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Winter 2016

Iron uptake in symbiosis: The role of siderophore in the association between Vibrio fischeri and uprE ymna scolopes

Evan DaSilva University of New Hampshire, Durham

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Recommended Citation DaSilva, Evan, "Iron uptake in symbiosis: The role of siderophore in the association between Vibrio fischeri and Euprymna scolopes" (2016). Master's Theses and Capstones. 1090. https://scholars.unh.edu/thesis/1090

This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. IRON UPTAKE IN SYMBIOSIS: THE ROLE OF SIDEROPHORE IN THE ASSOCIATION

BETWEEN VIBRIO FISCHERI AND EUPRYMNA SCOLOPES

BY

EVAN DASILVA

BA in Biology, Hiram College, 2009

THESIS

Submitted to the University of New Hampshire

in Partial Fulfillment of

the Requirements for the Degree of

Master of Science

in

Genetics

December, 2016 This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Master’s in Genetics by:

Thesis Director, Dr. Cheryl Whistler, Associate

Professor, Molecular, Cellular, & Biomedical Sciences

Dr. Estelle Hrabak, Associate Professor, Molecular,

Cellular, & Biomedical Sciences

Dr. William K. Thomas, Hubbard Endowed Chair and

Director, Hubbard Center for Genome Studies,

Professor, Molecular, Cellular, & Biomedical Sciences

On May 9, 2016

ii TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………...... v LIST OF FIGURES………………………………………………………………...... vi ABSTRACT……………………………………………………………………...... vii

CHAPTER PAGE

I. INTRODUCTION………………………………………………………………...... 1 Direct uptake of iron ………………………………………………. ……....2 Iron uptake facilitated by extracellular ferric reductases ………………...... 4 Siderophore synthesis and uptake …………………………………………..5 Heme uptake in host-microbe interactions ………………………...... 11 TonB energy transduction facilitates ferric iron uptake in gram negative bacteria …………………………………………………………...11 Siderophore mediated evasion of host defenses …………………...... 12 Regulation of iron uptake …………………………………………..……...13 Alternative regulation of iron uptake ……………………………...... …....17 Anaerobic regulation of iron uptake …………………………...... 18 Use of the squid model for studying the role of iron uptake ………………19 Goals and hypotheses of thesis research ……………………………...…...20 II. METHODS ………………………………………………………………...... 23 III. RESULTS ………………………………………………………………………….31 Bioinformatics prediction of genes related to iron uptake and siderophore biosynthesis …………………………………………………..31 Identifying aerobactin biosynthesis and uptake mutants and defining the regulatory context for production, through phenomics analysis ………………………………………………………..36 Role and regulation of iron uptake in growth and protection against oxidative species …………………………………………………...... 41 The ΔiucA tonB1::Tn5erm mutant does not have a persistence

iii defect ……………………………………………………………….……..49 The siderophore uptake mutants, ED4D1 and ED4E1, do not exhibit a competitive defect in the light organ compared to the wildtype strain ES114 ……………………………………………………..50 G11 has a competitive defect in the light organ compared to strain ES114 ……………………………………………………………………...52 IV. DISCUSSION ……………………………………………………………...... 53 REFERENCES ……………………………………………………………………...... 60

iv LIST OF TABLES

TABLE 1. EXAMPLES OF DIRECT UPTAKE SYSTEMS FOR FERROUS (FE(II)) AND FERRIC (FE(III)) IRON ……...…...……………………………………...…...... 3

TABLE 2. CATECHOL TYPE SIDEROPHORES ...... 6

TABLE 3. HYDROXAMATE TYPE SIDEROPHORES …………………………………8

TABLE 4. CARBOXYLATE TYPE SIDEROPHORE ……………………………………9

TABLE 5. HYDROXY-CARBOXYLATE TYPE SIDEROPHORE ………………...... 9

TABLE 6. STRAINS USED ………………………………………………………………23

TABLE 7. PLASMIDS AND PRIMERS USED ………………………………………….24

TABLE 8. IRON UPTAKE SYSTEMS FOUND IN VIBRIO FISCHERI ……………….33

TABLE 9. TRANSPOSON INSERTIONS IN V. FISCHERI ES114 THAT RESULT IN A SIGNIFICANT SIDEROPHORE PHENOTYPE …………………………………...37

v LIST OF FIGURES

FIGURE 1. MODEL FOR IRON DEPENDENT REGULATION OF GENES THROUGH THE FERRIC UPTAKE REGULATOR ……………………………………14

FIGURE 2. THREE OF THE THEORIES OF HOW FUR BINDS TO FUR BOXES …..15

FIGURE 3. REGULATION OF THE FERRIC UPTAKE REGULATOR ……………....16

FIGURE 4. MODEL OF ALL IRON UPTAKE SYSTEMS AVAILABLE TO V. FISCHERI BASED ON A BIOINFORMATIC SEARCH ……………………………34

FIGURE 5. INDUCTION OF IRON REGULATION ……………………………………44

FIGURE 6. SIDEROPHORE PRODUCTION CAPACITY OF STRAINS OF INTEREST ………………………………………………………………...44

FIGURE 7. IRON UPTAKE CONTRIBUTES TO GROWTH WHEN IRON IS LIMITING ……………………………………………………………...45

FIGURE 8. ROLE OF SIDEROPHORE IN PEROXIDE RESISTANCE ……….…...... 46

FIGURE 9. HEME UPTAKE ABILITY OF STRAINS OF INTEREST …………………47

FIGURE 10. LIGHT ORGAN PERSISTENCE …………………………………………..49

FIGURE 11. RCI (RELATIVE COMPETITIVE INDEX) PLOTS OF LIGHT ORGAN COMPETITIONS ……………………………………………………….50

vi ABSTRACT

IRON UPTAKE IN SYMBIOSIS: THE ROLE OF SIDEROPHORE IN THE ASSOCIATION

BETWEEN VIBRIO FISCHERI AND EUPRYMNA SCOLOPES

by

Evan DaSilva

University of New Hampshire, December, 2016

Iron acquisition is well studied in pathogens, and successful virulence is often attributed to iron acquisition by siderophore and heme uptake; however, the role of iron uptake in mutual symbiotic interactions is not as well understood. The mutual symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a well-characterized system in which iron uptake has been implicated as a symbiotic factor. Four studies have implicated iron uptake in the symbiosis: 1) A TnLux reporter assay revealed that siderophore is more highly expressed by V. fischeri in the light organs of juvenile squid compared to V. fischeri in liquid culture; 2)

vii Microarray data showed that genes for siderophore production are upregulated in the light organs of adult squid; 3) A siderophore deficient glnD mutant of V. fischeri had a persistence defect in the light organ that was complemented by addition of iron to the seawater; and 4) A V. fischeri mutant in which the heme uptake locus was deleted had a persistence defect in the squid light organ that was apparent in competition with the ancestor strain V. fischeri ES114. I hypothesize that iron uptake by siderophore is necessary for persistence of V. fischeri in the squid light organ, complementary to heme uptake, and that due to the toxic nature of iron, sequestration by siderophore contributes oxidative stress response.

To assess the role of iron uptake in the interaction between V. fischeri and the Hawaiian bobtail squid we utilized several strategies: 1) I identified itron uptake systems available to V. fischeri by bioinformatically comparing known iron uptake systems against the genome; 2) To reveal potential avenues by which iron uptake is regulated in V. fischeri, we identified genes that influence siderophore biosynthesis by screening a transposon mutant library for siderophore phenotypes; 3) I assessed the physiological role, in growth and oxidative response, of several of the iron uptake genes previously identified; and 4) I directly assessed the symbiotic ability of mutants deficient in iron uptake.

The bioinformatic search revealed several siderophore uptake systems, as well as the previously described heme uptake system; however, only one siderophore biosynthesis system, for aerobactin, was identified. In screening the mutant library, I identified many genes in the flagellar locus and the cellular biosynthesis locus that positively influence siderophore production as well as two quorum sensing genes, AinS and RpoQ, and several cell wall biogenesis/oxidative sensing genes that negatively influence siderophore production. We determined that aerobactin biosynthesis does not contribute to oxidative stress response but does viii contribute to growth in iron limiting conditions, suggesting a purely nutritional role for siderophore in the symbiosis. When we tested the symbiotic ability of an iucA mutant deficient in siderophore, we could not demonstrate a persistence defect; however, we did find that two siderophore uptake mutants have a competitive defect 24 hours after inoculation, suggesting that siderophore contributes to symbiotic fitness. These findings suggest that regulation of iron uptake in V. fischeri involves more than just response to iron levels, and that iron uptake regulation is intertwined with symbiotically relevant traits. Due to the monospecific nature of the symbiosis, it is unlikely that the non-aerobactin uptake systems contribute to the symbiotic ability of V. fischeri; however, it is clear that aerobactin does contribute to symbiotic ability by conferring a growth advantage over other strains deficient in aerobactin uptake.

ix CHAPTER I: INTRODUCTION

Iron is an essential element for the growth of all organisms with rare exception (1). Due to the redox potential of iron, it is invaluable as a prosthetic group for many enzymes, including those involved in electron transport, nitrogen fixation, and oxidative stress response. In host environments, iron is typically limiting due to host sequestration, therefore high affinity iron uptake is essential for host-microbe interactions.

Iron acquisition is essential to the virulence of many known pathogens including Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Neisseria gonorrhoeae, Yersinia septica,

Yersinia pestis, E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and many more (2–8).

Even though some human symbionts play a role in preventing infection by pathogens, and there is at least one example where iron uptake is directly implicated, there has been a lack of focus on iron uptake in mutual symbiotic interactions (9). The mechanisms of iron uptake are not different between pathogens and mutualists, but whether iron uptake plays an equal role in pathogens and mutualists has yet to be sufficiently addressed. For my thesis I will determine the role of iron uptake in the mutual symbiosis between the Hawaiian Bobtail squid and the bioluminescent bacterium Vibrio fischeri. In order to understand the role of iron uptake in a host setting, it is helpful to understand the mechanisms by which iron is acquired by microbes, so I will give an in-depth overview of the different mechanisms for iron uptake, as well as how these mechanisms are regulated.

1 Direct uptake of iron

Under aerobic conditions and at a neutral pH, any iron not already biologically sequestered is primarily in the non-soluble ferric state (Fe(III)) which often forms biologically unusable oxides or hydroxides, yet under anaerobic or acidic conditions, available iron is typically in the soluble ferrous form (Fe(II))(10). Bacteria may employ direct uptake systems for both forms of iron, depending on the state of the available iron pool and examples of these uptake systems are listed in Table 1. All of the direct uptake systems for iron function by transporting either ferric or ferrous iron across the cytoplasmic membrane, and do not rely on active transport across the outer membrane. The passive transport of ferric iron across the outer membrane is peculiar, because it is not water soluble, however the Sfu system still functions even when TonB, which enables active transport of iron ligands across the outer membrane, is

"knocked out". This suggests that either un-chelated ferric iron can traverse the outer membrane in a passive manner, or that another system is enabling active transport across the outer membrane, yet the mechanism behind this is still unknown (11, 12).

