IDENTIFICATION AND CHARACTERIZATION OF BIOSYNTHESIS, TRANSPORT, AND REGULATION ELEMENTS AMONG BACTEROIDES SPECIES

BY

ZACHARY ANDREW COSTLIOW

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2018

Urbana, Illinois

Doctoral Committee:

Assistant Professor Patrick H. Degnan, Chair Associate Professor Carin K. Vanderpool, Co-Chair Professor Gary J. Olsen Professor James A. Imlay

ABSTRACT

Thiamine (vitamin B1) is an essential for all organisms. Humans primarily acquire thiamine through their diet and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood. In addition, little is known about how thiamine, the

Bacteroidetes ability to biosynthesize and transport thiamine, or the regulation of biosynthesis and transport impacts the stability of microbial gut communities and human health as a whole.

To investigate the role thiamine plays in the gut we leveraged in silico analyses of gut microbial species to determine prominent strategies utilized to attain thiamine. In addition, we have identified the genetic content and operon structure of thiamine transport and biosynthesis across the prominent gut phylum, . Along with the bioinformatic methods, RNAseq revealed differential responses to exogenous thiamine by three abundant Bacteroides species. This is highlighted by the global down- regulation of thiamine and amino acid biosynthesis, central, and purine metabolism when thiamine was present in Bacteroides thetaiotaomicron. In contrast Bacteroides uniformis and vulgatus show a much more reserved transcriptomic response to exogenous thiamine.

In order to build upon these data, we leveraged genetic mutants of thiamine biosynthesis and transport loci in B. thetaiotaomicron. These analyses determined both systems were critical for growth in thiamine-deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron. Along with the phenotypic analysis of

ii thiamine acquisition operons in B. thetaiotaomicron we investigated how these deletions impacted its fitness in thiamine deplete and replete conditions.

Building on the phenotypic analysis in B. thetaiotaomicron we turned our attention to the conserved regulatory TPP riboswitches preceding thiamine biosynthesis and transport in B. thetaiotaomicron, B. uniformis, and B. vulgatus utilizing transcriptional and translational reporter assays. These assays have shown a clear regulatory hierarchy between thiamine biosynthesis and transport pathways. In addition, trends in TPP riboswitch distance from their predicted regulon points to the mechanism of regulation (transcriptional or translational) and warrants further investigation.

Together, these data show that thiamine acquisition mechanisms and their regulation are critical to physiology and fitness among the Bacteroidetes. In addition future work may provide insight into modeling how other gut microbes respond to the shifting availability of thiamine in the gut and how to therapeutically alter the gut microbiota from a dysbiotic state to a non-disease state.

iii ACKNOWLEDGMENTS

I would like to thank my advisors Dr. Patrick Degnan and Dr. Cari Vanderpool for instructing me in the matter microbial and providing the opportunity for me to work in their laboratories over the course of my Ph.D. at the University of Illinois in Urbana-

Champaign. Patrick has been a driving force in developing my understanding of the gut microbiome and the development of my science communication skills and data presentation. I also owe Cari many thanks for fostering an interest in Microbiology and for encouraging me to pursue my doctorate, without her and the amazing community she has created in her laboratory I would not have made it this far. Both of my mentors have been crucial to my growth as a scientist and have encouraged me to pursue my dreams in the future. In addition to my advisors, I have to thank my committee members present and former, Dr. Gary Olsen, Dr. James Imlay, and Dr. Jeffrey Gardner, for the critical advice over the course of the year that helped shape my thesis over my time here at the

University of Illinois.

I am deeply thankful for all the members of the Degnan and Vanderpool laboratories for dealing with my daily shenanigans and being incredible people to talk about a wide variety of topics in the realms of life and science. In addition they provided a supportive and collaborative environment that enriched my time as a graduate student.

Finally I would not have been able to attain my Ph.D. without the unconditional support and love of my parents, sister, extended family, and friends. They have all been incredible and have enriched the many years I have spent at the University of Illinois with joy, happiness, and many memories that I will cherish for the rest of my life.

iv TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ...... 1

Thiamine and the microbiome: a crossroads of human health and disease ...... 1

The role of thiamine ...... 3

Thiamine acquisition and biosynthesis ...... 7

The microbiome ...... 14

Thiamine, the microbiome, and disease ...... 15

Conclusion ...... 20

Figures...... 21

References ...... 26

CHAPTER 2: THIAMINE ACQUISITION STRATEGIES IMPACT

METABOLISM AND COMPETITION IN THE GUT MICROBE

BACTEROIDES THETAIOTAOMICRON ...... 39

Abstract ...... 39

Importance ...... 40

Introduction ...... 40

Results ...... 43

Discussion ...... 56

Methods...... 60

Acknowledgments ...... 67

Figures...... 68

Tables ...... 84

References ...... 97

v CHAPTER 3: TRANSCRIPTIONAL AND TRANSLATIONAL ACTING TPP

RIBOSWITCHES IDENTIFIED AMONG BACTEROIDES SPECIES ...... 101

Abstract ...... 101

Importance ...... 102

Introduction ...... 103

Results ...... 106

Discussion ...... 114

Methods...... 119

Acknowledgements ...... 123

Figures...... 125

Tables ...... 133

References ...... 138

CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS ...... 143

Summary ...... 143

Future Directions ...... 146

References ...... 152

vi CHAPTER 1: INTRODUCTION

Thiamine and the microbiome: a crossroads of human health and disease:

Before scientists had shifted their attention to the microbiome in order to explain human ailments, vitamin deficiencies and human nutrition were widely studied due to the profound impact these topics had on human health. Deficiencies in key vitamins were shown to cause diseases such as scurvy, rickets, osteoporosis, neurodegenerative disorders, anemia, infertility, and a multitude of other physiological disorders in humans

(16, 24, 28, 52, 95, 100). In addition to the role of vitamins in human health, many other vitamin related topics have garnered interest by the scientific community. These topics include the synthesis and transport of vitamins in many organisms. Another topic consists of characterizing vitamin requirements of metabolic processes and how they change in different organisms. The studies of vitamins in the microbial world have often led to a better understanding of metabolism and antibiotic discovery (11, 98, 109, 113).

Vitamin B1, thiamine, has been investigated since the early 1900s and yielded fascinating results. Thiamine has been shown to be crucial in neurodegenerative disorders such as Wernicke-Korsakoff syndrome, and beriberi (56, 73). In addition, the dysregulation of thiamine metabolism has been shown to be a biomarker for Alzheimer's disease (73, 100). Thiamine levels have also been linked to other diseases such as cancer, diabetes, irritable bowel disease (IBD), and obesity (68, 74, 99). Thiamine has been extensively studied and shown to be important in human physiology. In addition, Vitamin

B1 has been studied in model microbial organisms, e.g. , , and Bacillus subtilis. The study of thiamine in these microbes has led to key insights into how thiamine is synthesized and transported (60, 87, 135). Characterization

1 of thiamine acquisition has led to the discovery of a highly conserved, RNA-based, regulatory mechanism that controls the acquisition of thiamine (135).

Another area of research that relates to human health is the study of the human gut microbiome. The gut microbiome consists of the , fungi, archaea, and viruses that colonize the human (121). Utilizing clinical samples and germ- free mouse models, researchers have linked the gut microbiome to diseases such as diabetes, obesity, cancer, IBD, and Alzheimer's (14, 25, 81,125, 136). These diseases are typically attributed to a disruption of the normal gut flora. The perturbations of these communities (dysbiosis) have been attributed to host diet, genetics, antibiotic treatments, and environmental factors (29, 40, 62, 102, 123). As of yet, only correlative studies exist for how dysbiotic states are affected and impact human health. The absence of mechanistic knowledge about the gut microbiota highlights the need to understand how the gut microbiome changes and impacts human health. Gaining a mechanistic understanding of the gut microbiome will teach us how to therapeutically alter the microbiome via prebiotics, probiotics, fecal microbiota transplants, and phage therapies.

The studies of how vitamins and the microbes that colonize the human gastrointestinal tract continue to progress separately, but the gaps between these two topics of research are beginning to blur. The advent of multi-omic approaches have shown that the gut microbiome is a dynamic system. Continuing work will lead to an understanding of the competition that permeates the gut, and the role competition plays in human health and disease. To date, these studies cast a wide net but little is known about mechanism of how a single metabolite, like thiamine, can impact a microbial community or individual gut species.

2 This work attempts to interrogate the role that thiamine and the microbiome play in human health by reviewing (i) literature on thiamine and the roles that thiamine plays.

We also investigated (ii) how thiamine is acquired and (iii) current microbiome research.

All of these topics were reviewed in an attempt to (iv) highlight the role both the gut microbiome and thiamine have in specific human diseases.

The role of thiamine:

Thiamine

Thiamine, while naturally occurring, was first synthesized in 1936 (66). Around this time thiamine was also recognized for the ability to treat beriberi. Ever since, thiamine has yielded a complex and rewarding body of research. Thiamine consists of two ring structures, a pyrimidine ring (2,5-dimethyl-6-aminopyrimidine) linked together by a carbon bridge to a thiazolium ring (4-methyl-5-hydroxyethyl thiazole) (FIG 1.1A).

Thiamine is primarily acquired through dietary means in humans (66). Thankfully, thiamine is abundant in dietary staples such as rice, meat, beans, and wheat (66). In contrast to humans, microbes such as Salmonella, Escherichia coli, and Bacillus posses the ability to synthesize thiamine de novo (6, 88, 107). Thiamine in a phosphate free form

(FIG 1.1A) does not possess an immediate biological role. However free thiamine is physiologically important, as certain transporters in humans, yeast, and microbes can only import the free form of thiamine (23, 37, 43, 99).

3 Thiamine monophosphate

Thiamine monophosphate (TMP) (FIG 1.1B) has not been found to serve any particular biological role. Unlike free thiamine, TMP does not allow for transport through certain transporters. Transport of TMP is less efficient or all together blocked by the phosphate group. In addition, TMP does not serve the role of a cofactor like the diphosphorylated form of thiamine. However, TMP does make up an appreciable percentage (5-15%) of a cells internal thiamine pool (5, 23). TMP is also detectable in human blood serum (90). Together these data show, that despite no defined biological purpose, TMP is an important intermediate between thiamine and (TPP). TMP is also a flux point in the de novo biosynthesis of TPP. In most biosynthetic pathways, TMP is synthesized and then phosphorylated to the cofactor form, TPP.

Thiamine pyrophosphate

Across all domains of life TPP is an essential cofactor. TPP (FIG 1.1C) is primarily utilized in decarboxylation reactions. TPP is required, in central metabolism, for the synthesis of Acetyl-CoA and α-ketoglutarate (72). In addition, the pentose phosphate shunt employs a transketolase that requires TPP to be active (17). TPP also catalyzes the synthesis of branched-chain amino acids (leucine, isoleucine, and valine) through decarboxylation (67). TPP is also a cofactor in many other processes that were not covered above.

While pyrophosphorylation of thiamine is required to perform catalytic functions; the pyrophosphate is not involved in the decarboxylation reaction. TPP catalyzes

4 reactions by carrying a charge on the thiazolium ring of thiamine pyrophosphate. The thiazolium ring contains a tetravalent that acts as an electron sink to allow for the formation of a carbanion (26, 105). This carbanion then attacks a carbonyl group of the . In addition, the thiazolium ring forms a double bond between TPP and the carbonyl group of the substrate. This is done by rolling electrons from the substrate onto the tetravalent nitrogen. The carbonyl group is then broken from the substrate by reversing the double bond and regenerating the cofactor (26, 82, 105) (FIG 1.2).

Thiamine triphosphate

Thiamine triphosphate (TTP) (FIG 1.1D) was originally thought to only serve a biological role in vertebrates. In vertebrates, TTP activates anion channels in neuroblastoma cells (10). TTP also phosphorylates specific involved in nicotinic acetylcholine binding (83). More recent studies have found detectable levels of TTP fungi, plants, bacteria, and invertebrates (71). The presence of TTP across all of these organisms suggests that it may play a larger role than originally believed. Further work has shown that in bacteria, specifically E. coli, TTP acts as a signaling molecule. In E. coli TTP creates a regulatory phosphate cascade that down regulates metabolism in response to amino acid starvation (44).

In addition to studies striving to identify the biological purposes of TTP, considerable time has be spent on how TTP is generated. As of yet, no dedicated to generate this molecule has been identified in any organism. Early studies in mammals found that TTP was created by adenyl 1. This reaction is not the primary function of adenyl kinase 1, but nutrient starvation may decrease the level of the primary substrate.

5 Reduced levels of the primary adenyl kinase 1 substrate may allow for TPP to be converted to TTP (42). In bacteria, TTP is generated by ATP synthase in response to metabolic stress and amino acid starvation (45). Similar to vertebrates TTP generation is not the primary function of ATP synthase. In both vertebrates and bacteria, TTP can only be generated by metabolic when the primary substrate is in low abundance. The fact that TTP generation can only occur as a secondary function lends strength to the argument that it acts as a starvation response signal.

Synthetic thiamine analogs

In addition to the thiamine species highlighted above, scientists have created a multitude of synthetic thiamine analogs. These synthetic analogs were created to act as a more bioavailable version of thiamine to combat thiamine deficiencies. In addition, many of these analogs were also found to be promising antimicrobial and chemotherapeutic compounds. The first synthetic thiamine analog, allithiamine (FIG 1.1E), was created in

1965 in Japan. Allithiamine was created by synthesizing a disulfide bridge between thiamine and a hydroxyethyl group. This compound was synthesized to be a more bioavailable version of thiamine to combat beriberi (66). In the years since 1965, many other compounds such as oxythiamine, amprolium, pyrithiamine, and benfotiamine (FIG

1.1F-I) have been synthesized. All of these compounds were synthesized for similar reasons as allithiamine. In addition to the intended purpose, these synthetic compounds have all been shown to down regulate thiamine biosynthesis and transport in pathogens.

This down regulation creates a bacteriostatic effect by impairing the dissociation of these compounds from a highly conserved regulatory mechanism (33). In addition, these

6 compounds have also been used in the treatment of cancer (129). These compounds take advantage of a cancer cells immense thiamine requirements to act as a therapeutic. The synthetic thiamine analogs compete with thiamine for the active sites of metabolic enzymes in a cancer cell slowing the uncontrolled growth.

Thiamine acquisition and biosynthesis:

Transport

Due to the essentiality of thiamine pyrophosphate, many organisms transport some version of thiamine. The transport of TPP, TMP, thiamine, and thiamine precursors are important; especially when the fact that many organisms lack the ability to biosynthesize thiamine de novo is considered. Across all three domains there is a diversity of thiamine, TPP, and thiamine precursor transporters. Most of these transporters have a high affinity for the substrate. These high affinity thiamine transporters allow for organisms to acquire the essential cofactor when environmental concentrations are low.

In humans transport is especially important, because humans cannot synthesize thiamine. Humans can only change the phosphorylation state thiamine, typically converting free thiamine to TPP (1, 100). To this end, thiamine must be acquired through dietary means in humans. The majority of thiamine is absorbed in the upper gastrointestinal tract (1, 79, 100), but thiamine transporters are found throughout the gastrointestinal tract. The presence of distal gut thiamine transporters suggests that significant levels of thiamine must be present across the gastrointestinal tract (79, 115).

