially disrupt the equilibrium in the gut. This work has also established an experimental model for future testing of siderophores, as outlined by figures 1A, 2A, and 3A. Bacillibactin deficient bacteria were not any more or less effective than wild type bacteria in rescuing worm volume or iron levels, making it unlikely that bacillibactin is an essential metabolite for C. elegans (Fig. 3D, 3E). However, since the assay that demonstrated this result was performed on plates that had been soaked with ampicillin, a replicate without antibiotics should be obtained. While not demonstrated, it seems likely that the excessive spread of B. subtilis across the plates was due to its ability to sporulate, a response triggered by starvation, high cell density, or even xenosiderophores (Grandchamp, Caro, and Shank 2017). In order to obtain this result in an antibiotic-free environment, a key gene required for sporulation could be targeted for knockout. The master regulator of sporulation, Spo0A (Piggot and Hilbert 2004), could be targeted. Another potential candidate for this knockout is the gene encoding isoleucyl tRNA-synthetase, ileS. B. subtilis mutants with an ileS knockout were severely attenuated in effective sporulation, but these mutants retained the ability to grow normally (Kermgard et al. 2017). These mutants could potentially be used for the assay described (Fig. 3), but the ability of C. elegans to use this bacteria as food would have to be tested again. Salmochelin, the other siderophore supplement that yielded worms with a significantly higher volume than the mock, should also be tested further, along with all E. coli produced siderophores. Since E. coli makes up the largest proportion of the C. elegans microbiome (Zhang et al. 2017; Berg et al. 2016) it is reasonable to expect the two species have evolved a mutually beneficial relationship. E. coli produces three other siderophores, salmochelin, yersiniabactin, and aerobactin (Searle et al. 2015). K12, however, does not synthesize these siderophores (“EcoCyc: Encyclopedia of E. Coli Genes and Metabolic Pathways” n.d.). In order to test these E. coli produced siderophores further, a strain encoding the genes required for the synthesis of these siderophores would have to be obtained, and knockouts would have to be generated. Many of these experiments be repeated, since the data shown includes only one replicate. The inconclusive trials (Table 1) should also be repeated. The siderophores tested in this work hardly represent a comprehensive list of all siderophores produced in the gut of C. elegans. The other flora commonly present in the gut should also be explored. 5. Acknowledgements I thank Min Han for proposing and supporting this work, Bitta Kahangi for her supervision, advice, instruction, and obtaining of strains and reagents, Aileen Sewell and Bin Qi for instruction and advice, and all members of the Han lab for their feedback. 6. References Andrews, Simon C., Andrea K. Robinson, and Francisco Rodríguez-Quiñones. 2003. “Bacterial Iron Homeostasis.” FEMS Microbiology Reviews 27 (2–3): 215–37. https://doi.org/10.1016/S0168-6445(03)00055-X. Baba, Tomoya, Takeshi Ara, Miki Hasegawa, Yuki Takai, Yoshiko Okumura, Miki Baba, Kirill A. Datsenko, Masaru Tomita, Barry L. Wanner, and Hirotada Mori. 2006. “Construction of Escherichia Coli K-12 in-Frame, Single-Gene Knockout Mutants: The Keio Collection.” Molecular Systems Biology 2: 2006.0008. https://doi.org/10.1038/msb4100050. Berg, Maureen, Ben Stenuit, Joshua Ho, Andrew Wang, Caitlin Parke, Matthew Knight, Lisa Alvarez-Cohen, and Michael Shapira. 2016. “Assembly of the Caenorhabditis Elegans Gut Microbiota from Diverse Soil Microbial Environments.” The ISME Journal 10 (8): 1998–2009. https://doi.org/10.1038/ismej.2015.253. Brestoff, Jonathan R, and David Artis. 