Out of all of the uptake systems specific to ferrous iron, FeoABC is the only system that is utilized by a wide range of bacteria, however despite its ubiquity, little is known about how it functions. The Feo system typically consists of three proteins: FeoA, FeoB, and FeoC (YhgG).

For Gram negative bacteria, it is theorized that ferrous iron diffuses into the periplasm through unidentified porins and is then carried across the cytoplasmic membrane by the iron permease

FeoB, driven by hydrolysis of GTP. FeoB consists of two major domains: the hydrophilic N- terminal domain, also called the G-domain, is thought to be responsible for hydrolysis of GTP and transduces the energy to the membrane, while the C-terminal membrane domain is responsible for actively transporting the iron into the cell. The roles of FeoA and FeoC are

2 unclear, however it is suggested that the FeoA protein may interact with the G-domain of FeoB, and with the core region of FeoB; and FeoC may act as a transcriptional regulator of the Feo operon (13–15).

Table 1. Examples of direct uptake systems for ferrous (Fe(II)) and ferric (Fe(III)) iron. Iron uptake representative system metal transported organism(s) Ref. YfeABCD divalent metals Yersinia pestis 16 MntH Mn(II), Fe(II) S. typhimurium, 17 E. coli ZupT divalent metals E. coli 18 EfeUOB Fe(II) E. coli Nissle 1917 19 FeoABC Fe(II) E. coli 13, 15 SitABCD Mn(II), Fe(II) S. typhimurium 20 FutABC Fe(III) Synechocystis sp. 6803 21 SfuABC Fe(III) Serratia marcescens 11 FbpABC Fe(III) Neisseria gonorrhoeae 22 HitABCD Fe(III) Haemophilus influenzae 23

Iron uptake facilitated by extracellular ferric reductases

In cooperation with direct iron uptake, extracellular iron reductases can assist in iron acquisition under conditions that favor the ferric state of iron or conditions in which iron is being sequestered by host proteins such as transferrin. Iron reductases are enzymes that catalyze the reduction of ferric iron to ferrous, and are more efficient than chelators, such as siderophores, at releasing iron from host proteins. Free ferrous iron is then oxidized and chelated by siderophores or taken into the cell via direct ferrous uptake (10, 24, 25). Because these extracellular reductases are constitutively expressed, Cowart hypothesized that siderophores may be secondary in function for iron uptake since, as secondary metabolites, siderophores are only expressed during stationary/death phase under iron limiting conditions (10). However, the phase or growth

3 conditions under which siderophores are expressed has not been widely examined thereby challenging this assertion as dogma.

Siderophore synthesis and uptake

Under iron limiting conditions, siderophores are utilized for the acquisition of ferric iron.

Siderophores are low molecular weight, iron chelating compounds that have high affinity for ferric iron and are utilized by organisms in all three domains of life (carboxylate and hydroxamate type siderophores have been detected from archaea, but none have yet been structurally characterized) (Tables 2-5). In bacteria, the genes that encode the proteins responsible for siderophore synthesis are often organized in an operon proximal to or in the same operon as the genes responsible for the uptake of that siderophore. There are many known types of bacterial siderophores that differ in structure, and the various types of siderophores can differ in iron affinity, rate of secretion and rate of uptake. For example, the catechol siderophore enterobactin has very high affinity for ferric iron in vitro, but is not utilized as efficiently in vivo as the α-hydroxycarboxylate siderophore aerobactin (Table 5)(26). In the case of salmochelin, the addition of two sugar groups onto the enterobactin structure, which is catalyzed by two enzymes encoded by the iroB and iroE genes of the IroA gene cluster, allows the siderophore to evade host lipocalins (Table 2).

Siderophores are bound by siderophore-specific receptors on the outside of the cell that actively transport the siderophore into the periplasm (in gram negative bacteria), or the cytoplasm (in gram positive bacteria). In gram negative bacteria, after transport into the periplasm, the siderophore is then bound by a periplasmic binding protein, such as FhuD, which delivers the siderophore to an ABC transporter, such as FhuBC, which transports the siderophore into the cytoplasm. Once in the cytoplasm, depending on the siderophore type, the siderophore is

4 either degraded to release the iron (catechol siderophores) or a ferric reductase releases the iron and the siderophore is recycled (hydroxamate siderophores). Siderophore receptors are more specific to the siderophore than the cognate periplasmic ABC transport systems. For example, the FhuBCD system can transport both aerobactin and ferrichrome (Table 3, Table 5), while the aerobactin specific receptor IutA does not recognize ferrichrome, and the ferrichrome specific receptor, FhuA, does not recognize aerobactin. A bacterial strain may harbor siderophore receptors that recognize siderophores not produced by that strain, and the overlap of the periplasmic transport systems may keep the strain from expending energy on a separate ABC transporter specific for each receptor. Harboring receptors for xenobiotic siderophores can greatly benefit bacteria as it allows acquisition of iron without an incurred cost of siderophore production, and it may also broaden habitat range of a strain.

5 29 27 28 erences 30, 31 Ref coli E. E. Coli V. cholerae Organism(s) V. anguillarum S. typhimurium fes iroD viuB Genes Genes for siderophore utilization fepA fepB fepDGC fepE iroCiroN fatDCBA viuAvctPDGC Genes Genes for siderophore uptake ctional ctional aregroups highlighted entF entCEBAH entD entS iroB iroE angB/G angRTHX vibBDEFH Genes Genes for siderophore biosynthesis . The iron binding fun Vibriobactin Salmochelin Anguibactin Siderophore Enterobactin atechol type siderophores C Structure Table 2.Table

6 6 32, 33 References Y. pestis Organism(s) V. vulnificus vuuB (viuB) Genes Genes for siderophore utilization ybtPQX fatB vuuA psn Genes Genes for siderophore uptake 0831, 0836, 0840, - - - VV1_3173, VV2_0830 VV2_0834 VV2_0838 VV2_0844 irp1 irp2 ybtETU ybtS Genes Genes for siderophore biosynthesis Vulnibactin Yersiniabactin Siderophore (continued) Structure Table 2.Table

7 37 36 erences 34, 35 Ref (Fungus) (Fungus) Organism(s) Streptomyces pilosus Ustilago sphaerogena Rhodotorula pilimanae Streptomyces coelicolor ed fhuAfhuCDB fhuE fhuCDB fhuE fhuCDB Genes Genes for siderophore uptake + 2 d ,si + 1 d si desABCD Genes Genes for siderophore biosynthesis he iron bindinghe functional aregroups highlight T . hodotorulic Acid R Ferrichrome Desferoxamine Siderophore Structure Hydroxamate type siderophores . 3 Table

8 38 39 References (Fungus) Organism(s) microspores Escherichia coli The iron binding functional groups Rhizopus Aerobacter aerogenes iutA fhuCDB Genes Genes for siderophore uptake groups aregroups highlighted iucABCD Genes Genes for siderophore biosynthesis functional (contains both hydroxamic acid and carboxylate groups) . Aerobactin* Rhizoferrin Siderophore . The iron binding carboxylate siderophore - Carboxylate type siderophore . Hydroxy 4 . Structure Table Table 5 Table are highlighted.

9 Heme uptake in host-microbe interactions

In a mammalian host environment, free ferric iron is not readily available, however hemoglobin and haptoglobin are abundant sources of heme-iron that many bacteria have the ability to utilize (40). There are two types of outer membrane receptors for heme: those that employ a hemophore such as the HasA/HasR system, and those that directly uptake host hemoproteins (hemoglobin, haptoglobin, hemopexin, etc.) as well as heme itself, such as the

Hmu and Hut systems. In either type of heme uptake system, neither the hemoproteins or hemophores are carried across the outer membrane, instead the heme is released to the receptor and is then brought into the cell via active transport and the hemophores are subsequently recycled (40–42). In Gram negative bacteria, heme in the periplasm is bound by a periplasmic binding protein (PBP) and delivered to an inner membrane-bound ABC transporter that carries the heme-Fe(III) complex into the cytoplasm where it is degraded to release the iron (43, 44).

TonB energy transduction facilitates ferric iron uptake in gram negative bacteria

In gram negative bacteria, where the outer membrane is un-energized, transport of siderophores, heme, and hemophores is problematic. The bacterial solution to this problem is the

TonB energy transduction system in which a TonB protein spans the periplasm to carry an electron to a receptor in the outer membrane with the help of ExbB and ExbD, thereby energizing the receptor and allowing active transport of the iron ligand into the periplasm. The iron ligand is then carried across the inner membrane via ligand-specific ABC transport. One study suggested that the TonB protein also acts as a scaffold to catalyze the rendezvous between the iron ligand and periplasmic binding protein of the ABC transport system. Some gram negative bacteria harbor up to three paralogs of TonB, called TonB1, TonB2, and TonB3. The latter two systems utilize an additional protein called TtpC that spans the inner membrane and

10 aids in the interaction between TonB2 or TonB3 and the outer membrane receptor (45, 46). In some organisms heme uptake solely relies on the TonB1 system, while siderophore uptake systems can also utilize TonB2 and TonB3.

Siderophore-mediated evasion of host defenses

Siderophore-mediated iron sequestration can pose a problem to a host, as pathogenic bacteria particularly will take advantage of available iron. Mammals address this problem by producing siderocalins or lipocalins, which are siderophore-binding proteins that sequester bacterial siderophores and starve the bacteria of iron (47, 48). In response, bacteria have evolved simple ways of skirting these host defenses. The most well-studied example of this is in

Salmonella enterica serotype Typhimurium, which produces an enterobactin-derived siderophore known as salmochelin that is not bound by lipocalin-2. Linearization and di-glucosylation of the enterobactin siderophore by the gene products of iroE and iroB, respectively, results in the production of salmochelin, and this minor change to the siderophore is enough to abolish binding by lipocalin-2. In the mammalian gut, production of salmochelin confers a measurable advantage over other gut microbes and is often associated with pathogenic enteric bacteria including strains of E. coli and Klebsiella pneumoniae (7, 9, 49, 50). Although it is usually associated with pathogenic strains, salmochelin is sometimes utilized by "good bacteria", the probiotic E. coli strain Nissle 1917 biosynthesizes several different siderophores including salmochelin and aerobactin, which both evade lipocalin. Due to this phenomenal iron uptake suite, Nissle 1917 is able to out-compete S. enterica Serovar Typhimurium in the gut and prevents infection by this strain and other enteric pathogens (51, 52).