7 The dietary thiamine available throughout the gastrointestinal tract is absorbed by humans and microbes alike.

The first characterized thiamine transporters in humans are members of the solute carrier protein super family (SLC). Specifically, these transporters are encoded by the genes SLC19A2 and SLC19A3. These genes encode thiamine transport protein 1 and 2 respectively (72). While both transporters are ~48% identical to one another at the amino acid level; they have been shown to be expressed differentially across the human body. In addition, SLC19A2 and SLC19A3 have different apparent kinetics for importing free thiamine (34). SLC19A2 is commonly found to be expressed in skeletal muscle throughout the human body. In addition, SLC19A2 has an apparent Km of 2.5 µM for free thiamine transport. In contrast, SLC19A3 encodes the primary thiamine transporter found in the upper intestinal tract. It possess an affinity for thiamine in in low nanomolar concentrations. This shows that thiamine transporter 2 is the high affinity thiamine transporter in the gut (49). The apparent kinetics of these proteins are likely beneficial for the specific regions they are expressed. Both proteins utilize an ion exchange mechanism that harnesses a concentration gradient to import free thiamine. This mechanism is similar to the one utilized by energy coupling factor (ECF) transporters found in bacteria.

Among ECF transporters, free thiamine is pyrophosphorylated to the active form of TPP as it is imported (39). In addition to the two previously characterized free thiamine transporters, a human TPP transporter has also been identified (79). This transporter

(SLC44A4, hTPPT) is exclusively expressed in the colon. SLC44A4 is believed to solely transport microbially derived TPP, but little else is known about it (79). Humans can also acquire free thiamine through spontaneous diffusion or nonspecific transport when the

8 concentration of free thiamine is greater than 2 µM (48, 101). This phenomena has also been shown to be true in microbes that have had their thiamine transporters deleted (23).

Interestingly, 2 µM is noted to be the highest end of physiological thiamine concentrations found in the human gastrointestinal tract (100).

Compared to vertebrates, the microbial world has developed a diverse set of importers to acquire thiamine, TPP, and its precursors. The microbial thiamine transporter most similar to the human thiamine transporters are the ECF-type transporters found among Lactobacillus species, ThiT. ThiT was even utilized as a model for the crystallization of the human transporters despite sharing less than 20% amino acid identity (72, 132). Similar to SLC19A3, ThiT has been characterized to operate in the low nanomolar range (76). Another ECF-type thiamine transporter has recently been discovered and lightly characterized. This ECF-type thiamine transporter, PnuT, shares a striking level of identity with other pyridine nucleotide uptake transporters. PnuT can be distinguished from other pnu transporters by the pyrophosphokinase that is typically downstream of pnuT. In addition, this pyrophosphokinase is believed to be involved in powering the transport of thiamine across the membrane (36, 96). PnuT transporters are widely conserved among gut microbes. For many of these microbes PnuT is also the only thiamine transporter. PnuT has the ability to transport free thiamine at low nanomolar to low micromolar concentrations (23, 43, 54). This high specificity for free thiamine may be especially beneficial for competing with the host to acquire thiamine throughout the gastrointestinal tract.

While ECF-type thiamine transporters are common across gut microbes, other types of thiamine transporters are much more widely conserved and utilized. These

9 transporters belong to the ABC transporter superfamily, specifically ThiPQ-TbpA in

Gram negative bacteria and YkoEDC in Gram positive bacteria (9, 55, 63, 133). These transporters utilize ATP to power the import of thiamine and the phosphorylated moieties of thiamine. ABC-type transporters have the ability to transport TMP and TPP in addition to free thiamine (45, 133). The ability to transport the phosphorylated versions of thiamine comes from the utilization of ATP to power transport. This is in contrast to the how ECF-transporters power transport e.g. harnessing gradients or the phosphorylation of free thiamine. ATP dependent thiamine transporters transport all three varieties of thiamine with similar apparent Kms to the microbial ECF-type transporters (ThiT and

PnuT). Studies have shown ABC thiamine transporters having a functional range between low nanomolar and low micromolar concentrations of thiamine (133).

In addition to the numerous thiamine, TMP, and TPP transporters there are additional microbial transporters that can transport thiamine precursors such as 2,5- dimethyl-6-aminopyrimidine (HMP-P) that make up part of the ring structure in thiamine

(36, 97). Transporters such as ThiXYZ, CytX, and ThiW have all be characterized to transport thiamine precursors (4, 99, 101). Thiamine precursor transporters are associated with a loss of synthetic genes for the specific molecule they now import (4, 33, 68). This suggests a strong link between thiamine biosynthesis and transport exists in many organisms.

Biosynthesis

While humans lack the ability to biosynthesize thiamine de novo, many organisms possess this ability. Often times biosynthesis exists in addition to common transport

10 pathways. Perhaps the best studied versions of de novo thiamine biosynthesis are the pathways found in E. coli and B. subtilis. To this end, thiamine biosynthesis typically consists of the same steps carried out by a different suite of enzymes.

Thiamine biosynthesis is a bifurcated pathway in which the two major precursors, pyrimidine ring (HMP-P) and thiazole (cThz-P), can be synthesized independently of each other (FIG 1.3). The formation of HMP-P is conserved across bacteria in that a single enzyme, ThiC (E. coli) or ThiA (B. subtilis), through an uncharacterized mechanism will convert 5-aminoimidazole ribonucleotide (AIR) into HMP-P (87, 128,

138). HMP-P will then be phosphorylated to HMP-PP, in preparation for thiamine condensation by ThiD (19).

The other initial step, synthesizing cThz-P, has a much more diverse series of steps between Gram negative and Gram positive bacteria. In Gram negative bacteria,

ThiS creates a thioester group by mobilizing sulfur from cysteine or another sulfur carrying molecule. This thioester group on ThiS is then chaperoned by ThiF to ThiH.

ThiH then catabolizes tyrosine in order to stabilize the thioester carried by ThiSH. Then

Dxs will combine pyruvate and glyceraldehyde-3-phosphate to form deoxy-D-xylulose 5- phosphate (DXP). ThiG will then combine the reactive thioester group synthesized by

ThiSHF and DXP, synthesized by Dxs, to form the thiazole ring (cThz-P) (91). In Gram positive bacteria, ThiH is replaced by ThiO. ThiO in contrast to ThiH utilizes glycine instead of tyrosine (7). In addition to the ability to biosynthesize thiazole de novo, many bacteria also possess the thiazole recycling enzyme, ThiK (Gram positive) or ThiM

(Gram negative). These enzymes can phosphorylate and convert thiazole from alternative sources into cThz-P (110). These two ring structures are then condensed together by ThiE

11 or YjbQ creating TMP (3, 110). TMP is then phosphorylated to the active TPP form by the TMP kinase, ThiL (134).

In addition to these complex pathways performed by bacteria, there is also a thiamine synthesis pathway in yeast. This pathway is still bifurcated similar to the bacterial mechanism. In contrast, HMP-PP synthesis performed by Thi5D, instead of

ThiCD. Thi5D utilizes histidine and pyridoxal in place of AIR (119). Likewise, the thiazole synthesis suite of genes are replaced by Thi4 in yeast. The thiazole synthesis genes of yeast utilize NAD+, cysteine, and glycine in place of the common bacterial precursors (75, 117).

Regulation

Thiamine biosynthesis and transport genes in all three domains of life are regulated by cis-acting RNA regulatory elements (TPP riboswitches) (13, 78, 94,135).

TPP riboswitches are highly conserved and maintain a pitch-fork like structure. In bacterial species, TPP riboswitches occur in the 5’-UTR of (135). In contrast, eukaryotes have TPP riboswitches in splice junctions (12). In all species these riboswitches impact the expression of thiamine acquisition genes.

In one branch of the pitch-fork like structure of the TPP riboswitch, there is a conserved sequence of 5’-UGAGA-3’ that binds the pyrimidine domain of thiamine,

TMP, and TPP through Watson-Crick base stacking interactions (80, 108). In addition to the pyrimidine binding domain, there is a pyrophosphate binding domain in TPP riboswitches (FIG 1.4). This pyrophosphate binding domain utilizes the secondary structure in coordination with divalent metals, such as magnesium and manganese, to

12 stabilize and bind TPP (108). This pyrophosphate binding domain aids in the sensing of thiamine. This domain makes TPP riboswitches 100-fold more likely to bind TPP than

TMP. The phosphate binding domain also confers a 1,000-fold higher affinity for TPP than free thiamine (122).

In bacteria, there are two mechanisms by which TPP riboswitches regulate their regulon. It is canonically known that TPP riboswitches in Gram negative bacteria regulate by ribosomal (RBS) occlusion. This is affected by the stable binding of TPP, which creates a conformational change that now blocks the RBS from being bound by the ribosome (108) preventing translation. In Gram positive bacteria, TPP riboswitches block transcription of their regulon by creating a stem-loop. This stem-loop causes the RNA to fall off, blocking the transcription of thiamine biosynthesis and transport genes (135). While these mechanisms are believed to be canonical and exclusive to their Gram type; there is an emerging trend of dual acting TPP riboswitches that act at the level of both transcription and translation (15). In addition, some gut microbes appear to have independent translational and transcriptional acting riboswitches (23).

In eukaryotes, TPP riboswitches are found near splice sites of thiamine biosynthesis genes (yeast) or in transport genes (18). Alternative splicing allows for the hiding of splice sites that create different mature thiamine acquisition proteins. When thiamine is replete, splicing prevents the maturation of high affinity thiamine transporter mRNA. Instead, the TPP bound riboswitch causes the maturation of a lower affinity thiamine transporter (18).

13 The microbiome:

The human microbiome is defined as the bacteria, fungi, viruses, and archaea that colonize the human body. It has also been shown that microbial cells outnumber human cells in the human body (127). As an example, bacterial cells can number as high as 1011-

1012 cells/mL in the distal colon (8). In addition, microbially derived transcripts and proteins are 1,000-10,000 times more abundant than human derived transcripts and proteins (38, 139). There are numerous types of microbiomes across the human body, but the most studied microbiome is that of the human gut. The gut microbiome is linked to human development, metabolic function, and disease (2, 41, 85, 111). However, most of these studies are correlative in nature. While many studies exist, there is a lack of mechanistic understanding about how the gastrointestinal microbiota impacts human health and disease. The gut microbiome has been extensively reviewed in many places, but a brief overview will be provided here. This overview is provided to emphasize the impact this biome plays in human development, immune modulation, and metabolic modulation.

Microbial communities begin impacting human health at the time of birth. This symbioses continues through the life of the host. After birth the gastrointestinal tract is immediately colonized by Bifidobacteria. This colonization is favored because

Bifidobacteria are adept at breaking down and utilizing the nutrients of the infants milk- based diet (22, 116). The gut community then shifts to being dominated by and Bacteroidetes when solid food is introduced (35, 57). This “healthy” microbiome and its establishment is crucial for the maturation of gut cilia and the immune system (46,

51). The early development of a “healthy” gut microbiome is important for long term

14 health. However, data shows that later in life interventions can have a positive impact on the severity and development of diseases such as diabetes, obesity, and Alzheimer's disease (21, 53). Due to the importance of the gut microbiome, identifying mechanisms to change the microbiome are of the utmost importance. The most common way gut microbiomes are modulated is through the clearing of most microbial species by broad spectrum antibiotics (21, 53, 64). This has recently been regarded as detrimental, as it routinely opens the intestinal tract up to being colonized by such as

Clostridium difficile (120). Other strategies, such as changes in diet and fecal microbiota transplants, have been utilized as more subtle mechanisms to affect change and improve human health (20, 32, 58, 86, 104).

The bacterial phyla that typically dominate the human gastrointestinal tract are the

Firmicutes and Bacteroidetes. The high abundance these phyla have led many to believe that both have an immense impact on the health of the human host. Shifts in either phylum have been correlated to diseases such as IBD, obesity, and diabetes (14, 29, 84).

However, little is known about how to selectively and therapeutically change the abundances of these phyla. It is hypothesized that altering host diet (e.g. metabolite intake) could create beneficial changes that mitigate many microbiome associated diseases.

Thiamine, the microbiome, and disease:

Both thiamine and the gut microbiome have been linked to human health.

Imbalances in either variable have been linked to many diseases, such as Alzheimer's,

15 Parkinson's, irritable bowel disease, obesity, and various types of cancer. Unfortunately, little consideration has been given to how both coordinately impact the development of these diseases. This section attempts to link the role thiamine and the native flora of the gastrointestinal tract play in human health.

Alzheimer’s disease

Alzheimer’s disease is a neurodegenerative disorder characterized by the accumulation of β-amyloid particles, fibrils, and oligomers in the brain. In addition to an over accumulation of these particles, they are typically misfolded. This misfolding leads to the particles no longer being able to perform their intended biological function (30,

70). Alzheimer’s disease typically impacts the elderly because this age group has a decreased ability to clear the misfolded amyloids (130). To date, there are no recorded therapies for Alzheimer’s disease. However, the intrinsic link of the gut microbiota and thiamine metabolism in Alzheimer’s disease suggests that both could be modulated as a therapy.

The gut microbiome is well known for the expression of lipopolysaccharides.

Consequently, gut microbes also produce many other metabolites, including amyloid particles (31, 106). These microbially derived amyloids become increasingly important as the host ages and the gut barrier becomes more permeable them. This increased leakiness may be caused by dysbiotic states that decrease the amount of short chain fatty acids

(SCFAs) produced by the gut community. The decrease in SCFAs leads to increases in inflammation and permeability of the gut membrane (27). In addition, certain pathogenic bacteria have been noted to excrete these amyloid-like particles. The increase in

16 microbially derived amyloids may seed the formation of human amyloids that cause

Alzheimer’s disease. Many of these pathogenic species can only gain prominence during dysbiotic states. Consequently these pathogenic bacteria may be a source of excess amyloid particles that lead to the non-clearing accumulation noted in Alzheimer’s disease

(50, 106). In addition to the pathogenic bacteria that have been associated with pro-

Alzheimer’s disease states, many beneficial microbes have been correlated with an anti-

Alzheimer’s disease state. As an example, the Bacteroidetes have been correlated with decreased amyloid particles (112). Other probiotic species, such as the Bifidobacteria, have been shown to be protective against amyloid-producing (131).

Considering the impact that the microbiota can have in preventing or causing

Alzheimer’s disease, it has become important to find modulatory therapies of the microbiome.

In addition to the role the microbiota plays in Alzheimer’s disease, thiamine has also been correlated with the pathology of the disease. It has been noticed that patients with Alzheimer’s disease have hypometabolism in the brain (103). This hypometabolic state has been correlated with decreased thiamine concentrations in the bloodstream.

Decreased concentrations of thiamine would lead to a down regulation of central metabolism, due to the role thiamine plays as a cofactor in this pathway (77). Thiamine levels do not correlate to the buildup of amyloid fibers; however the supplementation of thiamine or benfotiamine has been associated with the mitigation of Alzheimer’s disease symptoms (77, 89).

Altering the microbiome and thiamine availability could be used in coordination to treat Alzheimer’s disease. Consequently, similar therapies could be utilized to treat

17 other neurodegenerative disorders such as Parkinson’s disease. In addition, thiamine and benfotiamine supplementation may have a profound impact on the microbiome and also warrants further study.

Diabetes and obesity

Diabetes is a metabolic disorder that has both genetic and environmental factors that impact its pathology and severity in the host. Type I diabetes is attributed more to host genetics; while type II has more of an environmental based origin. Both however are immune modulated and have components related to the microbiome. The microbiome can initiate an inflammatory response that causes a signaling cascade creating insulin sensitivity in the host (47). Obesity is often a precursor to the onset of diabetes. Similarly, obesity is linked to the microbiota that inhabits the gastrointestinal tract. Diabetes may be mediated by metabolic dysregulation through dysbiosis (65). Thiamine has also been implicated in the development of diabetes and obesity. This implication is due to the role thiamine plays in the utilization of dietary carbohydrates. Studies of thiamine and the gut microbiome suggest that they both play a role in the prevention and treatment of diabetes.