2013. “Commensal Bacteria at the Interface of Host Metabolism and the Immune System.” Nature Immunology 14 (7): 676–84. https://doi.org/10.1038/ni.2640. Caspi, Ron. 2008. “Bacillus Subtilis Subtilis 168 Bacillibactin Biosynthesis.” BioCyc. April 25, 2008. https://biocyc.org/BSUB/NEW-IMAGE?type=PATHWAY&object=PWY- 5903&detail-level=2#. Cassat, James E., and Eric P. Skaar. 2013. “Iron in Infection and Immunity.” Cell Host & Microbe 13 (5): 509–19. https://doi.org/10.1016/j.chom.2013.04.010. Coleman, William H., De Chen, Yong-qing Li, Ann E. Cowan, and Peter Setlow. 2007. “How Moist Heat Kills Spores of Bacillus Subtilis.” Journal of Bacteriology 189 (23): 8458–66. https://doi.org/10.1128/JB.01242-07. de Benoist B et al., eds. 2008. “WHO | Global Anaemia Prevalence and Number of Individuals Affected.” WHO. 2008. https://www.who.int/vmnis/anaemia/prevalence/summary/anaemia_data_status_t2/en/. Deng, Peng, Adam Foxfire, Jianhong Xu, Sonya M. Baird, Jiayuan Jia, Keren H. Delgado, Ronald Shin, Leif Smith, and Shi-En Lu. 2017. “The Siderophore Product Ornibactin Is Required for the Bactericidal Activity of Burkholderia Contaminans MS14.” Appl. Environ. Microbiol. 83 (8): e00051-17. https://doi.org/10.1128/AEM.00051-17. Dinan, Timothy, and John Cryan. 2017. “Brain-Gut-Microbiota Axis and Mental Health.” Psychosomatic Medicine 79 (8): 920–26. https://doi.org/10.1097/PSY.0000000000000519. “EcoCyc: Encyclopedia of E. Coli Genes and Metabolic Pathways.” n.d. Accessed March 31, 2019. https://ecocyc.org/. Ellermann, Melissa, and Janelle C. Arthur. 2017. “Siderophore-Mediated Iron Acquisition and Modulation of Host-Bacterial Interactions.” Free Radical Biology & Medicine 105 (April): 68–78. https://doi.org/10.1016/j.freeradbiomed.2016.10.489. Eschner, Kat. 2018. “E. Coli Doesn’t Just Give You Food Poisoning—it Can Also Help You Get Essential Nutrients.” Popular Science. August 27, 2018. https://www.popsci.com/gut- microbiome-iron. Fisher, Anna EO, and Declan P Naughton. 2004. “Iron Supplements: The Quick Fix with Long- Term Consequences.” Nutrition Journal 3 (January): 2. https://doi.org/10.1186/1475- 2891-3-2. Gómez-Orte, Eva, Eric Cornes, Angelina Zheleva, Beatriz Sáenz-Narciso, María de Toro, María Iñiguez, Rosario López, et al. 2017. “Effect of the Diet Type and Temperature on the C. Elegans Transcriptome.” Oncotarget 9 (11): 9556–71. https://doi.org/10.18632/oncotarget.23563. Grandchamp, Gabrielle M., Lews Caro, and Elizabeth A. Shank. 2017. “Pirated Siderophores Promote Sporulation in Bacillus Subtilis.” Applied and Environmental Microbiology 83 (10). https://doi.org/10.1128/AEM.03293-16. Hooper, Lora V., Dan R. Littman, and Andrew J. Macpherson. 2012. “Interactions between the Microbiota and the Immune System.” Science (New York, N.Y.) 336 (6086): 1268–73. https://doi.org/10.1126/science.1223490. Hsiao, Elaine Y., Sara W. McBride, Sophia Hsien, Gil Sharon, Embriette R. Hyde, Tyler McCue, Julian A. Codelli, et al. 2013. “The Microbiota Modulates Gut Physiology and Behavioral Abnormalities Associated with Autism.” Cell 155 (7): 1451–63. https://doi.org/10.1016/j.cell.2013.11.024. Kermgard, Elizabeth, Zhou Yang, Annika-Marisa Michel, Rachel Simari, Jacqueline Wong, Michael Ibba, and Beth A. Lazazzera. 2017. “Quality Control by Isoleucyl-TRNA Synthetase of Bacillus Subtilis Is Required for Efficient Sporulation.” Scientific Reports 7 (January). https://doi.org/10.1038/srep41763. May, Jürgen J., Thomas M. Wendrich, and Mohamed A. Marahiel. 2001. “The Dhb Operon of Bacillus SubtilisEncodes the Biosynthetic Template for the Catecholic Siderophore 2,3- Dihydroxybenzoate-Glycine-Threonine Trimeric Ester Bacillibactin.” Journal of Biological Chemistry 276 (10): 7209–17. https://doi.org/10.1074/jbc.M009140200. Miethke, Marcus, and Mohamed A. Marahiel. 2007. “Siderophore-Based Iron Acquisition and Pathogen Control.” Microbiology and Molecular Biology Reviews : MMBR 71 (3): 413– 51. https://doi.org/10.1128/MMBR.00012-07. Pena-Rosas, Juan Pablo, Lisa Rogers, and Gretchen Stevens. 