11 Regulation of iron uptake

Although iron is necessary, free iron can be toxic for organisms due to Fenton chemistry, through which ferrous iron reacts with oxidative species such as peroxide to generate more harmful oxidative species such as hydroxyl radical and superoxide (53). Due to this duplicitous nature, intracellular iron levels must be monitored and the uptake regulated tightly. For this purpose, many bacteria utilize the iron-sensing ferric uptake regulator (Fur) which binds to intracellular ferrous iron, causing a conformational change that allows binding to Fur recognition sites (Fur boxes or iron boxes) in the genome, resulting in repression of downstream genes (54)

(Fig. 1).

Classically, the Fur-Fe(II) complex has been linked to repression of genes involved in iron uptake, however it is now known that Fur- Fe(II)is involved in repressing a wide array of genes seemingly not involved in iron metabolism (Fig. 1A)(55), and even divalent metal independent repression by Fur has even been demonstrated in Helicobacter pylor i (56). In many organisms, Fur indirectly upregulates several genes under iron replete conditions through the repression of a small inhibitory RNA called ryhB (Fig. 1B). Through ryhB, Fur controls expression of the succinate dehydrogenase operon sdhCDAB, aconitase and fumarase (acnA, fumA), ferritin and bacterioferritin (ftnA, bfr), the iron sulfur cluster biosynthesis system encoded by iscRSUA, and the iron superoxide dismutase SodB (57–60).

There are more than a few theories as to how Fur recognizes the fur-box motif (Fig. 2).

The classical theory is that a Fur dimer binds to a 19 bp palindromic region characterized by the consensus 5’GATAATGATAATCATTATC-3’. A second theory is that a fur box is composed of at least three 6 bp motifs of 5’GATAAT-3’ where the third motif is inverted and separated from the other two by a single base pair. A third theory is that a fur dimer recognizes a 7-1-7 12 motif of 5’TGATAATGATAATCA-3’ or 5’TGATAATCATTATCA-3’, the 19 bp consensus contains two overlapping 7-1-7 motifs classified as a (7-1-7)2 motif, which was shown to bind even more strongly than a lone 7-1-7 motif (61–64).

There are several pathways through which the expression of Fur is regulated, adding to the complexity and sensitivity of iron regulation. To start, fur transcription is activated by the cAMP-CAP system and is also moderately repressed by Fur-Fe(II)(Fig. 3B, 3C) (65). In this model, Fur expression would be higher under iron limitation when an abundance of the protein is not necessary. Večerek et. al. showed that Fur is also negatively regulated post-transcriptionally by ryhB (Fig. 3D), keeping the Fur levels in check during iron limitation (66). Under certain conditions, an increased sensitivity of iron surveillance resulting from increased levels of Fur may be beneficial for the cell. In E. coli, OxyR and SoxRS, as well as RpoS, all activate transcription of Fur, increasing sensitivity to iron levels during periods of oxidative stress and during stationary phase, which may protect against the toxicity of iron in the presence of oxidative species (Fig. 3A)(67, 68).

Fur activity is also modulated (post-translationally) in response to several environmental cues, either through general inhibition, inhibition at specific binding sites, or activation at specific sites, broadening the cues involved in regulation of iron uptake. Under conditions of nitric oxide stress, iron bound to Fur becomes nitrosylated, which renders the Fur dimers inactive

(Fig. 3E). This nitrosylation globally relieves Fur repression, including repression of hmp which encodes the NO-detoxifying flavohemoglobin (69). During early growth phase, methylation of

DNA within fur boxes by dam can block de-novo Fur binding, causing constitutive expression of certain transcripts, likewise, bound Fur can block methylation of dam sites, interrupting activation of certain genes (Fig. 3F) (70). Many of the environmental cues that influence Fur are

13 relevant in host-microbe interactions, suggesting an important role for Fur and iron uptake in symbiosis.

14 15 Alternative regulation of iron uptake

In α-proteobacteria it has been suggested that Fur homologs only play a secondary role in the regulation of iron uptake, the primary role being manganese uptake regulation, while regulation of iron uptake appears to be relegated to Irr and RirA (71, 72). In high GC-content gram positives (e.g, Corynebacterium, Mycobacterium, Streptomyces), the role of Fur is played by DtxR (diphtheria toxin regulator), homologous to IdeR and SirR, however DtxR is only analogous in function, suggesting that iron dependent regulation has arisen more than once (73–

16 76). In an organism with a very high GC content, it is possible that the classic Fur box sequence is rare, so another regulator had filled the role of Fur.

Interestingly, some marine bacteria isolated from sea sponges activate their iron uptake mechanisms in response to heterologous siderophores produced by other species (77) and possibly in tandem with the C8-HSL quorum sensing molecule (78); however it is not clear whether this activation by siderophores is in addition to, or instead of, repression by Fur. This type of regulation makes sense for bacteria that occasionally encounter environments in which the iron levels are so low that siderophore production may not be fruitful and thus costly for the organism, but it also relies on other constituents of a bacterial community to supply the initial signal, so it is not likely that bacteria in isolated mono-specific cultures would utilize this system.

Anaerobic regulation of iron uptake

Under anaerobic conditions, such as those found in a mammal gut, the iron pool is in the ferrous form, meaning that the uptake of ferric iron becomes less useful than the uptake of ferrous iron, and in response, the iron uptake systems are regulated to favor uptake of ferrous iron. Fnr is a transcriptional repressor/activator that is responsible for regulating anaerobic respiration in response to anaerobiosis, and anaerobic activation of the feoABC operon is also attributed to Fnr (13). The negative regulation of feoABC by Fur in response to iron, coupled with positive regulation by Fnr in response to anaerobiosis, ensures that ferrous iron uptake is only upregulated under conditions that favor ferrous iron, yet still protects the cell from iron toxicity. It was also discovered that TonB is more heavily repressed by Fur under anaerobic conditions than under aerobic conditions, another oxygen sensing regulatory element is likely to be involved but has not yet been identified (79). Because the iron ligands that TonB is involved in importing are either not present under anaerobic conditions or not needed under anaerobic

17 conditions, there is no need for TonB to be expressed during anaerobiosis, unless the available iron has been sequestered by host iron chelators.

Use of the squid model for studying the role of iron uptake

The bioluminescent bacterium Vibrio fischeri enters into a mutual monospecific symbiosis with the squid Euprymna scolopes and provides the squid with a type of camouflage called counter-illumination (80, 81). At night, light produced by V. fischeri is projected out from the squid’s light organ on the ventral surface of the animal, thereby reducing the silhouette made by the hunting squid against the moonlight. There are three stages of a successful symbiosis between the squid and V. fischeri: initiation, colonization (or accommodation), and persistence.

The squid juveniles are hatched apo-symbiotically (without light organ symbionts) and they selectively recruit symbionts from the surrounding environment. In order to successfully initiate a symbiotic relationship with the squid, potential symbionts must successfully aggregate in the mucus that is produced around the light organ appendages in response to bacterial peptidoglycan.

It has been shown that only live gram negative cells are capable of aggregation in the mucus and, despite being a rare constituent of the surrounding environment, symbiotic strains of Vibrio fischeri dominate the aggregate population (81–85). Next, the potential symbionts migrate toward and into the pores at the base of the larger light organ appendage and travel down the ducts that lead to the light organ crypts. Strains deficient in motility, the outer membrane protein

OmpU, gene regulation by GacA and RscS, and quorum sensing are defective at initiating symbiosis and these mutants either show an inability to enter the light organ or are delayed in colonization of the light organ (86–93). Once inside the light organ crypts, Vibrio fischeri must grow to a high enough cell density to induce the quorum-sensing-controlled bioluminescence.

Strains deficient in amino acid synthesis of lysine, GacA mutants (94, 95), and

18 lipopolysaccharide Pgm mutants (96) are defective in their ability to colonize the light organ.

After the initial colonization, every subsequent morning the squid vents roughly 95% of its light organ contents into the surrounding water and, in order to persist in the light organ, the remaining symbionts must repopulate before nightfall.

Goals and hypotheses of thesis research:

Iron uptake by siderophore has been implicated as a symbiotic factor for V. fischeri in the light organ of the squid, with three separate studies providing supporting evidence. First, a V. fischeri glnD::TnCm mutant (SP301) which is deficient in siderophore production, has a persistence defect in the squid light organ, meaning that, after the initial venting, this strain is not able to sustain colonization within the light organ, and this persistence defect is complemented by addition of iron to the seawater (97). However, a caveat to this research is that, because glnD is a global regulator, a transposon insertion in this gene could be affecting other symbiotic traits.

A defined siderophore mutant was not tested for persistence ability, therefore this study only showed that, either general iron uptake is necessary for symbiosis or that glnD affects iron responsive regulation of symbiotic traits. Second, TnLuxAB insertions used to measure gene expression changes in vitro and in situ, revealed that aerobactin biosynthesis operon is expressed highly in the light organs of juvenile squid (98), indicating that siderophore production is a symbiotically regulated trait. Third, microarray data from adult squid revealed that the siderophore biosynthesis operon was highly expressed between 10:00 am and 4:00 pm, the initial period of regrowth after the squid vents its light organ contents (99). Fourth and finally, a deletion mutant of the heme uptake and utilization locus (ΔhmuTUVexbB1exbD1tonB1hutWXZ) is unable to persist in competition with V. fischeri strain ES114, the native squid isolate (100).

19 These findings together implicate iron uptake as important to successful persistence of V. fischeri within the light organ. I further elucidated the role of iron uptake in the Squid-Vibrio symbiosis and specifically addressed the role of siderophore in the symbiosis. I hypothesized that iron uptake by siderophore plays a crucial nutritional role in the association between V. fischeri and the squid E. scolopes, complementary to the role of heme uptake, and that siderophore also plays a role in protecting V. fischeri from oxidative stress.

In addressing these hypotheses, I devised several experimental goals, described in this thesis. These are:

Goal 1: Define the iron uptake systems in the V. fischeri genome.

I did this by bioinformatically comparing known siderophore and other iron uptake systems against the V. fischeri genome using BLAST, and by searching the genome for already annotated iron uptake systems. This analysis will provide insight into the iron uptake landscape of V. fischeri, and allow us to make predictions about the overall fitness of certain mutants under iron limiting and iron replete conditions as how those strains will fare in the light organ of the squid.