The microbiome of the human gastrointestinal tract has been linked to both diabetes and obesity by correlating differential abundances of certain types of bacteria

(61, 124, 126). While research on this topic is on going, it is unknown whether the composition of the microbiome or the disease occurs first. Both obesity and diabetes can be mitigated by altering the composition of the gut microbiome (114). In obesity, an increase in the abundances of Firmicutes species have been correlated with the ability to lose weight. In contrast, increases in Bacteroides species have been shown to be

18 detrimental to weight-loss (65, 114). These associations are likely due to the differential metabolic aptitudes of each phylum. These aptitudes impact energy utilization and storage in the host (126). The utilization of complex carbohydrates by the gut microbiota may prevent the host from absorbing and storing the excess energy (59). Diabetes is also linked to a change in microbially derived metabolite production. Both diabetes type I and

II are associated with a decrease in butyrate producing bacteria, such as Faecalibacterium prausnitzii (93, 140). Butyrate is a prominent microbially derived SCFA in the gut.

SCFAs have been shown to create an anti-inflammatory response in the gut. This anti- inflammatory response may mitigate the inflammation linked to diabetes and other autoimmune diseases (69, 93, 140). Both obesity and diabetes are diseases that may be managed or prevented by therapeutically altering or supplementing the gut flora.

Thiamine availability and plasma levels are associated with the prevention and treatment of diabetes and obesity (92, 118, 137). This may be due to excess utilization of thiamine by central metabolic enzymes. High expression of these enzymes may prevent insulin sensitivity and the accumulation of fat deposits (118). It has also been shown that thiamine supplementation can be beneficial to patients already suffering from diabetes or obesity (137). Thiamine is beneficial because it allows the breakdown of metabolites, such as glucose. This prevents storage and sensitivity to excess glucose in the host (92).

Both thiamine supplementation and the alteration of the gut microbiome have been shown to be beneficial in the treatment of diabetes and obesity. Therefore, the study of how these factors interact with one another could be beneficial to the prevention and treatment of diabetes and obesity.

19 Conclusion:

The microbiome and thiamine are intrinsically linked to human health and disease. The gut microbiome is also linked to other diseases such as cancer, inflammatory bowel disease, depression, and many more. In turn, thiamine deficiencies are strongly correlated to neurodegenerative disorders, cancer, diabetes, and obesity. Conflicting reports and studies have made it difficult to state that thiamine or the gut microbiome are definitive treatments of any disease. However, the evidence that both play a role warrants further investigation into how they impact human health.

Due to the importance of both thiamine and the microbiome, it is imperative that we study how they impact one another. It is likely that the microbiome affects the thiamine availability in the host gastrointestinal tract and blood stream. It is also likely that thiamine changes the abundances of keystone microbial species in the gut. Studying both variables and how they affect one another is crucial in studying the development and prevention of many diseases.

20 Figures:

A Thiamine B TMP C TPP

D TTP E Allithiamine F Oxythiamine

G Amprolium H Pyrithiamine I Benfotiamine

FIG 1.1. Structures of thiamine and various thiamine analogs

Structures for (A) thiamine, (B) TMP, (C) TPP, (D) TTP, (E)Allithiamine, (F)

Oxythiamine, (G) Amprolium, (H) Pyrithiamine, and (I) Benfotiamine were obtained via pubchem to illustrate differences in structure between these various moieties. (National

Center for Biotechnology Information. PubChem Compound Database; CID=6202, https://pubchem.ncbi.nlm.nih.gov/compound/6202 (accessed June 13, 2018).

21

FIG 1.2. General decarboxylation mechanism utilized by TPP

Decarboxylation of pyruvate by TPP is used as a model for this reaction. Charge on the thiazole ring of TPP carries out a nucleophilic attack on the substrate for decarboxylation

(pyruvate). This forms a double bond between pyruvate and TPP. Then the bound between TPP and the substrate is broken. This regenerates TPP and creates a decarboxylated (acetyl). Example reaction was obtained via https://en.wikipedia.org/wiki/Pyruvate_decarboxylase#/media/File:Pyruvate_decarboxyla se_mechanism.png

22

Glycolysis GAP Purine Vitamin B6 Metabolism Metabolism AIR PLP Dxs Pyruvate 2.2.1.7 Cys

ThiC THI5 4.1.99.17 -.-.-.- HMP-PP ThiG ThiI SufS 2.8.1.10 -.-.-.- 2.8.1.7 ThiD TenA 2.7.4.7 IscS-SH 2.7.1.49 cThz*-P TenI ThiF ThiE/ 5.3.99.10 2.7.7.73 YjbQ 2.5.1.3 ThiH ThiS-SH ThiM 4.1.99.19 -.-.-.- ThiO 2.7.1.50 1.4.3.19 ThiK Gly Tyr 2.7.1.89 TMP TH-1 ThiL 2.5.1.2 THI4 TenA NTPCR 2.7.4.16 Tnr3 2.8.2.10 3.5.99.2 3.6.1.15 2.7.6.2 NAD+ Tnr3 THTPA THI 2.7.6.2 TPP 3.6.1.28 TTP

FIG 1.3. General thiamine biosynthesis pathways

Thiamine biosynthesis pathways that are utilized by microbes and yeast are depicted above in a combined manner to show similarities in the pathways. Dedicated thiamine biosynthesis genes are highlighted in green. Enzymes required for thiamine biosynthesis, but that are not wholly dedicated to the pathway are highlight in blue. EC numbers are provided for enzymes that have been cataloged in the EC database.

23

FIG 1.4. Representative TPP riboswitch binding of TPP

Model of the coupling between TPP binding and structural rearrangements in the TPP riboswitch. (A) Summary of the contacts observed between the pyrophosphate (blue) and pyrimidine (pink) sensor helices, TPP, and the switch region. Watson-Crick base pairs are indicated in black, nonstandard base pairs are shown in green, and hydrogen bonds are indicated with thin black lines. TPP is schematically drawn in yellow: phosphates, triangles; thiazole ring, pentagon; pyrimidine base, hexagon. The Mg2+ ion is shown as a red dot. Contacts between nucleotides and magnesium-bound TPP are highlighted in red.

Red double-lines indicate stacking interactions with the ligand. Base A72, which forms the crucial assembly platform, is shown in yellow. (B) Upon TPP binding (1), bulges J2/3 and J4/5 rearrange, thereby closing the sensor helix clamp and forming new hydrogen bonds at the tip of loop L5. The TPP-induced parallel positioning of the sensor helices stabilizes the three-way junction with A72 at the core of the interaction platform (2) and promotes the formation of the switch helix P1 (3), which turns the riboswitch “off.”

24 FIG 1.4. (cont.)

Figure and legend taken from: Thore S, Leibundgut M, Ban N. 2006. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science

312:1208–1211. (122)

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38 CHAPTER 2: THIAMINE ACQUISITION STRATEGIES IMPACT

METABOLISM AND COMPETITION IN THE GUT MICROBE BACTEROIDES

THETAIOTAOMICRON

Abstract:

Thiamine (vitamin B1) is an essential cofactor for all organisms. Humans primarily acquire thiamine through their diet and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood and little is known about how thiamine impacts the stability of microbial gut communities. To investigate thiamine’s role in the gut we utilized the model gut microbe, Bacteroides thetaiotaomicron. RNAseq revealed a global down-regulation of thiamine and amino acid biosynthesis, glycolysis, and purine metabolism when thiamine was present. Using genetic mutants of thiamine biosynthesis and transport loci we determined both systems were critical for growth in thiamine- deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron.

Mutant phenotypes were recapitulated during pairwise competitions, reinforcing the importance of encoding versatile thiamine acquisition mechanisms when thiamine concentrations are variable. In addition, LC/MS analyses corroborate that exogenous thiamine levels affect the internal thiamine pool of B. thetaiotaomicron. Further, we computationally examined the ability of other gut microbes to acquire thiamine and identified lineage specific differences in thiamine acquisition strategies. Among the

Bacteroidetes the capacities for both thiamine transport and biosynthesis are common.

Together, these data show that thiamine acquisition mechanisms used by B.

39 thetaiotaomicron are not only critical for its physiology and fitness but provide the opportunity to model how other gut microbes may respond to the shifting availability of thiamine in the gut.

Importance:

Variation in the ability of gut microbes to transport, synthesize, and compete for

Vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota and ultimately this variation may have both direct and indirect effects on human health.

Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering presence or concentrations of water-soluble vitamins, such as thiamine, may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease.

Introduction:

Thiamine, a small metabolite vitamin is an essential cofactor in all three domains of life. Thiamine is necessary for glycolysis, the tricarboxylic acid (TCA) cycle, branched chain amino acid metabolism, and nucleotide metabolism (6). Deficiencies of thiamine in humans can lead to neurodegenerative disorders such as Wernicke’s encephalopathy and

40 beriberi, as well as heart failure, ataxia, and paralysis (14). In humans, thiamine is believed to be primarily derived from dietary sources such as protein rich foods as well as fortified grains (14) and absorbed throughout the small and large intestines (12).

However, there is evidence that thiamine availability is modulated by or acquired from the dense and diverse microbial communities present in the gastrointestinal tract (12, 18,

21).

Competition for and production of small metabolites such as vitamins, amino acids, and short chain fatty acids influence the composition (e.g., abundance and types) of microbes that inhabit the gastrointestinal tract (4, 5, 27). As such, small metabolites are therapeutic candidates for treating diseases associated with radical changes in gut microbial community composition such as Alzheimer’s, diabetes, and obesity (10, 37,

42). Indirect evidence suggests thiamine may influence gut community structure.

Metagenomic analysis of different aged human cohorts determined that not only did overall microbial communities change with age, but also the abundance of microbial thiamine biosynthesis genes increased with the age of the host (45). Conversely, in a separate study the activity of human intestinal thiamine transporters was observed to decrease with age (29). These observations may indicate a lower requirement for thiamine by the host with age or possibly an increase in thiamine availability due to microbial production in older individuals. While these correlative data exist, little is known mechanistically about how the competition for and production of thiamine impacts individual gut microbes like Bacteroides thetaiotaomicron, microbial gastrointestinal communities, or human health.

41 Microbial thiamine biosynthesis is a bifurcated pathway involving the synthesis of two precursors that are condensed to form thiamine (15). Microbes that lack complete de novo biosynthesis of thiamine often encode partial biosynthetic pathways of the precursors thiazole and hydroxymethyl pyrimidine phosphate or simply phosphorylate free thiamine into the biologically active form, thiamine pyrophosphate (TPP) (19).

Organisms that lack or have incomplete biosynthetic pathways require transporters for thiamine or its precursors. Humans exclusively transport free thiamine in the proximal small intestine as well as in the large intestine (29), while some microbes like Salmonella enterica use an ABC transporter to acquire thiamine and its phosphorylated forms (41).

However, despite our understanding of thiamine biosynthesis and metabolism in humans and a limited number of model organisms little is known of thiamine acquisition strategies among most beneficial gut microbes.

Initial comparative genomic analyses of several gut microbes suggested that the

Bacteroidetes, a phylum that can comprise as much as 50% of the gastrointestinal community (3), possesses a canonical thiamine biosynthesis pathway (28). In addition, a novel transport system was predicted due to the presence of cis-acting RNA regulatory elements preceding the putative transport genes, TPP riboswitches (28). However, limited investigation of thiamine acquisition in the Bacteroidetes has been attempted (18). Here, we have carried out a bioinformatic analysis of 641 gut associated microbes and performed a transcriptomic, genetic, and competitive characterization of the prominent gut microbe B. thetaiotaomicron in response to thiamine. In turn, we have characterized

(i) the effect of thiamine on the transcriptome, (ii) the predicted thiamine biosynthesis and transport genes, (iii) the competitive defects of biosynthesis and transport mutants,

42 and (iv) the affects that exogenous thiamine has on the internal thiamine pool of B. thetaiotaomicron.

Results:

Global gene expression response of B. thetaiotaomicron to thiamine availability

B. thetaiotaomicron, a prominent gut microbe, is typical of the Bacteroidetes in its thiamine acquisition strategy encoding both TPP riboswitch regulated biosynthesis

(thiSEGCHF-tenI, BT0653-BT0647), putative outer membrane transporter (OMthi,

BT2390) and putative inner membrane transporter and thiamine pyrophosphorylase

(pnuT-tnr3, BT2396-BT2397) operons (28). TPP riboswitches can act at the transcriptional or translational level (23) and it is uncertain how they function in the

Bacteroidetes. To investigate the response of B. thetaiotaomicron to the availability of thiamine, strand-specific RNAseq was performed on cultures grown in minimal medium supplemented with either 0 or 15 µM thiamine in duplicate. A total of 34,890,999 reads were recovered, ranging from approximately 8 million to 9.5 million reads per sample that were quality filtered, trimmed, and analyzed with Rockhopper (Table 2.1) (35). Of the 4,778 coding sequences in the B. thetaiotaomicron genome, 151 showed ≥ 2-fold expression change (q ≤ 0.05). Of the three putative TPP riboswitch regulated operons, the biosynthetic operon as a whole was significantly down regulated when thiamine was present (FIG 2.1A). No significant differential expression was observed at either of the predicted transport operons (FIG 2.1A). This is in striking contrast to the multiple vitamin B12 riboswitch-regulated B12 transporters in B. thetaiotaomicron, which exhibit a

43 strong transcriptional induction when grown in vitamin B12 limiting conditions (4).

However, this does not rule out their involvement in thiamine transport, particularly as the putative inner membrane transporter, PnuT, has been heterologously expressed in E. coli and demonstrated to transport thiamine (18).

In addition to the genes in the biosynthetic operon, 148 other genes were differentially expressed due to exogenous thiamine in the environment. In fact, 132 of the

151 differentially regulated genes were down regulated when 15 µM thiamine was present in the medium (FIG 2.1B). Using a gene set enrichment analysis (GSEA) we determined if specific pathways are differentially expressed due to thiamine availability

(34). Significantly down-regulated KEGG pathways in the presence of exogenous thiamine were observed in thiamine metabolism, amino acid biosynthesis, purine metabolism, the TCA cycle, coenzyme A biosynthesis, and pyruvate metabolism (FDR corrected p ≤ 0.001) (FIG 2.1C). This suggests that in the presence of excess exogenous thiamine, B. thetaiotaomicron down-regulates transcription of the thiamine biosynthetic pathway in order to maintain homeostatic levels of internal thiamine. In addition to this,

B. thetaiotaomicron also decreases transcription of thiamine dependent pathways such as branched chain amino acid biosynthesis, glycolysis, and the citric acid cycle when excess thiamine is present. This decreased expression in the presence of excess thiamine is counter intuitive to what was expected. Normally, it is expected that expression of cofactor dependent enzymes is positively correlated with the concentration of the cofactor (e.g., iron) (13). In the case of thiamine in B. thetaiotaomicron we observe the opposite. The expression of thiamine dependent enzymes was inversely correlated.

44 Re-analysis of microarray data

To test for evidence of transcriptional changes in response to thiamine availability we re-analyzed existing microarray data from B. thetaiotaomicron growing both in vitro and in vivo (33). Briefly, microarrays had been hybridized with total RNA recovered from wildtype B. thetaiotaomicron grown to mid-log phase in biological duplicate in minimal medium with no thiamine added (8h), in TYG that has excess thiamine (3.5h) or from the ceca of two monoassociated gnotobiotic NMRI mice (GSE2231) (33). Raw

GeneChip data were normalized and model-based expression values (PM-MM model) were compared using DNA-Chip Analyzer (v2011.05) as described (33). Mean and standard deviations of the normalized expression values were calculated and significantly differentially regulated genes were detected with t-test, p < 0.05 using FDR correction.