2015. “The Global Prevalence of Anaemia in 2011.” WHO. Pi, Hualiang, and John D. Helmann. 2018. “Genome-Wide Characterization of the Fur Regulatory Network Reveals a Link between Catechol Degradation and Bacillibactin Metabolism in Bacillus Subtilis.” MBio 9 (5). https://doi.org/10.1128/mBio.01451-18. Piggot, Patrick J, and David W Hilbert. 2004. “Sporulation of Bacillus Subtilis.” Current Opinion in Microbiology, Growth and development, 7 (6): 579–86. https://doi.org/10.1016/j.mib.2004.10.001. Qi, Bin, and Min Han. 2018. “Microbial Siderophore Enterobactin Promotes Mitochondrial Iron Uptake and Development of the Host via Interaction with ATP Synthase.” Cell, August. https://doi.org/10.1016/j.cell.2018.07.032. Qi, Bin, Marina Kniazeva, and Min Han. 2017. “A Vitamin-B2-Sensing Mechanism That Regulates Gut Protease Activity to Impact Animal’s Food Behavior and Growth.” ELife. June 1, 2017. https://doi.org/10.7554/eLife.26243. Searle, Laura J., Guillaume Méric, Ida Porcelli, Samuel K. Sheppard, and Sacha Lucchini. 2015. “Variation in Siderophore Biosynthetic Gene Distribution and Production across Environmental and Faecal Populations of Escherichia Coli.” PLoS ONE 10 (3). https://doi.org/10.1371/journal.pone.0117906. Shapira, Michael. 2017. “Host-Microbiota Interactions in Caenorhabditis Elegans and Their Significance.” Current Opinion in Microbiology 38 (August): 142–47. https://doi.org/10.1016/j.mib.2017.05.012. Sharon, Gil, Neha Garg, Justine Debelius, Rob Knight, Pieter C. Dorrestein, and Sarkis K. Mazmanian. 2014. “Specialized Metabolites from the Microbiome in Health and Disease.” Cell Metabolism 20 (5): 719–30. https://doi.org/10.1016/j.cmet.2014.10.016. Tettamanti, Mauro, Ugo Lucca, Francesca Gandini, Angela Recchia, Paola Mosconi, Giovanni Apolone, Alessandro Nobili, et al. 2010. “Prevalence, Incidence and Types of Mild Anemia in the Elderly: The ‘Health and Anemia’ Population-Based Study.” Haematologica 95 (11): 1849–56. https://doi.org/10.3324/haematol.2010.023101. Visser, M. B., S. Majumdar, E. Hani, and P. A. Sokol. 2004. “Importance of the Ornibactin and Pyochelin Siderophore Transport Systems in Burkholderia Cenocepacia Lung Infections.” Infection and Immunity 72 (5): 2850–57. https://doi.org/10.1128/IAI.72.5.2850-2857.2004. Wessling-Resnick, Marianne. 2017. “Excess Iron: Considerations Related to Development and Early Growth.” The American Journal of Clinical Nutrition 106 (Suppl 6): 1600S–1605S. https://doi.org/10.3945/ajcn.117.155879. Zhang, Fan, Maureen Berg, Katja Dierking, Marie-Anne Félix, Michael Shapira, Buck S. Samuel, and Hinrich Schulenburg. 2017. “Caenorhabditis Elegans as a Model for Microbiome Research.” Frontiers in Microbiology 8 (March). https://doi.org/10.3389/fmicb.2017.00485.

Supplemental Figures Figure S1

Figure S1. The functional groups of the siderophores used in this work are boxed (Miethke and Marahiel 2007; Stephan et al. 1993). The molar masses of the siderophores are given as well (PubChem n.d.). Structures courtesy of PubChem (PubChem n.d.).

Figure S4

Figure S4. A: XA 6901 pftn-2::gfp reporter worms were plated with full bacterial lawns and either enterobactin or a mock. They were scored after one day (data not shown), two days (B, C), and three days (D, E). B, D: Fluorescence was analyzed on days 2 and 3. One plate per group was washed to collect worms for Nomarski. C, E: Worm volume was scored on days 2 and 3. F-I: Images of the worms taken on day 3 are shown here. Top: Image of plates under dissecting microscope. Middle: Brightfield image of worms under Nomarski microscope. Bottom: Image of fluorescing worms under Nomarski microscope. F: C. elegans fed B. subtilis and DMSO. G: C. elegans fed B. subtilis and enterobactin. H: C. elegans fed Ent A- and DMSO. I: C. elegans fed Ent A- and enterobactin.