Goal 2: Identify genes and gene products that influence siderophore production in V. fischeri to gain insight into the context of iron metabolic regulation and obtain mutants of interest in these studies.

I utilized a quantitative liquid chrome-azurol-S (CAS) assay to screen a random transposon mutagenesis library (already sequenced and transposon insertion sites identified) for mutants with siderophore phenotypes. Identifying genes and gene products that influence siderophore production will help us to understand how siderophore regulation is intertwined with other traits and regulatory networks necessary for symbiosis.

20 Goal 3: Assess resistance to oxidation by a siderophore biosynthesis mutant

(iucA::TnErm) compared to the background strain ES114.

I tested the oxidative stress resistance of the iucA::TnErm mutant by placing a filter disk with hydrogen peroxide on top of a fresh bacterial lawn and measuring the resulting zone of clearing after overnight incubation. By comparing width of the zones of clearing against those for ES114 and oxyR::TnErm, I will be able to assess whether siderophore contributes to peroxide resistance.

Goal 4: Assess the role of siderophore production in the mutualism between V. fischeri and E. scolopes.

Using methods laid out by Graf and Ruby (97) and Septer et. al. (100), I assessed the ability of the siderophore production and uptake mutants from the transposon mutant library

(iucA::TnErm, iutA::TnErm, and fhuB::TnErm) to colonize and persist in the squid light organ. I also tested the deletion mutant ΔiucA and the double mutant ΔiucAtonB1::TnErm for colonization and persistence ability to assess the combined role of siderophore and heme uptake.

The results of these experiments will be useful in elucidating whether siderophore is absolutely necessary for successful symbiosis between V. fischeri and the squid, or if siderophore uptake is only accessory to iron acquisition by heme uptake in the light organ.

21 CHAPTER II: METHODS

Strains, Plasmids, Primers, and Culture Conditions

The strains, plasmids, and primers used in this study are listed in Table 6 and Table 7. V. fischeri strains were grown at 28°C on LBS plates or in LBS broth (0.05M Tris-HCl, 0.5% (w/v)

Bacto-Yeast Extract (BD Biosciences, Bedford, MA), 1% (w/v) Bacto-Tryptone (BD

Biosciences), 2% (w/v) NaCl) or Pipes Minimal Media (PMM, 100mM PIPES-NaOH pH 6.8,

0.0058% K2HPO4, 0.1% NH4Cl, 0.3%glycerol in Artificial Sea Water (ASW)[0.1M MgSO4,

9.85 mM CaCl2, 0.3 M NaCl, 10.05 mM KCl]). The PMM was made using iron-free MilliQ water and any glassware used was acid washed to remove iron. E. coli strains were grown at

37°C on LB plates or in LB broth (0.5% Bacto-Yeast extract, 1% Bacto-Tryptone, 1% NaCl).

For strain Π3813, 0.3mM thymidine was added to the medium. Antibiotics were added to the media when required, Kanamycin (Km) 100 μg mL-1 for V. fischeri and E.coli; Erythromycin

(Erm) 5 μg mL-1 for V. fischeri; and Chlor amphenicol (Cm) 1 μg mL-1 for ccdB mediated mutagenesis, 2.5 μg mL-1 for V. fischeri or 25 μg mL-1 for E. coli. For plating squid experiments,

Instant Ocean (Spectrum Brands, Blacksburg, VA) at a salinity of 33 ppm was used.

Bioinformatics search for iron uptake systems in V. fischeri

We identified genes involved in iron uptake in the V. fischeri genome using the gene search function in NCBI to identify already annotated iron uptake genes and pBLAST

(blast.ncbi.nlm.nih.gov) to identify genes in V. fischeri that are homologous to other known iron uptake genes.

22 Table 6. Strains used. name genotype source V. fischeri strains ES114 V. fischeri isolate from squid light organ (101) VCW6C5 ES114 iucA::Tn5erm (VF_A0165) this study ED1F2 ES114 tonB1::Tn5erm (VF_1225) this study ED4D1 ES114 iutA::Tn5erm (VF_A0161) this study ED4E1 ES114 fhuB::Tn5erm (VF_A0160) this study ED4H1 ES114 hutW::Tn5erm (VF_1226) this study ED1C4 ES114 oxyR::Tn5erm (VF_2299) this study ED4C1 ES114 luxA::Tn5erm (VF_A0921) this study G11 ES114 transductant, glnD1 (glnD::mTn5Cm) (97) AKD910 ES114 ΔVF_1220-1228 (100) (ΔtonB1exbBD1hmuTUVhutWXZ) YLW111 ES114 Δfur (VF_0810) (100) ED1I1 ES114 ΔiucA::Km (allele exchanged from pED1) this study ED1C2 ED1F2 ΔiucA::Km (allele exchanged from pED1) this study

E. coli strains One Shot® F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Invitrogen Top10 chemically ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU (Carlsbad, CA) competent cells galK rpsL (StrR) endA1 nupG Helper CC180λpir carrying pEVS104 Π3813 B462 ΔthyA::(erm-pir-116) (Ermr) (102)

23 Table 7. Plasmids and primers used.

name genotype/sequence source Plasmids pMKm pMTL24 cloning vector containing a PvuII (103) fragment with the aphA gene from pDSK509 conferring Km resistance

r pSW7848 oriVR6Kγ; oriTRP4; araC-PBADccdB (Cm ) (102) pED1 pSW7848 carrying ΔiucA::Km PstI/SalI fragment from pIucAkanTOPO pED1TOPO pCR2.1 TOPO vector with the ΔiucA::Km allele

Primers Soe "A" product

iucA SoeAF salI 5'-GTCGACTCGTCCCTTGTCCGCAC-3' this study

kan|iucA SoeAR 5'-CATCAGAGATTTTGAGACACATCACTCGCCCTATC-3' this study Soe "B" product

iucA|kan SoeBF 5'-GATAGGGCGAGTGATGTGTCTCAAAATCTCTGATG-3' this study

iucA|kan SoeBR 5'-CTTGGTATCTCGGATTCAAGTCAGCGTAATGCTCT-3' this study Soe "C" product

kan|iucA SoeCF 5'-AGAGCATTACGCTGACTTGAATCCGAGATACCAAG-3' this study

iucA SoeCR pstI 5'-CTGCAGCTCCATCACCACTGCTCC-3' this study

Construction of ΔiucA::aphA(Kanr) allele

We designed primers to amplify the regions flanking the iucA gene: iucASoeAFsalI, kan|iucASoeAR, kan|iucASoeCF, and iucASoeCRpstI (1490-227bp from the start codon and

152-909bp from the stop codon of iucA, respectively). The primers kan|iucASoeAR and

24 kan|iucASoeCF are complementary to the primers for amplifying the aphA gene for kanamycin resistance from the plasmid pMKm, iucA|kanSoeBF and iucA|kanSoeBR (Table 7). In the first round of PCR, we independently amplified each of the two regions flanking iucA as well as the aphA gene. In a second round of PCR, we combined all three products from the first round along with the primers iucASoeAFsalI and iucASoeCRpstI to splice the aphA gene in between the two iucA flanking regions by overlap extension (104). We cloned the new ΔiucA::aphA allele into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) by TA cloning reaction to construct the plasmid pIucAkanTOPO and chemically transformed into E. coli One Shot Top10 cells (Invitrogen,

Carlsbad, CA). We then digested pIucAkanTOPO and the suicide vector pSW7848 (102) with the restriction enzymes SalI and PstI, and we ligated the ΔiucA::aphA allele into the multiple cloning site (MCS) of pSW7848 to construct pIucAkan. The plasmid pSW7848 harbors a RP4 oriT allowing it to be transferred via tri-parental mating, as well as a R6Kγ oriV which requires the pir gene and is not maintained in V. fischeri. Plasmid pSW7848 also carries the cat gene conferring chloramphenicol (Cm) resistance and the toxin gene ccdB under the control of the glucose inhibited, arabinose inducible PBAD promoter.

Construction of strains ED1 and ED2 by allele swap with pIucAkan

The plasmid pIucAkan was chemically transformed into E. coli Π3813 cells and then mated into V. fischeri ES114 or V. fischeri tonB1::Tn5 via tri-parental mating by mixing the donor (Π3813), helper, and recipient cells (ES114 or tonB1::Tn5) and spotting the mixture onto a plate with 0.3mM thymidine and 1% glucose and incubating for 8 hours at 28°C. After 8 hours, the spot was restreaked onto an LBS plate containing 1% glucose and 1 μg mL-1 Cm, and incubated at room temperature (RT) for 1-2 days (until colonies could be distinguished) to select for V. fischeri cells harboring pIucAkan. We restreaked ex-conjugates onto LBS plates 25 containing 1% glucose and 1 μg mL-1 Cm to ensure integration of the plasmid in chromosome II by homologous recombination. After incubation overnight at 28°C, we restreaked onto LBS plates containing 1% glucose without antibiotics and incubated at 28°C overnight to ensure that any non-integrated pIucAkan was lost. We patched colonies from the non-selective plates onto

LBS agar containing 1% glucose and 1 μg mL-1 Cm to confirm integration, and at the same time we inoculated LBS broth containing 0.2% arabinose (to induce ccdB expression) with a representative colony and incubated the culture for 4-4.5 hours at 28°C. This step selects for cells in which pIucAkan has recombined twice with the chromosome and the integrated plasmid

(minus the region between recombination events) has been lost. After incubation the culture was diluted 1:100 and 100 μL plated onto a LBS plate containing 0.2% arabinose and incubated at

28°C overnight. After incubation, colonies were picked and restreaked onto plates containing

0.2% arabinose to ensure loss of the integrated plasmid. From these plate cultures, we picked single colonies and patched onto both LBS agar and LBS agar containing 1μg mL-1 Cm to confirm loss of the integrated plasmid. After patching, the same colonies were added to 50 uL of diH2O for colony PCR to confirm the loss of the plasmid.

Quantitative Chrome Azurol-S (CAS) assay for siderophore production

To test for siderophore production, strains were grown in PMM 0.3% casamino acids and

10 μM Dipyridyl (DP) until stationary phase (17-25 hours), and the optical density reading was recorded at 600 nm. The cultures were centrifuged at a 3220 X g for 15 minutes and the supernatant was added to an equal volume of CAS assay solution (Solution 1: 2.15 g Piperazine dissolved in 15 mL milliQ H2O, 3.4 mL of concentrated HCl added to bring to pH of 5.6;

Solution 2: 0.011 g HDTMA (CTAB) dissolved in 25 mL milliQ H2O (warm solution in water bath to dissolve); Solution 3: Mix 0.75 mL 1 mM FeCl3, 3.75 mL 2 mM Chrome Azurol-S. Add 26 solution 3 to solution 2, and mix gently, add solution 1 and mix gently. Before use, add 1 mL

0.2 M 5'-sulfosalicylic acid (light sensitive) and mix gently). After an hour of incubation in the dark at RT, the optical density was recorded at 630 nm. Percent siderophore units were calculated by subtracting the sample (AS) from the reference (PMM blank with CAS assay solution, AR), dividing by the reference, and then multiplying by 100: [(AR - AS)/AR]x100 (105), and then normalized to culture density (OD600).