Genetic analysis of thiamine acquisition genes in B. thetaiotaomicron

To assess the conditions in which thiamine biosynthesis and uptake are important for the growth of B. thetaiotaomicron and validate the role of the predicted transporters in thiamine uptake a series of in-frame genetic deletions were generated. We focused on the three TPP riboswitch regulated operons, removing the entirety of the downstream biosynthetic or putative transport coding sequences. The resulting mutant strains and their complements (Table 2.2) were evaluated for their ability to grow under a range of exogenous thiamine concentrations.

Wildtype B. thetaiotaomicron readily grows in media without added thiamine

(FIG 2.3A) (11), which corresponds with elevated expression of the TPP riboswitch regulated thiSEGCHF-tenI operon identified by RNAseq. Therefore, deletion of the

45 major thiamine biosynthetic operon (∆BioThi: BT0653–BT0647) is expected to completely inhibit growth in minimal medium without thiamine. Indeed, growth inhibition occurs in the ∆BioThi mutant, severely attenuating terminal cell densities (FIG

2.3A,B). Partial growth of the ∆BioThi mutant is recovered when ≥ 1 nM thiamine is supplemented in the medium (FIG 2.3B). The calculated EC50 (the effective concentration of thiamine for which the terminal OD600 of the mutant attains 50% of the wildtype)(Table 2.3) for the ∆BioThi mutant is 2.8 nM (Confidence Interval of 95%,

CI95, 1.4 – 5.5 nM) showing that biosynthesis is critical in thiamine deficient environments. This growth defect can be complemented when the thiSEGCHF-tenI operon and its native promoter are returned to the ∆BioThi mutant in trans (FIG 2.3B) alleviating the requirement for exogenous thiamine and returning the EC50 to 0 nM thiamine (Table 2.3). Together these data suggest that B. thetaiotaomicron does in fact encode thiamine transporters that likely function in the low nanomolar range. This is comparable to other characterized thiamine transporters such as ThiPQ-TbpA in

Salmonella typhimurium (41).

To determine if the predicted TPP riboswitch regulated inner (pnuT: BT2396) and outer (OMthi: BT2390) membrane transporters are responsible for transporting thiamine individual transporter deletions were constructed in the wildtype background (∆pnuT,

∆OMthi). Regardless of the thiamine concentration in the medium, growth of the single transporter mutants never significantly varied from wildtype (FIG 2.3B). This result was expected, as the thiamine biosynthesis pathway is fully intact. Therefore, to further investigate the role of these transporters, double mutants consisting of either the biosynthetic operon and a single transporter or both transporters were generated.

46 Deletion of the biosynthetic operon in conjunction with either the inner

(∆∆BioThi-pnuT) or outer membrane (∆∆BioThi-OMthi) transporters resulted in mutants that required ~10 times more thiamine (1,000 nM) than the ∆BioThi mutant to achieve growth equal to the wildtype (FIG 2.3C). The EC50 for the double mutants increased to

15.8 nM (CI95 8.1 – 31 nM) thiamine for the biosynthesis and outer membrane transporter mutant and to 10.9 nM (CI95 9.2 - 13 nM) for the double mutant of biosynthesis and inner membrane transporter compared to an EC50 of 2.8 nM (CI95 1.4 – 5.5 nM) in the biosynthesis mutant (Table 2.3). Thus, partially disrupting thiamine transport in the absence of a complete biosynthetic pathway further attenuates, but does not completely abolish thiamine dependent growth. Growth of either of these two mutant backgrounds can be fully complemented by restoring the major thiamine biosynthesis operon in trans

(FIG 2.3C).

Compared to the individual biosynthesis and transporter mutants, deletion of both transporters while the biosynthesis locus is fully intact (∆∆Transport) has a highly deleterious effect on growth (FIG 2.3D). This mutant requires at least 10 µM of thiamine to achieve wildtype growth and has an EC50 of 30.3 nM (CI95 22.8 – 40.3 nM, Table 2.3).

To ensure this phenotype was not due to an inability to transport cysteine, the only thiamine precursor present in the minimal medium, the double transport mutant was grown with an alternate sulfur source. The growth phenotype of the mutant in the modified medium was unchanged suggesting that neither transporter is likely involved in cysteine transport (see Supplemental Results, FIG 2.4).

Individual complementation constructs were generated for the inner and outer membrane components and introduced in trans into the double transport mutant

47 (∆∆Transport ppnuT, ∆∆Transport pOMthi). Although we did not observe full complementation by either of the two constructs, they did significantly improve the EC50 from 30.3 nM (CI95 22.8 – 40.3 nM) in the double transport mutant to 1.9 nM (CI95 0.8 –

4.4 nM) and 0.1 nM (CI95 0 – 1.9 nM, Table 2.3) in the outer and inner membrane transport complement respectively (t-test, p < 0.0001; FIG 2.3D). In addition to single transport complements, a double transport complement (∆∆Transport ppnuT+OMthi) was made and tested to similar results as the single transport complements. The double complement allowed for better fitness at low levels of thiamine achieving >35% of wildtype growth at 0 nM added thiamine and greater than 80% of wildtypes growth at 10 nM (FIG 2.5). It also significantly improved the ∆∆Transport mutants EC50 from 30.3 nM to 0.1 nM (CI95 0 – 0.2 nM, Table 2.3). While not uncommon, it is possible that the introduction of the complementation vectors in trans were not expressed at the same level as the wildtype or are missing key upstream elements preventing the strains from growing as well as the wildtype B. thetaiotaomicron. (FIG 2.3D).

A final triple mutant of all three TPP regulated loci was generated (∆∆∆). This strain demonstrated a similar level of growth inhibition as compared to the double transport mutant (FIG 2.3D). It required 10 µM of thiamine in the medium to attain 100% wildtype growth (EC50 of 51.9 nM CI95 42.4 – 63.6 nM, Table 2.3). It is important to note that growth defects only affected the terminal density of the mutants and the doubling times remained fairly constant (~2 hrs). Together these data confirm the crucial role these operons play in producing and acquiring thiamine.

48 Factors affecting the phenotype of the double transport mutant

To determine if dysregulation of biosynthesis played a role in the severe growth phenotype of the double transport mutant RT-qPCR was performed. Consistent with the

RNAseq data (FIG 2.1), expression of the major biosynthetic operon in wildtype B. thetaiotaomicron is up regulated in thiamine deficient conditions (0 & 100 nM) as compared to replete (10,000 nM) (FIG 2.6A,B). In a thiamine-deficient environment genes thiC (FIG 2.6A) is 9.2-fold higher expressed in 0nM thiamine as compared to its expression in 10,000 nM and 3.3-fold higher as compared to its expression in 100 nM thiamine (FIG 2.6A). Similarly the expression of thiS is expressed 2.7-fold to 2-fold higher in 0 nM thiamine as compared to 10,000 nM and 100 nM respectively (FIG 2.6B).

As noted above the ∆∆Transport mutant cannot be cultured in 0 nM thiamine, but thiC and thiS expression was determined in 100 and 10,000 nM thiamine. In marked contrast with the wildtype, thiC and thiS expression are repressed over 100-fold in media with 100 nM or 10,000 nM thiamine. These data suggest that transport and biosynthesis are linked and likely contribute to the severe growth defect in the ∆∆Transport mutant despite the presence of an intact biosynthetic operon.

In addition, we also performed RT-PCR on tnr3, which is encoded immediately downstream of pnuT, to ensure that the generation of the ∆∆Transport mutant did not have a polar effect (FIG 2.6C). The expression of tnr3 remains unchanged in wildtype and the ∆∆Transport mutant cells regardless of the thiamine concentration tested. Given this result we infer that the fitness defect is not due to a polar effect of deleting the transport genes.

49 Characterization of transport mutants in response to thiamine precursors

In order to ascertain whether our transport mutant phenotypes were due to a disruption in transport of thiamine precursors instead of complete thiamine molecule.

Apart from cysteine, none of the other immediate precursors or intermediates for thiamine biosynthesis are present in minimal medium. Cysteine is both the reducing agent and reduced sulfur source in the medium. Therefore, it is possible that these two genes do not transport thiamine, and instead is required for the acquisition of cysteine as a precursor to enable thiamine biosynthesis. We note that B. thetaiotaomicron encodes a putative alanine, serine, and cysteine transporter (BT1223) and the biosynthetic pathway for cysteine (cysE, cysK, cysM). Also, based on the RNAseq results, these cysteine- associated genes are expressed in cells grown in minimal medium in the presence or absence of thiamine HCl. However, if OMthi and pnuT are involved in cysteine transport providing an alternative sulfur source to enable de novo biosynthesis of cysteine should reverse the apparent thiamine growth defect. Thus, the cysteine in the medium was replaced with sodium thioglycolate as a reducing agent and sodium sulfide as the sulfur source (FIG 2.4) (33). This change had no effect on the growth of wildtype and the single

OMthi and pnuT mutants indicating that B. thetaiotaomicron can use sodium sulfide to biosynthesize cysteine. Moreover, thiamine dependent phenotypes of the double mutants were recapitulated when cysteine was replaced with sodium sulfide as the sulfur source.

Together these data indicate that neither of the transporters is involved in cysteine transport.

B. thetaiotaomicron thiamine acquisition mutants were also tested to determine if thiamine precursors or moieties could rescue growth. Similar to the growth assays

50 performed before thiazole, tyrosine, and GAP were supplemented in the media. Wildtype and the single transport mutants grew normally, exhibiting no growth improvement or impairment in the presence of these metabolites. In addition, in the absence of thiamine none of these metabolites rescued the growth of the double transport mutants at the concentrations tested (0 – 10 µM) (FIG 2.4). The failure to rescue the double transport mutant suggests B. thetaiotaomicron lacks the ability to import the precursors necessary to biosynthesize thiamine or that some other feedback mechanism exists between biosynthesis and transport.

The responses of B. thetaiotaomicron mutants to the phosphorylated thiamine moieties were also evaluated for their ability to alter growth phenotypes. B. thetaiotaomicron mutants were supplemented with either thiamine monophosphate

(TMP) or thiamine pyrophosphate (TPP) and grown as before (Table 2.3). Neither TMP, nor TPP rescued growth of the mutants to wildtype levels at a concentration below 1 µM, which is reflected in the EC50 values of each mutant increasing on either TMP or TPP.

These data suggest that B. thetaiotaomicron, like other organisms that rely solely on pnuT type transporters (e.g. and Pseudomonas putida) cannot transport phosphorylated thiamine moieties. It is likely that the poor growth is due to the presence of dephosphorylated thiamine impurities in commercial TPP and TMP preparations.

Consequences of thiamine acquisition on competition

We investigated the competitive capacities of the B. thetaiotaomicron thiamine acquisition mutants in pairwise competitions with wildtype B. thetaiotaomicron. To

51 determine if co-culture increased (competition) or decreased (cross-feeding) the individual phenotypes mixed cultures were serially passaged for five days in media with

0, 10, 100, and 10,000 nM exogenous thiamine and strain abundances were monitored through time by qPCR (FIG 2.7, FIG 2.8). The single transport mutants (∆pnuT and

∆OMthi) successfully coexisted with wildtype B. thetaiotaomicron in every condition tested (FIG 2.7A,B). The mutants maintained 60-80% of the population throughout the five days of serial passaging. Although the abundance of the mutants fluctuated during passaging this was likely due to stochastic effects during the daily inoculations (FIG 2.8).

Together, these results extend the individual growth findings and show that cells lacking either the inner or outer membrane transporters are not at a disadvantage when grown in competition with the wildtype strain that encodes both fully intact transporters.

In contrast, the ∆BioThi mutant was rapidly outcompeted in the absence of thiamine, falling to < 0.1% of the population by day five despite initially making up 90% of the population. As exogenous thiamine levels were increased the fitness defect was alleviated. At 10 nM thiamine, the wildtype dominated the competition by day five.

However, the ∆BioThi only dropped to 44% of the population. As the concentrations of thiamine increased to 100 nM and 10,000 nM, the ∆BioThi mutant showed no fitness defect, maintaining ≥ 80% of the population throughout the competition (FIG 2.7C).

Similarly, ∆∆BioThi-pnuT and ∆∆BioThi-OMthi are dramatically outcompeted in 0 nM and 10 nM of added thiamine, to the level that they are not even detectable. As thiamine is increased in concentration the abundance of the double biosynthesis and transporter mutants stabilize to ~55% of the population for the ∆∆BioThi-OMthi and ~66% of the population for the ∆∆BioThi-pnuT mutant respectively (FIG 2.7D,E). The ∆∆Transport

52 and ∆∆∆ mutants were also interrogated for their competitive abilities. We observed severe phenotypes with both mutants in 0, 10, and 100 nM thiamine (FIG 2.7F,G). The mutants were rapidly outcompeted by the wildtype within the first three days and driven below the level of detection by day 5 (FIG 2.8). Only in 10,000 nM did the ∆∆Transport mutant and the ∆∆∆ mutant coexist with the wildtype; maintaining 40% and 80% respectively at day five. These results essentially mirror the individual growth data for the mutants, and suggest that wildtype B. thetaiotaomicron does not cooperate with or facilitate the growth of these mutants, indicating that B. thetaiotaomicron does not appear release thiamine to the environment under in vitro growth conditions.

Quantification of cell-associated thiamine

Intracellular concentrations of thiamine, TMP and TPP were measured after wildtype B. thetaiotaomicron cultures were grown in four different concentrations of thiamine (0, 10, 100, and 10,000 nM). In all the B. thetaiotaomicron cultures tested the cofactor form TPP is the predominant moiety, averaging between 100,000 and 250,000 molecules per cell (FIG 2.9A) and constituting 70 – 87% of the total thiamine (FIG

2.9B). Therefore, despite a 10,000x increase in the amount of exogenous thiamine, the internal concentration changes no more than 2.5-fold. As such, thiamine moieties appear to be maintained within some homeostatic range. However, as exogenous thiamine levels increase we did detected marginally significant increases in thiamine and TMP concentrations (FIG 2.9A). While TMP maintains 9 – 14% of the thiamine pool in spite of its increased concentrations, thiamine itself increases from ~2 to 16% of the total pool

(FIG 2.9B). These data suggest thiamine may be entering the cell via non-specific

53 mechanisms, the same mechanisms responsible for the ability of the ∆∆Transport and

∆∆∆ mutant to grow in 10,000 nM thiamine. As a result, this excess thiamine may impede or exceed the capacity of the thiamine pyrophosphorylase, Tnr3.

Identification of thiamine acquisition genes in gut Bacteroidetes

To expand upon the knowledge gained from our experiments in B. thetaiotaomicron we interrogated 641 genomes from 5 Phyla and 11 Classes of gut bacteria for the presence of thiamine-dependent enzymes. Every one of the 641 genomes encoded at least one thiamine-dependent enzyme (FIG 2.10A). We then determined the route by which these bacteria acquire thiamine by examining their genome’s ability to synthesize, transport, or recycle thiamine. Only 48% (307/641) genomes investigated have readily identifiable, complete thiamine biosynthesis pathways, implying that many gut microbes likely compete with one another and/or the host for this essential cofactor

(FIG 2.10A).

All of the gut Bacteroidetes genomes examined exhibit evidence of requiring thiamine (n=114). This includes encoding one or more putatively thiamine dependent enzymes (� = 13 ± 2 genes) or a thiamine binding riboswitch (� = 2 ± 1). As such, thiamine biosynthesis and transport genes occur in 94% (107/114) of gut associated

Bacteroidetes (FIG 2.10B). A minority of sequenced Bacteroidetes from the gut encode biosynthesis (n=2) or transport (n=5) alone.

Thiamine biosynthesis occurs in one of two configurations in gut associated

Bacteroidetes. The majority of genomes including B. thetaiotaomicron have a pathway in

54 which 4-amino-2-methyl-5-diphosphomethylpyrimidine (HMP-PP) is synthesized by

ThiC and ThiD (Blue in FIG 2.10B,C) and 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate (cThz-P) is produced by ThiSFGH-TenI (Orange in FIG 2.10B,C) (15). These precursors are condensed into thiamine monophosphate by ThiE or YjbQ (Tan in FIG

2.10B,C), which is then phosphorylated into the biologically active form of thiamine pyrophosphate by ThiL (Green in FIG 2.10B,C). A handful of gut associated

Bacteroidetes have replaced the canonical cThz-P biosynthetic branch with a THI4-like thiazole synthase enzyme (Fuchsia in FIG 2.10B,C) (13/109; E.C. 2.8.1.10) (FIG

2.10B,C). Regardless, in all 109 genomes encoding a major biosynthetic operon also encode a TPP riboswitch immediately upstream (28).

None of the gut associated Bacteroidetes possess a recognizable thiamine ABC transport system similar to the characterized ThiQP-TbpA of Gram negative bacteria or

YkoEDC of Gram positive bacteria (28, 41). Moreover, Bacteroidetes lack the ThiT thiamine transporter and the CytX and ThiW precursor transport proteins (28). Thus like

B. thetaiotaomicron, the previously uncharacterized inner and outer membrane transporters (PnuT and OMthi) are widely conserved in gut Bacteroidetes with more than three quarters (91/114) of the genomes analyzed encoding both OMthi and pnuT (FIG

2.10B). However, heterogeneity in the presence and genomic organization of these genes exists. While B. thetaiotaomicron encodes a split operon most genomes encode OMthi, pnuT, and tnr3 as a single operon and others have apparently lost some or all of these genes (e.g., B. coprocola). In most instances the TPP riboswitch is retained and sometimes duplicated when the operon is split.

55 Discussion:

Thiamine is an essential cofactor for all living organisms; as such its availability impacts human health as well as that of our microbiota. Here, we have identified substantial genetic diversity within and among gut bacterial phyla for acquiring thiamine suggesting diverse strategies are employed leading to opportunities for cooperation and/or competition (FIG 2.10A). Among the Bacteroidetes a ubiquitous and abundant gut phylum responsible for carbohydrate fermentation, fatty acid production, and immune function (17, 44), we identified three distinct strategies for thiamine acquisition: biosynthesis and transport, biosynthesis alone, and transport alone. We analyzed the expression profiles, dissected the genetics, and the competitive advantages of these strategies in B. thetaiotaomicron demonstrating the importance of both thiamine biosynthesis and transport.

Our results demonstrate that the biosynthesis of thiamine is essential for growth and competition of B. thetaiotaomicron in environments with limited thiamine availability. Like many Bacteroidetes, B. thetaiotaomicron encodes a major biosynthetic gene operon (BioThi: BT0653-BT0647), which includes essential enzymes for both

HMP-PP and cThz-P production and condensation (FIG 2.10B, FIG 2.1A). Our genome- wide expression profiling identified evidence of thiamine-dependent regulation of this locus that we attribute to the TPP binding riboswitch preceding this operon. This expression response is consistent with re-analysis of microarray data from B. thetaiotaomicron grown both in vitro and in vivo under thiamine variable conditions (FIG

2.2) (33). Subsequent deletion of the entire biosynthetic operon in B. thetaiotaomicron renders it auxotrophic for thiamine, severely impairing growth in media with less than 10

56 nM of thiamine. Both the genetic organization of the locus and a similar growth defect of a thiC deletion mutant are seen in S. enterica (25). Moreover, the defect in B. thetaiotaomicron, which can be complemented by restoring the entire biosynthetic operon in trans, is recapitulated during co-culture with wildtype cells. Competitions result in the ∆BioThi mutant being outcompeted by the wildtype cells during a multi-day co-culture experiment in low or no thiamine concentrations. We note that thiamine availability in the gut is expected to fluctuate due to factors including host genetics, diet, and intestinal adsorption. Measurements of thiamine in the intestinal lumen range between 20 – 2,000 nM (29). As such, a gut microbe’s ability to synthesize its own thiamine is likely most important during transient drops in thiamine availability to enable the microbe's own growth. We predict that gut microbes that we’ve identified that are wholly dependent on thiamine transport such as members of the genus Alistipes and many members of the Bacilli (FIG 2.10), to be adversely affected during such drops in thiamine availability.

Our global transcriptomic analysis revealed differential responses of thiamine acquisition operons to exogenous thiamine. In conditions of excess thiamine, we’ve shown that B. thetaiotaomicron down-regulates the BioThi operon. However, we found that transcripts for the putative inner (pnuT) and outer membrane (OMthi) transporter genes adjacent to TPP binding riboswitches show minimal expression differences (FIG

2.1A). These proteins were previously predicted to function in thiamine transport (28) and PnuT was recently shown to transport thiamine in a heterologous host (18).

Curiously, individual deletions of these transporters have no effect on the growth of mutants individually or in co-culture with wildtype cells. However, deletion of both

57 transport genes in tandem yielded a severe growth defect (FIG 2.3). After ruling out an additional role of these transporters for importing a critical precursor (FIG 2.4), we speculate that they may play a role in thiamine recycling or that there is an uncharacterized connection between biosynthesis and transport of thiamine. When we measured expression of the BioThi operon in the double transport mutant using RT-qPCR we found expression levels of thiC and thiS were down regulated (FIG 2.6A,B). This raises the possibility that an unknown protein regulatory mechanism may be dysregulated and repressing biosynthesis in the ∆∆Transport mutant. Regardless, further investigations are warranted to disentangle thiamine biosynthesis, transport, and their interconnected pathways.

The dual transport and biosynthesis pathways appear to facilitate the ability of wildtype B. thetaiotaomicron to maintain a fairly stable intracellular concentration of the cofactor TPP (< 3-fold variation) despite radical changes in extracellular thiamine concentrations. However, these increases in intracellular concentrations of the thiamine moieties are sufficient to lead to the down-regulation of the BioThi operon in addition to a suite of thiamine-dependent pathways including amino acid metabolism and nucleotide biosynthesis. It is possible that the increased proportion of thiamine to TPP may provide a signal sensed by unknown regulatory mechanisms, outside of the regulatory riboswitches that control biosynthesis and transport genes. These additional regulatory factors are likely important for modulating responses to the stress of variable levels of exogenous thiamine.

We observed that competitions between B. thetaiotaomicron and thiamine acquisition mutants yielded similar results to growth of the mutants in isolation (FIG 2.3,

58 FIG 2.7) suggesting that B. thetaiotaomicron does not release thiamine into the environment. Confirming this hypothesis, no thiamine was detected in the supernatant of spent cultures of B. thetaiotaomicron grown in thiamine free medium. This is not in line with previously published data in which thiamine was still detectable in the feces of a select group of humans were that were completely thiamine starved (21). It was inferred that the thiamine detectable in these patients was derived from their microbial communities (21). This leads us to speculate that other gut microbes including divergent

Bacteroidetes or release thiamine, as they are the most prevalent thiamine synthesizers. Alternatively, host-driven cell lysis (40), microbe-microbe antagonism and/or intoxication (43), or some gut-specific signal results in the liberation of this essential cofactor.

As an essential small molecule, thiamine appears to have an important role in the persistence of microbes in the gut. Given the universal requirement for this cofactor and the diverse strategies for acquisition results in scenarios that would force competition or cooperation among gut microbes. Among B. thetaiotaomicron mutants encoding a single acquisition strategy we have observed competitive defects, similar to what has been seen for B12 transport (4). However, more complex communities may be crucial to enabling cooperation, thus modeling the dynamics of thiamine in natural or reconstructed gut communities will be a way forward to understand these dynamics. It thus remains possible that cofactors such as thiamine may be utilized to manipulate gut communities.

Ultimately such manipulation may play a role in the combating of disease such as diabetes, obesity, and cancer.

59 Methods:

Bacterial culturing and genetic manipulation

Routine culturing of B. thetaiotaomicron VPI-5482 occurred anaerobically at

37°C in liquid tryptone yeast extract glucose (TYG) medium (11) or Difco brain heart infusion (BHI) agar with the addition of 10% defibrinated horse blood (QuadFive,

Ryegate, MT, USA). Cultures were grown anaerobically within a vinyl anaerobic chamber with an input gas mix consisting of 70% nitrogen, 20% carbon dioxide, and 10% hydrogen (Coy Laboratory Products, Grass Lake, MI, USA). Escherichia coli S17-1 λ pir strains used for cloning and conjugation of suicide vectors were grown in LB medium at

37°C aerobically. Antibiotics were added to the medium when appropriate in the concentrations as follows: ampicillin 100 µg/mL, gentamicin 200 µg/mL, erythromycin

25 µg/mL, tetracycline 2 µg/mL, and 5-fluoro-2’-deoxyuridine (FUdR) 200 µg/mL.

Markerless deletions were generated via allelic exchange and confirmed by PCR

(4, 16). Briefly, HiFi Taq MasterMix (KAPA Biosystems, Wilmington, MA, USA) was used to amplify right and left flanks of thiamine associated loci by PCR, which were then combined using splicing by overlap extension (SOE) PCR. Purified SOE products were restriction digested and cloned into pExchange_tdk vector using standard methods (30).

Sequence confirmed vectors were then conjugated from donor E. coli S17-1 λ pir strains into recipient B. thetaiotaomicron strains. Transconjugants were colony purified and screened for successful mutants by PCR (Table 2.2). Complementation and barcoded vectors were all constructed in pNBU2 vectors and were introduced into the genome in a single copy (4, 20). Construction and conjugation of pNBU2 vectors was done by the

60 same methods as pExchange_tdk vectors. A complete list of primers and vectors used for this study are provided (Table 2.4).

Thiamine transcriptomic response

A modified minimal medium was use for all thiamine growth assays (after Varel and Bryant (39), Scholle et al. (31)). The modified minimal medium consisted of glucose

(27.8 mM), ammonium chloride (6. 0 mM), L-cysteine free-base (4.1 mM), sodium carbonate (3.8 mM), iron sulfate (0.3 mM), vitamin B12 (5.8 µM), a histidine-hematin solution (0.1% of 1.9 mM hematin in 0.2 M histidine), 1 M potassium phosphate buffer at a pH of 7.2 (2%), and a premixed mineral solution (5%). The mineral solution consisted of 3 M sodium chloride, 3.6 mM of calcium chloride dehydrate, 1.6 mM of magnesium sulfate, 1 mM of manganese chloride tetrahydrate, and 84.1 µM of cobalt chloride hexahydrate. Replicate cultures of wildtype B. thetaiotaomicron were grown overnight in

5 mL minimal medium supplemented with 10,000 nM thiamine HCl (THI, > 99% pure)

(Sigma Aldrich, St. Louis, MO, USA). Aliquots of each culture were pelleted by centrifugation (1 min at 13,300 x g), culture supernatants were decanted and the cells were washed 4 times in minimal medium with 0 nM thiamine. Cells were inoculated into

10 mL of minimal medium with either 0 or 15 µM thiamine HCl at a final dilution of

1:2,000 in biological duplicate. Cell growth was monitored and cells were harvested between OD600 0.4 and 0.6 on a UV spectrometer. Total RNA was extracted using the

Qiagen RNeasy kit and stored at -80°C. Total RNA was DNAse treated with DNase I

(Thermo Fisher, Waltham, MA, USA) and NEB DNase treatment protocol. DNAse treated RNAs were re-cleaned using the Qiagen RNeasy kit (Hilden, Germany) and

61 quantitated using a Qubit 2.0 (Life Technologies, Carlsbad, CA, USA) and stored at -

80°C.

RNA was submitted for integrity analysis, ribosomal RNA depletion, and library construction at the W.M. Keck Center for Comparative and Functional Genomics at the

University of Illinois at Urbana-Champaign.

For targeted gene expression analysis, total RNA was purified as above and used to generate cDNA libraries using the first strand cDNA synthesis kit (Thermo Fisher,

Waltham, MA, USA). RT-qPCR was performed on a Biorad CFX connect instrument

(Biorad, Hercules, CA, USA) and SYBR Fast MasterMix 2x Universal (Kapa

Biosystems, Wilmington, MA, USA) following the manufacturer’s instructions for triplicate biological samples in technical triplicate. Four genes were amplified 16 ribosomal RNA, thiC, thiS and tnr3. 16S rRNA primers were used as the control gene (4) and novel primers for thiC, thiS and tnr3 were designed using Primer3 (38). Standard curves were used to evaluate the efficiency of the amplification; all 4 genes had R2 ≥

0.985 and slope between -3.33 and -3.40. Relative expression changes were calculated using the ∆∆Cq method (2).

RNA sequencing analysis

RNAseq reads from each sample were quality filtered using a custom R script and fastx_clipper (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Filtered RNAseq data were analyzed using the Rockhopper program (35) to identify differentially expressed genes between thiamine replete and deficient samples. Reads were aligned to B.

62 thetaiotaomicron VPI-5482 genome and significantly differentially expressed genes were identified (≥ 2-fold change and q value ≤ 0.05).

Re-analysis of microarray data

To test for evidence of transcriptional changes in response to thiamine availability we re-analyzed existing microarray data from B. thetaiotaomicron growing both in vitro and in vivo (33). Briefly, microarrays had been hybridized with total RNA recovered from wildtype B. thetaiotaomicron grown to mid-log phase in biological duplicate in minimal medium with no thiamine added (8 h), in TYG that has excess thiamine (3.5 h) or from the ceca of two monoassociated gnotobiotic NMRI mice (GSE2231) (33). Raw

GeneChip data were normalized and model-based expression values (PM-MM model) were compared using DNA-Chip Analyzer (v2011.05) as described (33). Mean and standard deviations of the normalized expression values were calculated and significantly differentially regulated genes were detected with t-test, p < 0.05 using FDR correction.

Assaying thiamine dependent growth phenotypes

B. thetaiotaomicron wildtype, mutant, and complemented strains were grown and washed in minimal medium 0 µM thiamine as above for RNAseq. Cells were normalized, diluted to 0.0004 OD600 and dispensed into 96 well plates with thiamine HCl, thiamine monophosphate HCl (TMP, > 95% pure) (Sigma Aldrich, St. Louis, MO, USA), and thiamine pyrophosphate chloride (TPP, > 98%) (Thermo Fisher Scientific, Waltham,

MA, USA) at concentrations ranging from 0-10,000 nM (modified from Degnan et al.

(4)). Cell growth was measured over the course of 25 hours using BioTek Synergy HTX

63 Multi-Mode Microplate Reader in conjunction with a BioStack3 microplate stacker

(BioTek, Winooski, VT, USA). All phenotypic assays were averaged from three to nine biological replicates with three technical replicates each. All phenotypes were calculated as a percentage of WT growth achieved in minimal medium + 10,000 nM THI at 17.5 hours. Pairwise student t-tests were carried out using GraphPad Prism v6 and p values ≤

0.05 were considered significant.

In vitro competition assays

Cells were grown for 16 hours in minimal medium supplemented with 10,000 nM

Thiamine HCl. Cells were pelleted and washed 4 times with minimal medium. Cell

OD600 were normalized to 0.01. Cells were inoculated 1:100 into an Axygen deep-well plate (Corning Inc., Corning, NY, USA) in biological triplicate for each mutant at 4 different concentrations (0, 10, 100, and 10,000 nM) of thiamine HCl in minimal medium. Cultures were incubated anaerobically at 37°C for 24 hours and passaged

1:1,000 into the same media type they were previously grown in (4, 20). Time points were taken for 4-5 days and saved in media +20% glycerol at -80°C. Samples were prepped for genomic DNA (gDNA) using the HotShot method (36) and relative abundance of each barcoded strain was determined using qPCR in reference to a standard curve on a Biorad CFX connect instrument (Biorad, Hercules, CA, USA) and SYBR Fast

MasterMix 2x Universal (Kapa Biosystems, Wilmington, MA, USA) (4). Data were analyzed using the efficiency corrected ∆Cq method (2).