Iron uptake growth assay

We inoculated the strains ES114, ED1, ED2, tonB1::TnErm, iutA::TnErm, fhuB::TnErm,

AKD910, and G11 into PMM. After incubating with shaking overnight, 1 μL of each strain was inoculated into three 200 μL wells each of PMM, PMM 10 μM Fe(III)Cl3, and PMM 50 μM 2-2- dipyridyl. We read the absorbance of the cultures @ 600 nm every hour for 26 hours using a

Tecan Infinite 3000 plate reader.

Heme uptake growth assay

Strains ES114, ED1, ED2, tonB1::TnErm, iutA::TnErm, AKD910 and G11 were inoculated into PMM and after incubating with shaking overnight, we inoculated 1 μL of each strain into five 200 μL wells each of PMM 50 μM 2-2-dipyridyl and PMM 50 μM 2-2-dipyridyl

50 mg mL-1 hemin. We read the absorbance of each culture at 600 nm every hour for 30 hours using a Tecan Infinite 3000 plate readers (Tecan US, Chapel Hill, NC).

Oxidative stress resistance assay

We grew strains ES114, iucA::TnErm, and oxyR::TnErm in LBS overnight with shaking and spread 50 μL of each culture onto 4 LBS plates and 4 LBS plates with 10 μM Fe(III)Cl3. We

27 placed filter discs onto the center of each plate and pipetted 5 μL of H2O2 onto the center of each filter disc. After incubating overnight, we recorded the average radius of each zone of clearing from two measurements.

Squid persistence assay

V. fischeri strains ES114 and ΔiucA were grown in PMM for ~44 hours before exposing groups of squid to 1-5*103 cfu mL-1 of one strain each. Each day after inoculating the squid, we recorded the luminescence output from each squid. At 22 hours post-inoculation, after recording luminescence, we flash froze half of the animals and stored them in individual tubes at -80° C for later use, and the same was done for the rest of the animals at 94 hours post-inoculation.

Squid competition assay

We grew strains ES114, iutA::TnErm, fhuB::TnErm, and G11 in PMM for ~44 hours before exposing groups of squid to paired strains. One experiment was performed in which we exposed squid to a paired inoculum of 1658 cfu/mL of ES114 and 2400 cfu/mL of iutA::TnErm.

One experiment was performed in which we exposed squid to a paired inoculum of 260 cfu/mL of ES114 and 440 cfu/mL of strain G11. Two experiments were performed in which we exposed squid to a paired inoculum of ES114 and fhuB::TnErm, with inoculum values of 938 and 3600 cfu/mL for ES114 respectively, and 2692 and 8396 cfu/mL for fhuB::TnErm respectively.

Roughly 22 hours post-inoculation, we recorded the luminescence output and flash froze each squid in individual tubes for storage at -80° C.

Determination of light organ cfu

28 We thawed each squid on ice and suspended in Instant Ocean before crushing to release the light organ contents. We made dilutions of each light organ suspension and spread each dilution onto two LBS plates. For the competition experiments, we also spread dilutions onto

LBS 5 μg/mL Erm for iutA::TnErm and fhuB::TnErm, or onto LBS 1 μg/mL Cm for strain G11.

After incubating overnight, we recorded the number of colonies on each plate.

29 CHAPTER III: RESULTS

Bioinformatics prediction of genes related to iron uptake and siderophore biosynthesis

To predict the genomic potential for encoding different iron acquisition and utilization systems that may contribute to squid symbiotic colonization by Vibrio fischeri, we used BLAST

(106) and the gene search tool in NCBI to compare known iron uptake systems (46, 73, 107–

109) against the complete strain ES114 genome (Tables 2-5, 8, and Fig. 4). A single siderophore biosynthesis operon for the hydroxamate-carboxylate siderophore, aerobactin, was encoded on chromosome II (iucABCD; VFA_0161-0164), and the TonB-dependent aerobactin receptor

(iutA; VFA_0165) was adjacent to this operon but presumably controlled by its own promoter

(110). The ABC transport system fhuCDB (VF_A0158-0160) for the transport of hydroxamate siderophores across the cytoplasmic membrane was located adjacent to iucABCDiutA in the genome and is likely to be involved in the transport of aerobactin. ES114 also encodes a system for the uptake of ferrichrome, a hydroxamate siderophore, with the ferrichrome receptor FhuA

(VF_0784) adjacent to another FhuCDB ABC transport system (VF_A0781-0783), with a ttpCexbB2exbD2tonB2 energy transduction system (VFA_0776-0779) close by. The receptor

FhuE (VF_A0191), for ferric coprogen, ferrioxamine B, and rhodotorulic acid, which are all hydroxamate type siderophores, and a ttpCexbB2exbD2tonB2 energy transduction system

(VF_A0193-0196) were also located close by. The lack of an ABC transport system proximal to the FhuE receptor likely means that this receptor utilizes one of the other two FhuCDB systems available. In order to utilize the iron brought into the cell via hydroxamate siderophore, bacteria must release the iron from the siderophore by reducing it with a ferric iron reductase (73), and

30 there is a predicted ferric reductase on chromosome I (VF_1219), which may be used for this purpose. A complete heme uptake system is also encoded in ES114, with the receptor HutA

(VF_1234), the exbB1exbD1tonB1 energy transduction system (VF_1223-1225), hemin ABC transport system hmuTUV (VF_1220-1222), and hutWXZ (VF_1226-1228) which dismantle heme, once in the cytoplasm, to release the iron (44). The heme uptake and utilization system has previously been studied in ES114, and the function of the heme uptake locus has been confirmed through a heme uptake assay (100). ES114 also encodes an ABC transport system for catechol siderophore VctPDGC (VF_A0824-0827) as well as a catechol utilization protein ViuB

(VF_A0823) that is responsible for cleaving the catechol siderophore open to release the ferric iron (111), however, genes predicted to encode the necessary catechol receptor were apparently absent from the genome. There are three TonB-dependent receptors (VFA_0672, VFA_0059,

VFA_0332) for which the ligand is not identified, so there is a possibility that one or more of these receptors could be specific to catechol siderophore uptake, or that ES114 has lost its ability to capture these types of siderophores. There is also an uptake system for vitamin B12 with the receptor BtuB (VF_2435) and the ABC transport system BtuCDE (VF_A0971 - 0973). Vitamin

B12 does not provide any iron for the cell, however, the ABC transport for B12 very closely resembles the ABC transport for ferric dicitrate and is annotated as such in NCBI. No protein resembling the ferric dicitrate receptor FecA was located, nor were any proteins resembling FecR or FecI, which are the anti-sigma and sigma factors commonly associated with ferric dicitrate uptake (112). Due to the apparent non-specificity of the ABC transporters involved in iron uptake, and the specificity of the outer membrane ligand receptors, it is more likely that the genes VF_A0971-0973 are involved in vitamin B12 uptake than ferric dicitrate uptake. There were two systems for direct iron uptake, including the FeoABC system for ferrous iron

31 (VF_0833-0835), as well as the SfuABC system for ferric iron uptake (VF_2149-2151), along with the iron storage protein ferritin (FtnA, VF_0084). As expected, ES114 also encodes two highly conserved global regulators, including the ferric uptake regulator Fur (VF_0810), as well as the ryhB sRNA (VF_2578). This analysis indicates that V. fischeri is potentially endowed with a diversity of uptake systems for this micronutrient from many different biological contexts in which these organisms must survive, only some of which are likely to contribute to symbiosis.

32 Table 8. Iron uptake systems found in Vibrio fischeri.

TonB V. fischeri locus % ID$ dependent? Iron regulation Ferric Uptake Regulator (Fur) VF_0810 79 ryhB small inhibitory RNA VF_2578 97 TonB systems TonB1 energy transduction system (tonB1exbB1exbD1) VF_1223-1225 32 TonB2 energy transduction system (ttpCexbB2exbD2tonB2) VF_A0776-0779 33§ VF_A0193-0196 38§ Aerobactin yes Aerobactin biosynthesis operon (iucABCD)** VF_A0161-0164 41 Ferric aerobactin receptor precursor (iutA)** VF_A0165 44 Ferric hydroxamate uptake ABC transport operon (fhuCDB) VF_A0158-0160 53 Ferrichrome yes ferrichrome outer membrane receptor (fhuA) VF_A0784 35 Ferrichrome uptake ABC transport operons (fhuCDB) VF_A0781-0783 34 Coprogen/Ferrioxamine B/Rhodotorulic Acid yes coprogen/ferrioxamine B/rhodotorulic acid receptor (fhuE) VF_A0191 25 Catechol Siderophore yes Ferric anguibactin transport operon (vctPDGC) VF_A0824-0827 62## Vibriobactin utilization (viuB) VF_A0823 30## Heme TonB1** Heme receptor (hutA)** VF_1234 25 hemin ABC transport (hmuTUV)** VF_1220-1222 41 heme utilization (hutWXZ) VF_1226-1228 37 Vitamin B12/cobalamin* yes vitamin B12/cobalamin receptor (btuB) VF_2435 34 vitamine B12/cobalamin ABC transport (btuCDE)# VF_A0971 - 0973 33 Ferrous Iron no Ferrous iron transport (feoABC) VF_0833-0835 43 Ferric Iron no iron(III) ABC transport system (sfuABC) VF_2149-2151 47@ Hydroxamate siderophore utilization no Siderophore ferric iron reductase (hypothetical) VF_A0156 predicted ferric reductase VF_1219 29 ** supported by experimental evidence in V. fischeri; * not involved in iron uptake, but TonB dependent; § compared to Shewanella oneidensis MR-1; ## compared to V. cholerae O1 El Tor; #more closely related to the fecCDE operon; $ % amino acid Identity compared against same protein in E. coli; @ compared to Y. pestis CO92

33 34 Identifying aerobactin biosynthesis and uptake mutants, and defining the regulatory context for production, through phenomics analysis

In the previous study by Graf and Ruby (97), a siderophore deficiency was implicated in the persistence defect of a strain with a transposon insertion in the nitrogen utilization regulator

GlnD, and another study showed that loss of the GacA global regulator of symbiotic traits decreases expression of siderophore (90). Two other studies demonstrated that siderophore expression is upregulated in the light organ of the squid (98, 99). These findings indicate that siderophore is controlled within a complex regulatory network contributing to symbiosis. To help tease out the regulatory networks that may be contributing to siderophore biosynthesis we capitalized on a defined transposon mutant library consisting of 2,318 independent TnErm mutations generated in the V. fischeri ES114 background (approx. 61% coverage), and evaluated each of these mutants for siderophore biosynthesis ability using a quantitative liquid CAS assay.