64 Quantification of thiamine in B. thetaiotaomicron and medium

B. thetaiotaomicron was grown in either 0 nM, 10 nM, 100 nM, or 10,000 nM of thiamine HCl were grown to mid log (OD600 ~0.5). Cells were pelleted at 10,000-x g for

2 minutes. Cells were washed three times with PBS buffer. Cells and media aliquots were submitted to the Roy J. Carver Biotechnology Center’s Metabolomics Center at the

University of Illinois at Urbana-Champaign for quantification of thiamine, thiamine monophosphate, and thiamine diphosphate. Samples were analyzed with the 5500

QTRAP LC/MS/MS system (Sciex, Framingham, MA, USA). Software Analyst 1.6.2 was used for data acquisition and analysis. The 1200 series HPLC system (Agilent

Technologies, Santa Clara, CA, USA) includes a degasser, an autosampler, and a binary pump. The LC separation was performed on a Phenomenex Polar column (4.6 x 100 mm,

4 µm) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). The flow rate was 0.4 mL/min. The linear gradient was as follows: 0-3 min, 95%A; 6-9 min, 0%A; 10-15 min, 95%A. The autosampler was set at

10°C. The injection volume was 100 µL. Mass spectra were acquired under positive electrospray ionization (ESI) with the ion spray voltage of +5000 V. The source temperature was 450°C. The curtain gas, ion source gas 1, and ion source gas 2 were 33,

50, and 65, respectively. Multiple reaction monitoring (MRM) was used for quantitation: thiamine m/z 265.0 m/z 122.0; thiamine monophosphate m/z 345.1 m/z 122.0; and thiamine diphosphate m/z 425.1 m/z 122.0.

65 Computational prediction of thiamine-associated genes

Thiamine biosynthesis, transport, and dependent genes were identified searching for gene specific TIGRFAM (32) when available or PFAM (7) hidden Markov models with HMMR (--tc_cut) (Table 2.5) in 641 gut bacteria including 114 Bacteroidetes genomes available in RefSeq from the Human Microbiome Project (Table 2.6) (24, 26).

Predicted annotations were confirmed with BLAST and PFAM hidden Markov models

(7, 1). Homologs of B. thetaiotaomicron VPI-5482 loci were confirmed and compared among the other 113 Bacteroidetes genomes using a reciprocal best BLAST approach. In addition, Infernal 2.0 was used to detect putative TPP riboswitches adjacent to thiamine- associated genes (RF00059) (9, 22). The presence or absence of biosynthetic and transporter genes were used to categorize the route of thiamine acquisition. Microbes capable of complete ‘biosynthesis’ have the ability to synthesize both HMP-PP (thiC or thi5 and thiD), cThz-P (thiGH or thi4 in addition to tenI), possesses thiamine condensation genes (thiE and/or yjbQ), and a thiamine kinase. Microbes categorized as capable of ‘recycling’ lack the ability to completely synthesize either thiazole and/or

HMP-PP de novo, but retain the thiamine kinase enzymes. Microbes were categorized as capable of ‘transport’ if any gene predicted to transport thiamine was identified. Finally, microbes were categorized as ‘incomplete/uncertain’ if we were unable to identify any thiamine transporters and/or sufficient genes to carry out thiamine biosynthesis or recycling. Additionally, using an identical approach as described in Degnan et al. (4) a set of 13 core genes conserved among all three domains of life were identified in the 641 gut microbial genomes, aligned and a phylogeny was reconstructed using the maximum likelihood method.

66 Acknowledgements:

We thank members of the Degnan and A. K. Hansen labs for comments on the manuscript. We also thank Zhong (Lucas) Li from the Roy J. Carver Biotechnology

Center’s Metabolomics Center and Alvaro Hernandez from the W.M. Keck Center for

Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign for their technical services.

67 Figures:

FIG 2.1. Transcriptomic response of B. thetaiotaomicron to exogenous thiamine

(A) Schematic of TPP riboswitch regulated operons is shown with expression fold changes indicated and colored according to the heat map. Asterisks (*) indicate individual genes that are significantly down regulated (q value ≤ 0.05) in the presence of thiamine. (B) Genome-wide expression response of B. thetaiotaomicron to thiamine.

Gray circles indicate genes that are not significantly up- or down-regulated, red circles are genes that are significantly down regulated in the presence of thiamine and green circle are significantly up regulated (q value ≤ 0.05). (C) KEGG gene clusters that contain genes that are significantly down regulated. Numbers to the right of each KEGG pathway denote the number of genes down regulated in each category. Underlined categories indicate KEGG pathways that are significantly down regulated through GSEA analysis (p value ≤ 0.05).

68

FIG 2.2. Microarray data of thiamine biosynthesis and transport genes

Expression of B. thetaiotaomicron genes involved in thiamine biosynthesis and transport during growth with known or assumed thiamine availability. Asterisks and brackets indicated significant expression differences between samples (FDR corrected t-test, p <

0.05) and shaded areas indicate genes encoded in the same operon.

69

FIG 2.3. Growth phenotypes of B. thetaiotaomicron thiamine acquisition mutant

Thiamine acquisition operons have differential effects on the ability of B. thetaiotaomicron to grow in thiamine limiting conditions. (A) A representative growth curve of wildtype and ∆BioThi showing growth defects of ∆BioThi in a thiamine deficient environment (0 nM) and a thiamine replete environment (10,000 nM). Arrow indicates maximal growth point at ~17.5 hrs. used to calculate percent growth of WT in minimal medium supplemented with 10,000 nM thiamine to compare growth phenotypes across concentrations of thiamine in panels B-D. (B) Single mutants of the outer membrane transporter (∆OMthi), and inner membrane transporter (∆pnuT) loci showed no defect, while ∆BioThi strains were variably attenuated in thiamine concentrations less than 100 nM and a complement of ∆BioThi with the entire loci with the native promoter expressed in trans can fully complement the mutant. (C) Mutants lacking biosynthesis

70 FIG 2.3. (cont.)

(∆BioThi) and a single transport gene (pnuT or OMthi) have severe growth phenotypes below 100 nM thiamine, but can complemented with the biosynthesis locus under its native promoter to wildtype or near wildtype levels. (D) The double transport mutant

(∆∆Transport) and triple locus mutant (∆∆∆) were both highly attenuated in thiamine concentrations less than 1,000 nM. Complement of single transporters in the ∆∆Transport background can partially rescue the severe growth phenotype.

71 FIG 2.4. Putative B. thetaiotaomicron thiamine transporters are specific for thiamine

The specificity of the thiamine dependent phenotypes of the thiamine acquisition mutants were tested using media supplemented with thiamine precursors. To disentangle cysteine’s role as a sulfur source and reducing agent, growth analyses were carried out with medium supplemented (A) cysteine and thioglycolate or (B) Na2S and thioglycolate.

Other commercially available thiamine precursors (C) Tyrosine, (D) GAP, and

72 FIG 2.4. (cont.)

(E) Thiazole were also analyzed to determine if they could rescue thiamine growth phenotypes. In all panels thiamine was either at (+) 10,000 nM or (-) 0 nM in the and experiments were performed in biological duplicate.

73

FIG 2.5. Growth phenotype of ∆∆Transport double transport Complement

∆∆Transport mutant was complemented with both the inner (pnuT) and the outer membrane transporter (OMthi) under the natural promoter of pnuT and expressed at a non-native locus in the ∆∆Transport mutant. The double complement allows for a marked improvement in growth at low nanomolar concentrations of thiamine and partially relieves the fitness defect that we observe in the ∆∆Transport mutant.

74

FIG 2.6. Analysis of thiamine biosynthesis in wildtype and double transport mutant

75 FIG 2.6. (cont.)

Expression of biosynthesis was quantitated via RT-qPCR of (A) thiC and (B) thiS in both wildtype and double transport mutant backgrounds of B. thetaiotaomicron at 0, 100, and

10,000 nM concentrations of thiamine for wildtype and 100 and 10,000 nM concentrations of thiamine for the double transport mutant. Expression of tnr3 was also measured in wildtype and double transport mutant at 0 and 10,000 nM thiamine (C) in order to ensure that the double transport mutant did not have a polar effect on downstream gene. N.G. means No Growth was achieved to carry out the experiment.

N.T. means Not Tested at a certain concentration.

76

FIG 2.7. Single competitions between wildtype and thiamine acquisition mutants

Competitions between wildtype B. thetaiotaomicron and thiamine acquisition mutants

(A) ∆OMthi, (B) ∆pnuT, (C), ∆BioThi, (D) ∆∆BioThi-OMthi, (E) ∆∆BioThi-pnuT, (E)

∆∆Transport, and (F) ∆∆∆ mutant were performed over 4 or 5 days in defined thiamine concentrations.

77 FIG 2.7. (cont.)

Strain abundances were quantified by qPCR and represented as a percentage of the entire community at 4 concentrations of thiamine (0, 10, 100, and 10,000 nM). Error bars represent standard error of the mean for three biological replicates.

78

FIG 2.8. Barcoded competitions of individual acquisition mutants and wildtype cells

79 FIG 2.8. (cont.)

Abundance of wildtype (WT) and thiamine acquisition mutants during multiday competitions was tracked using qPCR. Wildtype B. thetaiotaomicron was competed against (A) ∆BioThi, (B) ∆pnuT, (C) ∆OMthi, (D) ∆∆Transport, (E) ∆∆∆ mutants, (F)

∆∆BioThi-OMthi, and (G) ∆∆BioThi-pnuT in 0 nM, 10 nM, 100 nM, and 10,000 nM thiamine. All competitions were performed in biological triplicate and error bars represent standard error of the mean.

80

FIG 2.9. Quantification of thiamine pools in B. thetaiotaomicron

(A) Intercellular quantification of thiamine and its phosphorylated moieties indicate that total quantities of thiamine, TMP, and TPP increase to varying degrees as exogenous thiamine is increased. Values represent the average and standard error of the mean for six biological replicates. Letters for individual moieties indicate groups that are not significantly different from each other (p value ≤ 0.05, Tukey’s HSD test). (B) The proportions of thiamine and TMP increase as the concentration of exogenous thiamine increases, while the proportion of TPP decreases.

81 Route of A Require Thiamine Require Thiamine Thiamine Acqusition No Thiamine Bacteroidia (114) Required

Deltaproteobacteria (6) Transport only Transport & Recycling (112) Transport & Biosynthesis Biosynthesis only (12) Incomplete/Uncertain (93)

Fusobacteria (19)

Negativicutes (10)

Clostridia (109)

Erysipelotrichi (12)

Bacilli (109)

Actinobacteria (45)

THI B TPP Biosynthesis Transport thi4 thiC thiD thi5 thiS thiH thiG tenI yjbQ thiE thiL tnr3 pnuT OMthi Bacteroides fragilis (16) B. nordii B. salyersiae B. thetaiotaomicron (5) B. faecis B. caccae (2) B. finegoldii (2) B. ovatus/xylanisolvens (15) B. oleiciplenus B. intestinalis B. cellulosilyticus (3) B. eggerthii (2) B. stercoris B. clarus B. uniformis (5)* B. fluxus B. helcogenes B. coprophilus B. plebeius B. salanitronis B. coprocola B. vulgatus (5) B. dorei (7) Paraprevotella xylaniphila Pp. clara Prevotella salivae P. copri P. stercorea Parabacteroides merdae (3) Pb. johnsonii (2) Pb. goldsteinii Pb. distasonis (9) Tannerella forsythia Porphyromonas gingivalis (3) Tannerella. sp. mossii Odoribacter splanchnicus O. laneus Bacteroides sp. Alistipes indistinctus A. putredinis A. finegoldii (2) 0.2 a.a. substitutions Alistipes sp. per site A. shahii

Gene present for: Gene absent: HMP Biosynthesis Biosynthesis (Condensation) Bacterial Thiazole Biosynthesis Kinase / Phosphatase Yeast Thiazole Biosynthesis Transport

C Glycolysis

Purine Vitamin B6 GAP Metabolism Metabolism Dxs AIR PLP 2.2.1.7 Pyruvate Cys

ThiC THI5 4.1.99.17 -.-.-.- HMP-PP ThiG ThiI SufS 2.8.1.10 -.-.-.- 2.8.1.7 ThiD 2.7.4.7 IscS-SH TenA TenI 2.7.1.49 ThiE/YjbQ cThz*-P 5.3.99.10 ThiF 2.5.1.3 2.7.7.73 ThiH ThiS-SH 4.1.99.19 ThiM -.-.-.- 2.7.1.50 ThiK TMP Gly 2.7.1.89 Tyr ThiL TenA TH-1 2.7.4.16 THI4 3.5.99.2 2.5.1.2 NTPCR 2.8.2.10 3.6.1.15 Tnr3 2.7.6.2 NAD+ Tnr3 THTPA THI 2.7.6.2 TPP 3.6.1.28 TTP FIG 6 FIG 2.10. In silico predictions of thiamine acquisition pathways

82 FIG 2.10. (cont.)

(A) Schematic phylogeny of 11 classes of cultivable gut associated bacteria and corresponding pie charts indicating the proportion of genomes that encodes a thiamine dependent enzyme and predicted thiamine acquisition strategies. Parentheses represent number of genome sequences in each class. (B) Presence and absence of predicted thiamine biosynthesis and transport genes in diverse gut Bacteroidetes are indicated. The phylogeny represents a maximum likelihood reconstruction gut Bacteroidetes species.

Numbers in parentheses represent the number of individual strains analyzed and nodes marked with black circles indicate bootstrap values ≥ 75. The asterisk (*) indicates that one of the five B. uniformis genomes analyzed is missing pnuT and tnr3, but this may be an assembly artifact. (C) Schematic representation of thiamine biosynthesis in the

Bacteroidetes. Arrow colors correspond to the key in (B). Gene names in grey are missing and names in green are genes that are not solely dedicated to the biosynthesis of thiamine.

83 Tables:

TableTable S1. Samples2.1. usedSa mplesfor RNA sequencing used experiment. for RNA sequencing Quality Filtered Reads Mapped to Media/Replicate/Description Intial file name Raw Reads Reads % filtered NC_004663 % mapped Bt Minimal Media Replicate #1 8_4_GACGACAT_L002_R1_001.fastq 9,170,438 8,231,596 90% 7,712,374 94% Bt Minimal Media Replicate #2 9_4_TACAGCAT_L002_R1_001.fastq 8,258,740 7,454,637 90% 6,830,124 92% Bt Minimal Media plus 15 µM Thiamine Replicate #1 8_4T_TCGAAGAT_L002_R1_001.fastq 9,017,267 8,169,029 91% 7,783,046 95% Bt Minimal Media plus 15 µM Thiamine Replicate #2 9_4T_ATCACGAT_L002_R1_001.fastq 8,444,554 7,652,018 91% 7,144,413 93%

84 Table 2.2. Deletion loci and complements generated in this study

Description

'wildtype' deletion background, ∆BT2275, FUDRR, WT intact thiamine biosynthetic and transport operons (Martens et al. 2008)

Deletion mutants deletion of thiamine biosynthetic operon thiSEGCHF- ∆BioThi tenI, BT0647-0653 (797,661 - 803,616) deletion of putative outer membrane, TonB- ∆OMthi dependent thiamine transporter BT2390 (2,977,834 - 2,980,058) deletion of putative inner membrane thiamine ∆pnuT transporter BT2396 (2,992,218 - 2,992,778)

∆∆BioThi-OMthi deletion of BT0647-0653 and BT2390

∆∆BioThi-pnuT deletion of BT0647-0653 and BT2396

∆∆Transport deletion of BT2390 and BT2396

∆∆∆ deletion of BT0647-0653, BT2390, and BT2396

Complementation constructs

ΔBioThi + pBioThi BT0647-0653 complemented in trans

ΔΔBioThi-OMthi +pBioThi BT0647-0653 complemented in trans

ΔΔBioThi-pnuT + pBioThi BT0647-0653 complemented in trans

ΔΔTransport + pOMthi BT2390 complemented in trans ΔΔTransport + ppnuT BT2396 complemented in trans

85 Table 2.3. Effective concentrations for 50% WT growth represented in nM of thiamine requiredTable S2. Effective concentrations for 50% WT growth represented in nM of thiamine required.