This screen allowed identification of genes and gene products that influence siderophore production through measuring the extent to which iron is sequestered away from the CAS reagent by siderophores in the culture medium. A total of 78 transposon insertions resulted in attenuation of siderophore, while only 23 transposon insertions caused an increase in siderophore production (Table 9).

Several of the mutations localized to shared functions or operons. Disruption of any of ten genes within the flagellar biosynthesis locus independently resulted in attenuated siderophore production accounting for 43.5% of the genes in the locus that were hit. Disruption of six genes within the cellulose biosynthesis locus also led to attenuated siderophore production. As expected interruption of each gene in the aerobactin biosynthesis locus (VF_A0161-0164) abolished siderophore production, as did insertions in the genes for the type II citrate synthase 35 (gltA VF_0818) and the divalent anion/sodium symporter family protein YfbS (VF_0322).

Insertions in ainS and in rpoQ (VF_1037, VF_A1015) increased siderophore production, while insertions in luxO and luxP (VF_0937, VF_0707) attenuated siderophore production, indicating a potential link to regulation through quorum sensing. Interestingly a transposon insertion within glnD did not attenuate siderophore production, in contrast to what had been previously reported

((97)). The transposon insertion for this mutant was localized earlier in the ORF compared to the transposon insertion in strains SP301 and G11 from the previous work. This suggests that the mutation in SP301 and G11 only alters, but does not completely knock out, GlnD function, and that partial GlnD function is necessary to elicit a siderophore phenotype.

We identified that many of these mutants would be useful for our directed analysis of siderophore, and so utilized several of the insertion mutants from the library in in vitro and in vivo experiments to further elucidate the role of siderophore in growth of V. fischeri on defined media and within the light organ of the squid. The iucA library mutant was used in persistence experiments, to evaluate whether siderophore biosynthesis and uptake contributes to sustained colonization within the light organ, and the iutA, and fhuB library mutants were utilized in competition experiments with ES114 to assess whether siderophore uptake confers an advantage to ES114 in the squid light organ. We also swapped the entire WT iucA allele with a kanamycin

(Km) resistance cassette to remove any effect that might be caused by the presence of the transposon. We used the tonB1 library mutant to construct a siderophore biosynthesis/heme uptake double mutant that was utilized to elucidate the combined contribution of siderophore and heme uptake to persistence ability. These mutants were also used in vitro to confirm the roles of these gene products in iron uptake, through the use of growth experiments and the quantitative

CAS assay.

36 Table 9. Transposon insertions in V. fischeri ES114 that result in a significanta siderophore phenotype. The flagellar, cellulose biosynthesis, and siderophore biosynthesis loci are highlighted in light gray, and other genes of interest are highlighted in gray. sid- p- V.f. locus gene product value* value VF_0026 elongation factor yigZ 1.37 0.0008 VF_0034 thiamin (thiazole moiety) biosynthesis protein ThiF 0.39 <.0001 VF_0140 UDP-N-acetylglucosamine 4,6-dehydratase 1.75 <.0001 VF_0148 flagellin modification protein A PtmA 1.35 0.0038 VF_0169 dTDP-glucose-4,6-dehydratase RmlB 1.47 0.0109 VF_0174 beta-D-GlcNAc beta-1,3-galactosyltransferase 1.43 0.0037 VF_0201 UDP-glucose 4-epimerase 1.28 0.0211 VF_0322 divalent anion:sodium symporter family protein YfbS 0.05 <.0001 VF_0429 pyrroline-5-carboxylate reductase proC 1.34 0.0215 VF_0436 16S ribosomal RNA methyltransferase RsmE 0.58 0.0110 VF_0483 preprotein translocase subunit SecG 0.66 0.0141 VF_0498 DNA polymerase III, psi subunit holD 1.29 0.0082 VF_0537 aspartate kinase 0.57 0.0022 VF_0543 glutamate--cysteine ligase gshA 0.65 0.0349 VF_0707 periplasmic AI-2 binding protein LuxP 0.64 0.0240 VF_0714 flagellar motor protein PomA; motA1 0.13 <.0001 VF_0715 flagellar motor protein MotB 0.43 0.0015 VF_0784 short chain dehydrogenase ybbO 1.38 0.0052 VF_0797 ATP-dependent protease ATP-binding subunit ClpX 0.51 <.0001 VF_0818 type II citrate synthase GltA 0.04 <.0001 VF_0885 ABC transporter permease protein 0.67 0.0191 VF_0891 DNA-binding transcriptional regulator HexR; yebK 0.60 0.0030 VF_0904 leucine-responsive transcriptional regulator lrp 0.68 0.0307 VF_0937 autoinducer repressor protein LuxO 0.54 0.0005 VF_0966 hypothetical protein 1.57 <.0001 VF_1037 C8-HSL autoinducer synthesis protein AinS 1.30 0.0420 VF_1051 DNA topoisomerase I topA 0.46 <.0001 VF_1266 multidrug efflux system YeeO 1.48 0.0071 VF_1287 23S rRNA m(2)G2445 methyltransferase rlmL 0.60 0.0047 VF_1363 formate hydrogenlyase subunit 6 0.53 0.0003 VF_1428 sulfatase family protein 0.61 0.0005 VF_1446 ribose ABC transporter permease protein rbsC 0.55 0.0383 VF_1506 RTX repeat-containing calcium-binding cytotoxin RtxA1 0.66 0.0174 VF_1531 ferrochelatase 0.10 <.0001

37 VF_1565 cobalt transport ATP-binding protein CbiO 0.67 0.0256 VF_1679 hypothetical protein 0.64 0.0048 VF_1686 hypothetical protein ybaB 0.68 0.0217 VF_1689 proteolytic adapter for RpoS degradation by ClpXP (RssB, 0.57 <.0001 ExpM) VF_1834 flagellar biosynthesis sigma factor fliA ND ND VF_1835 flagellar synthesis regulator FlhG 1.07 1.0000 VF_1836 flagellar biosynthesis regulator FlhF ND ND VF_1837 lagellar biosynthesis protein FlhA 0.67 0.0692 VF_1839 flagellar biosynthesis protein FlhB 0.96 1.0000 VF_1840 flagellar biosynthesis protein FliR 0.51 ND VF_1841 flagellar biosynthesis protein FliQ 0.57 0.0002 VF_1842 flagellar biosynthesis protein FliP 0.92 1.0000 VF_1843 flagellar biosynthesis protein FliO ND ND VF_1844 flagellar motor switch protein fliN ND ND VF_1845 flagellar motor switch protein FliM 0.47 0.2429 VF_1846 flagellar basal body-associated protein FliL 0.43 0.2113 VF_1847 flagellar hook length control protein FliK ND ND VF_1848 flagellar biosynthesis chaperone fliJ ND ND VF_1849 flagellum-specific ATP synthase fliI ND ND VF_1850 flagellar assembly protein H fliH ND ND VF_1851 flagellar motor switch protein G fliG ND ND VF_1852 flagellar MS-ring protein FliF 0.45 <.0001 VF_1853 flagellar hook-basal body protein FliE 0.93 1.0000 VF_1854 two-component response regulator FlrC 0.57 0.0002 VF_1855 sensory histidine kinase FlrB 0.54 <.0001 VF_1856 sigma-54-dependent transcriptional activator FlrA ND ND VF_1858 flagellar protein FliS ND ND VF_1859 flagellar protein FlaI ND ND VF_1860 flagellar capping protein FliD 0.68 0.2712 VF_1861 flagellar protein FlaG ND ND VF_1862 flagellin FlaE 0.81 0.9858 VF_1863 flagellin FlaD ND ND VF_1864 flagellin FlaC 1.37 0.0008 VF_1865 flagellin FlaB ND ND VF_1866 flagellin FlaA 0.98 1.0000 VF_1867 flagellar hook-associated protein FlgL ND ND VF_1868 flagellar hook-associated protein FlgK 0.30 <.0001 VF_1869 flagellar rod assembly protein/muramidase FlgJ 1.03 0.5260 VF_1870 flagellar basal body P-ring protein FlgI 0.52 0.0007 VF_1871 flagellar basal body L-ring protein FlgH ND ND VF_1872 flagellar basal body rod protein FlgG 0.33 <.0001

38 VF_1873 flagellar basal body rod protein FlgF ND ND VF_1874 flagellar hook protein FlgE 0.33 <.0001 VF_1875 flagellar basal body rod modification protein FlgD 0.74 0.5510 VF_1876 flagellar basal body rod protein FlgC 0.33 <.0001 VF_1877 flagellar basal-body rod protein FlgB ND ND VF_1982 transcriptional regulator PhoU 0.59 0.0029 VF_1993 chaperone protein DnaJ 0.38 <.0001 VF_2102 hypothetical protein ygfB 0.09 <.0001 VF_2138 chitin sensor histidine kinase ChiS 1.18 0.0020 VF_2152 ammonium transporter AmtB 1.51 0.0010 VF_2166 poly(A) polymerase I PcnB 0.43 <.0001 VF_2244 multifunctional tRNA nucleotidyl transferase/2'3'-cyclic 0.69 0.0403 phosphodiesterase/2'nucleotidase/phosphatase cca VF_2256 long-chain-fatty-acid--CoA ligase 1.82 <.0001 VF_2317 flagellar motor protein MotX 0.55 <.0001 VF_2324 tRNA delta(2)-isopentenylpyrophosphate transferase MiaA 0.20 <.0001 VF_2445 chorismate--pyruvate lyase UbiC 1.26 0.0012 VF_2504 putative cytoplasmic protein 1.34 0.0152 VF_2505 permease 0.69 0.0277 VF_2576 sulfur carrier protein ThiS 0.26 <.0001 VF_A0078 MshD protein 0.69 0.0269 VF_A0161 aerobactin siderophore biosynthesis protein IucA 0.04 <.0001 VF_A0162 aerobactin siderophore synthesis protein IucB 0.06 <.0001 VF_A0163 aerobactin siderophore biosynthesis protein IucC 0.03 <.0001 VF_A0164 aerobactin siderophore biosynthesis protein IucD 0.05 <.0001 VF_A1178 hypothetical protein 0.70 0.0392 VF_A0215 hypothetical protein 0.47 <.0001 VF_A0216 two component response regulator 0.66 0.0139 VF_A0222 chromosome partitioning ATPase 0.36 <.0001 VF_A0233 hypothetical protein 0.21 <.0001 VF_A0235 glycerol uptake facilitator protein GlpF 0.55 0.0009 VF_A0238 glycerol-3-phosphate regulon repressor protein GlpR 0.60 0.0019 VF_A0307 peptide transport system permease protein SapC 0.29 <.0001 VF_A0314 hypothetical protein 0.72 0.0465 VF_A0372 mechanosensitive channel 0.70 0.0447 VF_A0405 inner membrane protein 1.35 0.0420 VF_A0625 hypothetical protein 0.67 0.0321 VF_A0689 hypothetical protein 0.71 0.0385 VF_A0731 trehalose-6-P hydrolase 0.67 0.0385 VF_A0741 hypothetical protein 0.63 0.0040 VF_A0811 transcriptional regulator MalT 0.74 0.0234 VF_A0881 cellulose synthase operon C protein 0.39 0.0002