1 EC50 [nM] (95% CI)

Strain THI TMP TPP WT 0.0 - 0.0 - 0.0 - ΔBioThi 2.8 (1.4 - 5.5) 89.7 (47.5 - 169.3) 108.9 (Wide) ΔBioThi pBioThi 0.0 - n.t. - n.t. - ∆pnuT 0.0 - 0.0 - 0.0 - ∆Omthi 0.0 - 0.0 - 0.0 - ΔΔBioThi-OMthi 15.8 (8.1 - 31) 91.6 (51.4 - 163) 139.9 (Wide) ΔΔBioThi-OMthi pBioThi 0.0 - n.t. - n.t. - ΔΔBioThi-pnuT 10.9 (9.2 - 13) 39.8 (27.4 - 58) 135.3 (90.1 - 203.5) ΔΔBioThi-pnuT pBioThi 0.0 - n.t. - n.t. - ΔΔTransport 30.3 (22.8 - 40.3) 141.2 (101.7 - 196.3) 221.4 (140.4 - 349.3) ΔΔTransport pOMthi 1.9 (0.8 - 4.4) n.t. - n.t. - ΔΔTransport ppnuT 0.1 (0 - 1.9) n.t. - n.t. - ΔΔTransport ppnuT+Omthi 0.1 (0 - 0.2) n.t. - n.t. - ΔΔΔ 51.9 (42.4 - 63.6) 103.9 (Wide) 566.3 (282.5 - 1135) 1n.t. = not tested, - = indeterminate, "Wide" = too broad to calculate

86 TableTable S3. Primers, 2.4. vectors, andPrimers, strains used in this study. vectors, and strains

Locus of Primer in Bt genome Primer, Plasmid, or Strain name Oligonucleotide sequence (5'-3')/Notes1 Citation (NC_004663) SOE Primers ZC129 / 5' RF BT0647-0653 XbaI atcggTCTAGAggtgcctttctttcaggcatatcgg This study 804720 - 804744 ZC130 / 3' LF BT0647-0653 BamHI gaacgGGATCCccgggtatcgctgttaaactgaacg This study 796651 - 796675 ZC131 / 5' LF BT0647-0653 SOE cgctctttgtatttactttcaacttattaatactgataggacaatgtaagaaatatcgattcgaatattatatacaatcataaataaagaaaccgctg This study 803617 - 803662 / 797607 - 797660 ZC132 / 3' RF BT0647-0653 SOE cagcggtttctttatttatgattgtatataatattcgaatcgatatttcttacattgtcctatcagtattaataagttgaaagtaaatacaaagagcg This study 797607 - 797660 / 803617 - 803662 ZC133 / 3' LF BT2390 BamHI tcaccGGATCCccccagtaatctgccgtattcacc This study 2976784 - 2976808 ZC134 / 3' LF BT2390 SOE ggcggcttctgtcttatctgtttgaatggttattgttacatcttgcttattaattagttctctacgc This study 2980059 - 2980097 / 2977805 - 2977833 ZC135 / 5' RF BT2390 SOE gcgtagagaactaattaataagcaagatgtaacaataaccattcaaacagataagacagaagccgcc This study 2977805 - 2977833 / 2980059 - 2980097 ZC136 / 3' RF BT2390 XbaI gatccTCTAGAgggggaatatgaaatggtacctgatcc This study 2980996 - 2981022 ZC137 / 5' LF BT2396 BamHI tcacgGGATCCgctacagtaacgaagaagtgaaaacttatttcacg This study 2991224 - 2991258 ZC138 / 3' LF BT2396 SOE gacggcttggggtatatagtgttcgtttatcatcatttccatatttgcttatttattcgttctctacgc This study 2992779 - 2992810 / 2992188 - 1992217 ZC139 / 5' RF BT2396 SOE gcgtagagaacgaataaataagcaaatatggaaatgatgataaacgaacactatataccccaagccgtc This study 2992188 - 1992217 / 2992779 - 2992810 ZC140 / 3' RF BT2396 XbaI aacgcTCTAGAgcatctttaagctctgcacgtaaacgc This study 2993749 - 2993775

Complementation Primers Bio_F_Nat_NdeI tcgcCATATGcggataatttgatgaatctttgtttctttttcgc This study 803790 - 803823 Bio_R_XbaI aatgTCTAGAttagtccgccaatttctttaactttttgaaatg This study 797658 - 797690 OMthi_F2_BamHI cgtcGGATCCatgaaaaaaatgatgatgatggccgtc This study 2977831 - 2977857 OMthi_R2_PstI ggccCTGCAGttaaaagcggaaagagacatggcc This study 2980039 - 2980062 pnuT_F2_BamHI tacgGGATCCatggaaatgaattatctggagatattcggtacg This study 2992215 - 2992247 pnuT_R2_PstI gtagCTGCAGtcattgcatgttcattatttttttccatttgaagtag This study 2992745 - 2992781

qPCR Primers ThiC_F gagcagctccgatggcagag This study 800577 - 800596 ThiC_R ggtccgggacacgtaccg This study 800710 - 800727 thiS_F ccattcggaacgtggaatca This study 803482 - 803501 thiS_R cctgcttctacccttacacaac This study 803559 - 803580 tnr3_F tgccaacggtgaatatcctg This study 2992816 - 2992835 tnr3_R gtctccatcgcctataatcacg This study 1882933 - 2992954

Competition Primers NBU2_att1_F cctttgcaccgctttcaacg Martens et al. 2008 6138201 - 6138220 NBU2_att1_R tcaactaaacatgagatactagc Martens et al. 2009 6139105 - 6129127 NBU2_att2_F tatcctattctttagagcgcac Martens et al. 2010 6216940 - 6216961 NBU2_att2_R ggtgtacctggcattgaagg Martens et al. 2011 6217369 - 6217288 UNIVR cacaatatgagcaacaaggaatcc Martens et al. 2008 BC03F ttatgaccagccgcaaatgaaaag This study BC04F ctccataaaggcgcataccgacta Degnan et al. 2014 BC06F gattacggcgtgatagattggtgt Degnan et al. 2014 BC08F cttcctgcccggctaaaaacacga Degnan et al. 2014 BC09F tcaaatccggggactgggcttaga This study BC21F atgttcgatcatcagttcagtagc This study

SOE Plasmids pExchange_tdk Construct carrying cloned tdk (BT2275) for counter-selection, AmpR, ErmR, FUDRS Koropatkin et al. 2008 pExchange_tdk_BT0647-0652_SOE This study pExchange_tdk_BT2390_SOE This study pExchange_tdk_BT2396_SOE This study

Complementation Plasmids pNBU2_erm Plasmids with cloned fragments integrates at NBU2 att1 and/or att2 sites, ErmR, AmpR Koropatkin et al. 2008 pNBU2_erm_NatP_BioThi This study pNBU2_erm_NatP_Omthi This study pNBU2_erm_NatP_pnuT This study

Competition Plasmids pNBU2_tet Plasmid integrates at NBU2 att1 and/or att2 sites, TetR, AmpR Martens et al. 2008 pNBU2_tet_BC03 Martens et al. 2008 pNBU2_tet_BC04 Martens et al. 2008 pNBU2_tet_BC06 Martens et al. 2008 pNBU2_tet_BC08 Martens et al. 2008 pNBU2_tet_BC09 Martens et al. 2008 pNBU2_tet_BC21 This Study

Parental Strains E. coli S17-1 λ pir thi pro hdsR hdsM+ recA, chromosomal insertion of RP4-2(Tc::Mu Km::Tn7) , AmpS Simon et al. 1983 Bt VPI-5482 ∆tdk 'wildtype' deletion background, BT2275–, FUDRR Koropatkin et al. 2008

Deletion Strains Bt VPI-5482 ∆tdk ∆BioThi ∆BioThi : deletion of BT0647-0653 This Study Bt VPI-5482 ∆tdk ∆OMthi ∆OMthi : deletion of BT2390 This Study Bt VPI-5482 ∆tdk ∆pnuT ∆pnuT : deletion of BT2396 This Study Bt VPI-5482 ∆tdk ∆∆BioThi-OMthi ∆∆BioThi-OMthi : deletion of BT0647-0653 in tandem with BT2390 This Study Bt VPI-5482 ∆tdk ∆∆BioThi-pnuT ∆∆BioThi-pnuT : deletion of BT0647-0653 in tandem with BT2396 This Study Bt VPI-5482 ∆tdk ∆∆Transport ∆∆Transport : deletion of BT2390 in tandem with BT2396 This Study Bt VPI-5482 ∆tdk ∆∆∆ ∆∆∆: deletion of BT0647-0653, BT2390, and BT2396 This Study

Complemented Strains Bt VPI-5482 ∆tdk ∆BioThi att2::pNBU2_erm_NatP_BioThi This Study Bt VPI-5482 ∆tdk ∆∆BioThi-Omthi att2::pNBU2_erm_NatP_BioThi This Study Bt VPI-5482 ∆tdk ∆∆BioThi-pnuT att2::pNBU2_erm_NatP_BioThi This Study Bt VPI-5482 ∆tdk ∆∆Transport att2::pNBU2_erm_NatP_Omthi This Study Bt VPI-5482 ∆tdk ∆∆Transport att2::pNBU2_erm_NatP_pnuT This Study

Barcoded Competition Strains Bt VPI-5482 ∆tdk att2::pNBU2_tet_BC03 This Study Bt VPI-5482 ∆tdk ∆BioThi att2::pNBU2_tet_BC04 This Study Bt VPI-5482 ∆tdk ∆Omthi att2::pNBU2_tet_BC06 This Study Bt VPI-5482 ∆tdk ∆pnuT att2::pNBU2_tet_BC08 This Study Bt VPI-5482 ∆tdk ∆∆Transport att2::pNBU2_tet_BC09 This Study Bt VPI-5482 ∆tdk ∆∆∆ att2::pNBU2_tet_BC21 This Study 1Bold denotes restriction enzymes

87 Table 2.5.Table S4.Search Search queries used used to to identifyidentify thiamine-dependent thiamine and thiamine-dependent acquisition genes and thiamine acquisition genes

HMM Description Gene EC Number Biosynthesis PF09084 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase THI5 TIGR00190 Hydroxylmethyl pryimidine synthase ThiC 4.1.99.17 TIGR00097 Hydroxylmethyl pyrimidine (phosphate) kinase ThiD 2.7.4.7 TIGR02352 Glycine oxidase ThiO 1.4.3.19 TIGR02351 Thiazole synthase ThiH 4.1.99.19 TIGR02006 Cysteine desulfurase IscS 2.8.1.7 TIGR01979 Cysteine desulfurase SufS 2.8.1.7 TIGR01683 Sulfur carrier protein ThiS TIGR02354, Adenyltransferase ThiF 2.7.7.73 TIGR02356 TIGR04271, Sulfur ThiI TIGR00342 TIGR00204 Deoxy-D-xylulose 5-phosphate synthase Dxs 2.2.1.7 PF05690 Thiazole synthase ThiG 2.8.1.10 PF02581 Thiazole Tautomerase TenI 5.3.99.10 TIGR00292 Thiamine thiazole synthase THI4 2.8.1.10 TIGR00694 HET transport, Thiazole kinase ThiM 2.7.1.50 TIGR00693 Thiamin phosphate synthase ThiE 2.5.1.3 TIGR00149 Secondary thiamine-phosphate synthase enzyme YjbQ 2.5.1.3 TIGR01379 Thiamin phosphate kinase ThiL 2.7.4.16 TIGR02721 Thiamine kinase ThiK 2.7.1.89 TIGR01378 Thiamin pyrophosphate kinase Tpk/Tnr3/ThiN/THI80 2.7.6.2 / 2.7.4.15 PF03266 Nucleoside-triphosphatase NTPCR 3.6.1.15 PF01928 Thiamine-triphosphatase THTPA 3.6.1.28 TIGR04541 Thiaminase I / Thiamine pyridinylase I THI1 2.5.1.2 TIGR04306 Thiaminase II / aminopyrimidine aminohydrolase THI20/TenA 2.7.4.7 / 2.7.1.49 / 3.5.99.2

Transport TIGR01528 Thiamine transporter PnuC/PnuT Thiamine outer membrane transporter OMthi TIGR02357 Thiamine transporter ThiT TIGR02358 HMP-P transport CytX TIGR02359 putative transporter of thiazole precursor ThiW TIGR01277 thiamin ABC transporter - ATP binding subunit ThiQ TIGR01253 thiamin ABC transporter - membrane subunit ThiP TIGR01276 Thiamine binding protein TbpA/ThiB TIGR00800 Thiamine transporter from Yeast THI7 TIGR00806 SLC19A2, 19 (thiamine transporter), member 2 THTR1 TIGR00806 SLC19A3, solute carrier family 19 (thiamine transporter), member 3 THTR2 solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), PF00153 SLC25A19 member 19 PF05805 Interaction w/ hTHTR-2 TM4SF4

Dependent TIGR00232 Transketolase TktA/TktB 2.2.1.1 TIGR00239 2-oxoglutarate dehydrogenase E1 component SucA 1.2.4.2 Dihydrolipoyllysine-residue succinyltransferase component of 2- TIGR01347 SucB 2.3.1.61 oxoglutarate dehydrogenase complex TIGR01350 Dihydrolipoyl dehydrogenase Lpd/PdhD/DLD 1.8.1.4 PF00676 2-oxoisovalerate dehydrogenase subunit alpha BfmBAA/BCKDHA 1.2.4.4 TIGR03393 Indole-3-pyruvate decarboxylase IpdC 4.1.1.74 TIGR00118 Acetolactate synthase (ALS) ILV2/IlvI/IlvG 2.2.1.6 TIGR03846 Sulfopyruvate decarboxylase, alpha subunit ComDE 4.1.1.79 TIGR03845 Sulfopyruvate decarboxylase, beta subunit ComDE 4.1.1.79 TIGR02720 Flavoprotein pyruvate oxidase Pox 1.2.3.3 TIGR02179 Phenylglyoxylate dehydrogenase (acylating) PadI 1.2.1.58 TIGR00759 (acetyl-transferring) PDK 1.2.4.1 TIGR03336 Indolepyruvate ferredoxin IorA 1.2.7.8 TIGR02179 Oxalate oxidoreductase Oxo 1.2.7.10 TIGR02177 2-oxoacid oxidoreductase (ferredoxin) PorB 1.2.7.1 TIGR03710 2-oxoacid oxidoreductase (ferredoxin) Por 1.2.7.11