39 VF_A0882 endo-1,4-D-glucanase bcsZ ND ND VF_A0883 cellulose synthase regulator protein bcsB 0.35 <.0001 VF_A0884 cellulose synthase catalytic subunit bcsA 0.54 0.0013 VF_A0885 hypothetical protein 0.45 0.0083 VF_A0886 hypothetical protein bcsE 0.54 0.0956 VF_A1191 hypothetical protein 0.26 <.0001 VF_A0887 cellulose synthase operon protein YhjU; bcsG 0.64 0.0041 VF_A0945 putative lipoprotein 1.44 0.0305 VF_A1006 DNA-binding transcriptional dual regulator 0.65 0.0167 VF_A1015 sigma-Q factor RpoQ 1.24 0.0114 VF_A1077 RIO1 protein 0.63 0.0423 VF_A1114 cobalt-zinc-cadmium resistance protein CzcD 0.44 <.0001 VF_A1196 hypothetical protein 1.52 0.0143 VF_A1140 hypothetical protein 1.45 0.0018 VF_A1163 outer membrane protein 0.58 0.0010 VF_A1165 ATP-binding protein, putative RTX transport secretion 0.48 <.0001 component RtxB

*%siderophore units normalized to wild-type; asignificance determined using Dunnet's test, comparing each mutant to WT

Role and regulation of iron uptake in growth and protection against oxidative species.

There are many studies that show that, in γ-proteobacteria, the ferric uptake regulator Fur regulates iron uptake systems in response to intracellular iron (113). As the intracellular concentration of iron rises, we expect activity of siderophore and other iron uptake systems to decrease. To verify that aerobactin production is regulated in response to iron in V. fischeri

ES114, we grew ES114 at increasing levels of added FeCl3 (0, 2.5, 5, 7.5, 10 and 20 µM added iron), and then tested these cultures for siderophore production using the quantitative liquid CAS assay. As expected there was a decrease in siderophore activity as the iron concentration rose.

Between 2.5 and 5 µM added iron, there was a steep decrease in siderophore production, and siderophore activity was completely abolished with 10 µM added iron (Fig. 5). The steep decrease between 2.5 and 5 µM added iron might indicate a rise in Fur activity with increased

40 iron, or there could be a critical point at which there is enough iron to be bound by most of the present Fur proteins.

To confirm the expected function of proteins encoded by genes of interest, we evaluated the siderophore phenotypes of strains ED1F2 (tonB1::Tn5erm), ED4D1 (iutA::Tn5erm), and

ED4E1 (fhuB::Tn5erm), as well as strains ED1I1 (ΔiucA, this study), ED1C2 (ΔiucA tonB1::TnErm, this study), G11 (glnD::TnCm, (97)), YLW111 (Δfur, (97)), and the heme uptake gene cluster deletion mutant, AKD910 (ΔVF_1220-1228, (100)). Given that iucA encodes the first gene in the aerobactin biosynthesis operon, and no other siderophore biosynthesis systems were found in the genome, we expected that strain ED1I1 would not elicit a color change on the

CAS assay, indicating a lack of siderophore activity, and by the same logic the siderophore/heme uptake double mutant ED1C2 should not elicit a color change. Assuming that the aerobactin uptake mutants, ED4D1 and ED4E1, do not have the ability to bring ferric-aerobactin into the cell, these mutants should elicit an equal or greater color change on the CAS assay, compared to

ES114. Because tonB1 and the rest of the heme uptake gene cluster do not encode genes thought to be involved in siderophore biosynthesis, uptake, or regulation, we expect that strains ED1F2 and AKD910 will exhibit similar siderophore production to ES114. Given that strain YLW111 is deficient in Fur, we expect that siderophore expression will be constitutive, giving a greater color change on the CAS assay than ES114. Previous work showed that strain G11 is deficient in siderophore production (97), so we expect that this strain will not produce a color change on the assay. As expected, strains ED1I1, ED1C2, and G11 did not display siderophore activity in the assay as indicated by the lack of a significant color change, while strains ED1F2, ED4D1,

ED4E1, and AKD910 elicited a similar color change to ES114, indicating that siderophore

41 activity is unaltered in these strains. As expected, strain YLW111 exhibited greater iron sequestration than ES114 as indicated by the more extensive color change (Fig. 6).

Given that iron contributes to growth, we expect that strains deficient in high affinity iron uptake (ED1I1, ED1C2, ED4D1, ED4E1, and G11) will have a growth defect under iron limiting conditions (50 µM 2'-2'-dipyridyl (DP)), compared to ES114, while strains that are deficient in heme uptake (ED1F2 and AKD910) will have growth similar to ES114. Under iron replete conditions (10 µM added FeCl3) no growth defects should be apparent, except for strain G11, which should show a growth defect under both iron replete and iron limiting conditions due to glutamine auxotrophy (97). Grown under iron replete conditions (10 µM added FeCl3) and baseline conditions (no added FeCl3 or DP), all strains tested grew comparably well to strain

ES114, except for the G11 strain which had impaired growth under all conditions (fig. 7). Under iron limiting conditions (50 µM DP), strains ED1I1, ED1C2, and ED4D1 were growth impaired when compared to ES114, while strains ED1F2 and AKD910 grew similarly to ES114.

Interestingly, strain ED4E1 was not growth defective, which indicates that the second copy of fhuB (VF_A0781) may be picking up the slack, or that the transposon insertion in fhuB has resulted in a leaky mutation (Fig. 7).

We hypothesized that in addition to meeting nutritional requirements, iron sequestration by siderophore could contribute to symbiosis by protecting against oxidative stress, by removing free iron that could catalyze the formation of harmful oxidative species. We subsequently tested the resistance of the iucA library mutant to oxidative stress using strain ES114 as a positive control and the oxyR library mutant as a negative control. We placed filter disks with hydrogen peroxide on top of bacterial lawns of each strain and after allowing the lawns to grow we measured the zones of clearing around the filter disks. Strain VCW6C5 (iucA::Tn5erm) had 42 similar zones of clearing compared to strain ES114, while the zones of clearing for strain ED1C4

(oxyR::TnErm) were almost twice as large as those for VCW6C5 (Fig. 8). Addition of 10 µM

FeCl3 to the seed culture medium had no effect on resistance to oxidative stress of any strain, suggesting that iron sequestration by siderophore does not contribute to oxidative stress resistance under these conditions and that ES114 has other ways of dealing with oxidative stress under iron replete conditions.

Previous work showed that heme uptake also contributes to persistence of V. fischeri in the squid light organ; however, the heme uptake locus mutant AKD910

(ΔtonB1exbBD1hmuTUVhutWXZ) exhibited a persistence defect that was not as extensive as the defect exhibited by the glnD mutants (SP301, G11)(97, 100). The persistence defect of the G11 mutant was complemented by exposure to iron in the seawater, indicating a general iron uptake defect, however, this strain was only assayed for siderophore production and not heme uptake.

We hypothesized that G11 and SP301 may also have a heme uptake defect, which, combined with the siderophore defect, contributed to the more extensive persistence defect of both strains.

We constructed a heme uptake/siderophore biosynthesis double mutant (ED1C2), to test whether heme uptake and siderophore utilization contribute to a combined role in persistence. To evaluate the heme uptake ability of strain G11 and to confirm the heme uptake defect conferred by the tonB1::TnErm mutation, we performed a heme-uptake assay. Added hemin greatly improved growth of strains ES114, ED1I1, and ED4D1, whereas it only slightly improved the growth of strains ED1C2, ED1F2, G11, and AKD910 (Fig. 9). These data suggest that the tonB1::TnErm mutation confers a heme uptake defect, equal to that of strain AKD910, and that strain G11 may also have a heme uptake defect.

43 44 45 46 47 The ΔiucA tonB1::Tn5erm mutant does not have a persistence defect.

Since the earlier studies on association of siderophore with symbiotic fitness have been challenged as providing only circumstantial evidence rather than proof, and more recent work has established the importance of heme uptake (100), we wished to utilize defined mutants in siderophore production and uptake to more directly test the hypothesis that aerobactin biosynthesis and uptake are necessary for successful persistence in the squid light organ. We performed a persistence experiment with strains ES114 (WT), ED1C2, and ED4C1

(luxA::Tn5erm, a strain known to exhibit a persistence defect) to determine whether the combined siderophore production and heme uptake abilities are necessary for strains to persist in the light organ (LO) up to 96 hours post inoculation. The light organ contents of animals inoculated with strain ED1C2 did not differ significantly between 24 and 96 hours post inoculation, that strain ED1C2 does not have a significant persistence defect (Fig. 10).

48 The siderophore uptake mutants, ED4D1 and ED4E1, do not exhibit a competitive defect in the light organ compared to the wildtype strain ES114.

Previous work demonstrated that some defects in symbiosis are only noticeable in a competitive context (114, 115). Due to the fact that siderophore is a common good shared between bacteria, we cannot test a siderophore production mutant in competition with strain

ES114 because they would share the siderophores produced by ES114. To test whether a siderophore deficient strain is less competitive in symbiosis we therefore utilized two strains that are deficient in their ability to transport siderophore into the cell, ED4D1 and ED4E1, thereby making the common good unattainable. In competition against strain ES114, neither of the siderophore uptake mutants exhibited symbiotic defects: strain ED4D1 had an average log(RCI)

49 value of -0.046, and strain ED4E1 had an average log(RCI) value of -0.274 (Fig. 11A,B). These data suggest that siderophore uptake does not contribute to successful symbiosis.

50 G11 has a competitive defect in the light organ compared to strain ES114

In persistence experiments utilizing strain G11 as a persistence deficient control, G11 colonized the squid much less frequently than strain ES114 (data not shown). This differed from previous reports with strain G11 (97). To further assess the symbiotic fitness of G11, we competed it against strain ES114 in the squid light organ where G11 had a strong competitive defect (average log(RCI) = -2.30) (Fig. 11C). There were low counts of G11 cells in the light organs of nearly all colonized squid, but not an absence, which could indicate an initiation defect in which the G11 cells are delayed in colonization of the light organ, or a colonization defect in which G11 cells are unable to reach a high quorum in the light organ. Since secondary mutations could contribute to this unexpected phenotype, whole genome re-sequencing of strain G11 could address this possibility.

51 CHAPTER IV. DISCUSSION

We identified many iron uptake systems in Vibrio fischeri ES114 that may contribute to symbiosis in the squid and may also enable ES114 to fill other niches. Due to the mono-specific nature of the association and the lack of any siderophore biosynthesis genes, other than the iuc operon, siderophore uptake systems other than the aerobactin uptake system encoded by fhuCDB do not likely contribute to iron acquisition in the squid light organ. The presence of two fhuCDB

ABC transport systems in the genome begs the question of why ES114 has retained both of these seemingly redundant systems. There are a few possible explanations. First, these two systems may have been independently acquired with enough evolutionary distance acquisitions (either in the source of acquisition or in actual time) that they have diverged to exclusively serve two different siderophore systems. Second, at least one of these two systems was recently acquired and not enough evolutionary time has passed to allow for one of these systems to be discarded.

Third, these two systems may be redundant in function, yet the presence of both systems confers a selective advantage. The two FhuB permeases from ES114 only share 37% amino acid identity, which supports the first explanation that they have diverged to serve two different functional roles. However, the fact that the fhuB mutant is not growth defective under the iron deplete condition argues against divergent functions for the two proteins. The best explanation for the redundancy may be that the presence of these two systems confers some advantage, which is supported by the lack of a growth defect for the fhuB mutant under the iron deplete condition.

The quantitative CAS assay screen of the transposon mutant library yielded some very interesting and diverse results. Not surprisingly, transposon mutations in the aerobactin

52 biosynthesis operon, iucABCD, resulted in abolished siderophore production. The substrate for aerobactin assembly by IucABCD is lysine, and citrate, so it was also not surprising that the CAS assay revealed GltA, the type II citrate synthatse. Many of the other gene products identified by this screen are less obviously linked to siderophore production revealing other potential avenues, by which, iron uptake is controlled in V. fischeri.

The most noticeable trend identified by this assay was the collection of flagellar genes that, when interrupted, cause a reduction in siderophore biosynthesis. This connection of flagellar biosynthesis and function to siderophore production is very interesting in light of previous work that shows a connection of iron regulation of flagellar and chemotaxis genes through Fur activation of the flagellar master regulator FlhD (116). One study links cell envelope perturbations, caused by inactivation of cell envelope proteins, to increased oxidative stress and altered expression of iron uptake genes (117). If the mutations in the flagellar locus are causing a similar cell envelope perturbation, leading to increased oxidation, it makes sense that iron uptake is being turned off, as oxidation can mediate release of iron from iron-sulfur clusters, thereby increasing the amount of free intracellular iron, which will signal the down-regulation of iron uptake. We can also attribute the siderophore defect caused by mutants of the cellulose synthase operon to this phenomenon, and it is also possible that several of the other mutations resulting in a down regulation of siderophore are caused by this cell envelope perturbation.

Mutations in four genes involved in quorum sensing and quorum sensing regulation were linked to siderophore phenotypes by the CAS assay screen. Insertions in luxO, encoding the autoinducer repressor, and luxP, the periplasmic AI-2 binding protein, lead to attenuation of siderophore, while insertions in the C8-HSL autoinducer synthase gene, ainS, and in rpoQ a potential regulator linked to quorum traits, resulted in increased siderophore production. Work in

53 Pseudomonas aeruginosa also reveals a link between quorum sensing and iron uptake, where the

VqsR quorum sensing regulator likely activates the pyoverdine synthesis gene pvdS (118). This link to quorum sensing may also explain the link between siderophore production and GacA, which is a global regulator that is known to influence quorum sensing in several different bacterial species (119–121).

Two genes involved in the proteolytic degradation of RpoS were interrupted by the transposon, and both caused a down-regulation of siderophore, suggesting a role for RpoS in activation of Fur, which is demonstrated in V. vulnificus (68). However, a transposon insertion in rpoS itself did not significantly affect siderophore production, which could suggest that increased or constitutive expression of RpoS is needed to elicit an effect on Fur expression, while Fur is expressed at normal levels, even in the absence of RpoS. This differential response to RpoS expression also suggests that other factors are involved in the activation of normal Fur expression in V. fischeri.

Evaluation of siderophore activity by ES114 at varying iron concentrations confirmed that siderophore expression is controlled in response to iron. Due to iron contamination in the minimal media, addition of the iron chelator 2'-2'dipryridyl was necessary to elicit a measurable phenotype, so the exact iron concentration at which siderophore expression is abolished, is indiscernible. However, the overall "shape" of iron regulation can tell us much more about cellular function than the fine details. In the siderophore induction experiment there was a shallow reduction of siderophore activity followed by a steep decrease in expression and then another shallow reduction leading to complete abolition of siderophore activity as the concentration of iron was increased (Fig. 5). This could suggest variable levels of Fur expression in response to the iron concentration, or this could just be indicative of normal Fur kinetics.

54 Protein function in relation to siderophore production for all of the mutants of interest was confirmed by CAS assay (Fig. 6). Strains ED1I1, ED1C2, and G11 caused only a slight color change on the assay, indicating that these strains did not produce siderophore in culture.

The result for strain ED1I1 confirms that aerobactin is the only detectable siderophore produced in culture, and the siderophore overproduction of strain YLW111 (Δfur) confirms that the iron response of ES114 is regulated through Fur.

In a time-point growth assay with the iron chelator 2'-2'-dipyridyl, iron uptake by siderophore contributes to growth under iron deplete conditions (Fig. 7). The absence of a growth defect for the fhuB mutant suggests that the other copy of fhuB (VF_A0783) may be filling the role, as mentioned above. This data indicates that iron acquisition by siderophore contributes to growth under iron limiting conditions, supporting a nutritional role for siderophore in the light organ.

The ultimate goal of this study was to evaluate the role of siderophore in the symbiosis between V. fischeri and the squid, and we hypothesized that siderophore is necessary for successful persistence of V. fischeri in the light organ after venting, and that siderophore may protect against oxidative stress in the light organ. To assess whether siderophore contributes to defense against oxidation we measured the resistance to oxidative stress of the siderophore deficient strain VCW6C5, and found that the resistance to oxidation was similar to that of ES114

(Fig. 8). Literature showing the induction of Fur by OxyR and SoxRS in response to oxidative stress suggests that iron uptake is more tightly controlled under oxidative stress, which makes sense considering the volatility of the free iron in the presence of oxidative radicals (67). To test whether siderophore biosynthesis contributes to persistence ability, in a preliminary experiment we inoculated squid with the siderophore deficient strain VCW6C5, and assayed for persistence

55 ability over 96 hours by sampling squid at 24, 48 and 96 hours post inoculation. Strain VCW6C5 did not have a persistence defect, suggesting that siderophore production is not required for successful persistence within the light organ (data not shown). This raised the question of what was contributing to the persistence defect of the glnD mutant SP301 if not siderophore?

In searching for an explanation for the persistence defect of the glnD mutant we devised the hypothesis that the persistence defect is due to a combined siderophore and heme uptake defect. Using a time-point growth assay with the iron chelator 2'-2'-dipyridyl, and heme as an iron source, we tested strains for heme uptake ability, and have confirmed that heme uptake can also contribute to growth under otherwise iron deplete conditions (Fig. 9). The growth improvement of the G11 strain was similar to that of AKD910, suggesting that G11 may have a heme uptake defect, however G11 has an inherent growth defect likely due to a glutamine auxotrophy, which may be confounding our results. This heme uptake defect is in support of the hypothesis that a combined siderophore and heme uptake defect is contributing to the persistence defect of strain SP301. To test the combined contribution of siderophore biosynthesis and heme uptake to persistence ability we constructed strain ED1C2 by moving the ΔiucA mutation into the tonB1::TnErm mutant background. When we tested the persistence ability of strain ED1C2 we were not able to exhibit a persistence defect, suggesting that siderophore production and heme uptake combined do not contribute to light organ persistence (Fig. 10).

Previous work highlights that some defects in the light organ only become apparent in competition with the native symbiont ES114 (100, 114, 115). To test whether siderophore uptake contributes to fitness of strains within the light organ we competed strains ED4D1, and

ED4E1 against ES114 in the light organ and found that these strains are not less fit. This finding

56 suggests that siderophore does not contribute to successful colonization of the light organ by

Vibrio fischeri (Fig. 11), however, we were able to show a competitive defect with strain G11.

Future work will focus on evaluating the combined role of siderophore and heme uptake in the squid-Vibrio association and we will assess whether a general iron uptake defect is influencing the more severe symbiotic defect of strains G11 and SP301. The fitness contribution of the other siderophore uptake systems should also be explored to determine whether other siderophores are available to the light organ population, and whether these other siderophore uptake systems significantly contribute to fitness outside the light organ.

Future work will also focus on exploring the regulatory networks that regulate iron uptake, as well as how these networks are intertwined with regulation of symbiotic traits. We have uncovered a potential link between GacA and GlnD in a CsrA binding site upstream of the glnD gene, and we will work to characterize this link using quantitative PCR, as well a glnD expression marker. We have also found a binding site for the integrative host factor (IHF) upstream of the iucABCD operon, and an IHF mutant that overproduces siderophore. Together these findings suggest that IHF may be mediating Fur binding. To explore this potential regulation by IHF we are developing multi-copy Fur-titration constructs to be used in IHF- and

IHF+ backgrounds, which will allow us to visualize whether IHF is influencing Fur binding.

Through this study, we have determined that iron uptake by siderophore and heme uptake contributes to growth in culture, and that siderophore does not protect against oxidation in culture. We have demonstrated that a siderophore and heme uptake double mutant does not have a persistence defect in the squid light organ and that mutations in siderophore uptake do not affect competitive fitness in the light organ, suggesting that high affinity iron uptake only plays a minor role in the symbiosis. So far, my work suggests that there is more of a role for high

57 affinity iron uptake in pathogenesis, however, further work should be done in other mutualistic models to determine a role for high affinity iron uptake in symbiosis in general.

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