88 Table 2.5. (cont.) TIGR00239 2-hydroxy-3-oxoadipate synthase/2-oxoglutarate decarboxylase Kgd 4.1.1.71/2.2.1.5 TIGR03457 Sulfoacetaldehyde acetyltransferase Xsc 2.3.3.15 TIGR04377 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase (decyclizing) IolD 3.7.1.22 TIGR03254 Oxalyl-CoA decarboxylase Oxc 4.1.1.8 TIGR01504 Tartronate-semialdehyde synthase Gcl 4.1.1.47 TIGR03297 Phosphonopyruvate decarboxylase Ppd 4.1.1.82 PF00205 Thiamine pyrophosphate enzyme, central domain multiple PF00456 Transketolase, thiamine diphosphate binding domain multiple PF01855 Pyruvate flavodoxin/ferredoxin oxidoreductase, thiamine diP-bdg multiple PF02775 Thiamine pyrophosphate enzyme, C-terminal TPP binding domain multiple PF02776 Thiamine pyrophosphate enzyme, N-terminal TPP binding domain multiple PF02779 Transketolase, pyrimidine binding domain multiple PF02780 Transketolase, C-terminal domain multiple

89 TableTable S5. Genomes 2.6. investigated Genomes for thiamine acquisition investigated and dependent genes. for thiamine acquisition and dependent genes

Genome Phylum Class Order Family Genus Refseq Bifidobacterium longum NCC2705 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_004307 Bifidobacterium longum DJO10A Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_010816 Bifidobacterium adolescentis ATCC 15703 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_008618 Collinsella aerofaciens ATCC 25986 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Collinsella NZ_AAVN00000000 Bifidobacterium animalis subsp. lactis AD011 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_011835 Bifidobacterium catenulatum DSM 16992 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABXY00000000 Bifidobacterium adolescentis L2-32 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_AAXD00000000 Bifidobacterium angulatum DSM 20098 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABYS00000000 Bifidobacterium gallicum DSM 20093 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABXB00000000 Collinsella intestinalis DSM 13280 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Collinsella NZ_ABXH00000000 Bifidobacterium breve DSM 20213 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ACCG00000000 Bifidobacterium animalis subsp. lactis HN019 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABOT00000000 Bifidobacterium pseudocatenulatum DSM 20438 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABXX00000000 Collinsella stercoris DSM 13279 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Collinsella NZ_ABXJ00000000 Actinomyces odontolyticus ATCC 17982 Actinobacteria Actinobacteria Actinomycetales Actinomycetaceae Actinomyces NZ_AAYI00000000 Bifidobacterium dentium Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_Bdentium Bifidobacterium sp. 12_1_47BFAA Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ADCN00000000 Bifidobacterium animalis subsp. lactis Bl-04 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_012814 Bifidobacterium animalis subsp. lactis DSM 10140 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_012815 Bifidobacterium bifidum NCIMB 41171 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABQP00000000 Bifidobacterium bifidum PRL2010 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_014638 Bifidobacterium bifidum S17 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_014616 Bifidobacterium dentium ATCC 27678 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABIX00000000 Bifidobacterium longum subsp. infantis 157F Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_015052 Bifidobacterium longum subsp. infantis ATCC 15697 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_011593 Bifidobacterium longum subsp. longum ATCC 55813 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ACHI00000000 Bifidobacterium longum subsp. infantis CCUG 52486 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_ABQQ00000000 Bifidobacterium longum subsp. longum JCM 1217 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_015067 Bifidobacterium longum subsp. longum JDM301 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_014169 Corynebacterium ammoniagenes DSM 20306 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae Corynebacterium NZ_ADNS00000000 Eggerthella sp. 1_3_56FAA Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Eggerthella NZ_ACWN00000000 Eggerthella sp. HGA1 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Eggerthella NZ_AEXR00000000 Eggerthella sp. YY7918 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Eggerthella NC_015738 Bifidobacterium animalis subsp. lactis BB-12 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_017214 Bifidobacterium animalis subsp. lactis V9 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_017217 Bifidobacterium bifidum BGN4 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_017999 Bifidobacterium longum subsp. longum BBMN68 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_014656 Collinsella tanakaei YIT 12063 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Collinsella NZ_ADLS00000000 Propionibacterium sp. 5_U_42AFAA Actinobacteria Actinobacteria Actinomycetales Propionibacteriaceae Propionibacterium NZ_ACUE00000000 Bifidobacterium longum subsp. longum 2-2B Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_AJTJ00000000 Bifidobacterium longum subsp. longum 44B Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NZ_AJTM00000000 Slackia piriformis YIT 12062 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Slackia NZ_ADMD00000000 Bifidobacterium longum subsp. infantis ATCC 15697 = JCM 1222 = DSM 20088 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_017219 Bifidobacterium longum subsp. longum F8 Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium NC_021008 Gordonibacter pamelaeae 7-10-1-b Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Gordonibacter NC_021021 Bacteroides caccae ATCC 43185 Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides NZ_AAVM00000000 Bacteroides thetaiotaomicron VPI-5482 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_004663 Bacteroides vulgatus ATCC 8482 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides 0 Bacteroides fragilis YCH46 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_006347 Bacteroides fragilis NCTC 9343 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_003228 Bacteroides ovatus ATCC 8483 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AAXF00000000 Bacteroides uniformis ATCC 8492 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AAYH00000000 Parabacteroides distasonis ATCC 8503 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NC_009615 Bacteroides dorei DSM 17855 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABWZ00000000 Alistipes putredinis DSM 17216 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NZ_ABFK00000000 Bacteroides coprocola DSM 17136 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABIY00000000 Bacteroides cellulosilyticus DSM 14838 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACCH00000000 Parabacteroides merdae ATCC 43184 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AAXE00000000 Bacteroides stercoris ATCC 43183 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABFZ00000000 Bacteroides intestinalis DSM 17393 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABJL00000000 Bacteroides plebeius DSM 17135 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABQC00000000 Bacteroides eggerthii DSM 20697 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABVO00000000 Bacteroides finegoldii DSM 17565 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABXI00000000 Parabacteroides johnsonii DSM 18315 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_ABYH00000000 Bacteroides fragilis 3_1_12 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABZX00000000 Bacteroides sp. D1 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACAB00000000 Prevotella copri DSM 18205 Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Prevotella NZ_ACBX00000000 Bacteroides sp. D2 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACGA00000000 Bacteroides sp. 1_1_6 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACIC00000000 Bacteroides coprophilus DSM 18228 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACBW00000000 Bacteroides WH2 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_BWH2 Bacteroides thetaiotaomicron 3731 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_Bthetaiotaomicron3731 Bacteroides thetaiotaomicron 7330 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_Bthetaiotaomicron7330 Bacteroides xylanisolvens XB1A Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_BxylanisolvensXB1A Alistipes sp. HGB5 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NZ_AENZ00000000 Bacteroides sp. 1_1_14 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACRP00000000 Bacteroides sp. 1_1_30 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADCL00000000 Bacteroides sp. 2_1_16 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACPP00000000 Bacteroides sp. 2_1_22 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACPQ00000000 Bacteroides sp. 2_1_33B Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACPR00000000 Bacteroides sp. 2_1_56FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACWI00000000 Bacteroides sp. 2_1_7 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABZY00000000 Bacteroides sp. 2_2_4 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ABZZ00000000 Bacteroides sp. 20_3 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACRQ00000000 Bacteroides sp. 3_1_19 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADCJ00000000 Bacteroides sp. 3_1_23 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACRS00000000 Bacteroides sp. 3_1_33FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACPS00000000 Bacteroides sp. 3_1_40A Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACRT00000000 Bacteroides sp. 3_2_5 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACIB00000000 Bacteroides sp. 4_1_36 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACTC00000000 Bacteroides sp. 4_3_47FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACDR00000000 Bacteroides sp. 9_1_42FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACAA00000000 Bacteroides clarus YIT 12056 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AFBM00000000 Bacteroides sp. D20 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACPT00000000 Bacteroides sp. D22 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADCK00000000

90 Table 2.6. (cont.)

Bacteroides dorei 5_1_36/D4 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACDI00000000 Bacteroides eggerthii 1_2_48FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACWG00000000 Bacteroides fluxus YIT 12057 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AFBN00000000 Bacteroides ovatus 3_8_47FAA Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ACWH00000000 Bacteroides ovatus SD CC 2a Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADMP00000000 Bacteroides ovatus SD CMC 3f Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADMO00000000 Bacteroides vulgatus PC510 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADKO00000000 Bacteroides xylanisolvens SD CC 1b Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADKP00000000 Dysgonomonas mossii DSM 22836 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Dysgonomonas NZ_ADLW00000000 Odoribacter splanchnicus DSM 20712 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Odoribacter NC_015160 Parabacteroides sp. D13 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_ACPW00000000 Paraprevotella xylaniphila YIT 11841 Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Paraprevotella NZ_AFBR00000000 Porphyromonas gingivalis ATCC 33277 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Porphyromonas NC_010729 Porphyromonas gingivalis TDC60 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Porphyromonas NC_015571 Porphyromonas gingivalis W83 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Porphyromonas NC_002950 Prevotella salivae DSM 15606 Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Prevotella NZ_AEQO00000000 Alistipes indistinctus YIT 12060 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NZ_ADLD00000000 Bacteroides fragilis 638R Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_016776 Odoribacter laneus YIT 12061 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Odoribacter NZ_ADMC00000000 Paraprevotella clara YIT 11840 Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Paraprevotella NZ_AFFY00000000 Prevotella stercorea DSM 18206 Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Prevotella NZ_AFZZ00000000 Tannerella sp. 6_1_58FAA_CT1 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Tannerella NZ_ACWX00000000 Alistipes finegoldii DSM 17242 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NC_018011 Alistipes shahii WAL 8301 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NC_021030 Alistipes sp. JC136 Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes NZ_CAEG00000000 Bacteroides caccae CL03T12C61 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXF00000000 Bacteroides cellulosilyticus CL02T12C19 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXG00000000 Bacteroides dorei CL02T00C15 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXH00000000 Bacteroides dorei CL02T12C06 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXJ00000000 Bacteroides dorei CL03T12C01 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXI00000000 Bacteroides faecis MAJ27 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGDG00000000 Bacteroides finegoldii CL09T03C10 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXW00000000 Bacteroides fragilis CL03T00C08 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXK00000000 Bacteroides fragilis CL03T12C07 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXL00000000 Bacteroides fragilis CL05T00C42 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXO00000000 Bacteroides fragilis CL05T12C13 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXP00000000 Bacteroides fragilis CL07T00C01 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXM00000000 Bacteroides fragilis CL07T12C05 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXN00000000 Bacteroides fragilis HMW 610 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXQ00000000 Bacteroides fragilis HMW 615 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXR00000000 Bacteroides fragilis HMW 616 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ALOB00000000 Bacteroides helcogenes P 36-108 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_014933 Bacteroides nordii CL02T12C05 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXS00000000 Bacteroides oleiciplenus YIT 12058 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ADLF00000000 Bacteroides ovatus CL02T12C04 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXT00000000 Bacteroides ovatus CL03T12C18 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXU00000000 Bacteroides salanitronis DSM 18170 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NC_015164 Bacteroides salyersiae CL02T12C01 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXV00000000 Bacteroides sp. CF50 Bacteroidetes Bacteroidia Bacteroidales NC_022526 Bacteroides uniformis CL03T00C23 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXX00000000 Bacteroides uniformis CL03T12C37 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXY00000000 Bacteroides vulgatus CL09T03C04 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXZ00000000 Bacteroides xylanisolvens CL03T12C04 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_AGXE00000000 Parabacteroides distasonis CL03T12C09 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AGZM00000000 Parabacteroides distasonis CL09T03C24 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AGZN00000000 Parabacteroides goldsteinii CL02T12C30 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AGZO00000000 Parabacteroides johnsonii CL02T12C29 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AGZP00000000 Parabacteroides merdae CL03T12C32 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AGZQ00000000 Parabacteroides merdae CL09T00C40 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_AJPU00000000 Parabacteroides sp. D25 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides NZ_ACUA00000000 Tannerella forsythia ATCC 43037 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Tannerella NC_016610 Dysgonomonas gadei ATCC BAA-286 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Dysgonomonas NZ_ADLV00000000 Bacteroides cellulosilyticus WH2 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides NZ_ATFI00000000 Barnesiella intestinihominis YIT 11860 Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Barnesiella NZ_ADLE00000000 Lactobacillus reuteri DSM 20016 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_009513 Lactococcus lactis subsp. lactis Il1403 Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus NC_002662 Streptococcus thermophilus LMG 18311 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NC_006448 Streptococcus thermophilus CNRZ1066 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NC_006449 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_008054 Lactobacillus casei ATCC 334 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_008526 Lactococcus lactis subsp. cremoris SK11 Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus NC_008527 Streptococcus thermophilus LMD-9 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NC_008532 Lactococcus lactis subsp. cremoris MG1363 Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus NC_009004 Streptococcus infantarius subsp. infantarius ATCC BAA-102 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NZ_ABJK00000000 Bacillus cereus AH187 Firmicutes Bacilli Bacillales Bacillaceae Bacillus NC_011658 Bacillus cereus G9842 Firmicutes Bacilli Bacillales Bacillaceae Bacillus NC_011772 Enterococcus faecalis PC1.1 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ADKN00000000 Enterococcus faecium PC4.1 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ADMM00000000 Enterococcus faecalis TX0104 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACGL00000000 Enterococcus faecalis TX1322 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACGM00000000 Enterococcus faecalis TX2134 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBW00000000 Enterococcus faecalis V583 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NC_004668 Enterococcus faecalis X98 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACAW00000000 Enterococcus faecium 1_141_733 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACAZ00000000 Enterococcus faecium 1_231_501 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACAY00000000 Enterococcus faecium Com12 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACBC00000000 Enterococcus faecium Com15 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACBD00000000 Enterococcus faecium DO Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AAAK00000000 Enterococcus faecium E1071 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ABQI00000000 Enterococcus faecium TX1330 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ACHL00000000 Lactobacillus acidophilus ATCC 4796 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACHN00000000 Lactobacillus acidophilus NCFM Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_006814 Lactobacillus amylolyticus DSM 11664 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ADNY00000000 Lactobacillus antri DSM 16041 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACLL00000000 Lactobacillus brevis subsp. gravesensis ATCC 27305 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGG00000000 Lactobacillus buchneri ATCC 11577 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGH00000000 Lactobacillus casei BL23 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_010999 Lactobacillus crispatus 125-2-CHN Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACPV00000000 Lactobacillus delbrueckii subsp. bulgaricus ND02 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_014727

91 Table 2.6. (cont.)

Lactobacillus fermentum ATCC 14931 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGI00000000 Lactobacillus fermentum IFO 3956 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_010610 Lactobacillus gasseri ATCC 33323 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_008530 Lactobacillus helveticus DSM 20075 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACLM00000000 Lactobacillus hilgardii ATCC 8290 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGP00000000 Lactobacillus johnsonii NCC 533 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_005362 Lactobacillus paracasei subsp. paracasei 8700_2 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ABQV00000000 Lactobacillus paracasei subsp. paracasei ATCC 25302 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGY00000000 Lactobacillus plantarum subsp. plantarum ATCC 14917 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGZ00000000 Lactobacillus plantarum JDM1 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_012984 Lactobacillus reuteri 100-23 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_AAPZ00000000 Lactobacillus reuteri CF48-3A Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACHG00000000 Lactobacillus reuteri JCM 1112 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_010609 Lactobacillus reuteri MM2-3 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACLB00000000 Lactobacillus reuteri MM4-1A Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGX00000000 Lactobacillus reuteri SD2112 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_015697 Lactobacillus rhamnosus GG Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_013198 Lactobacillus rhamnosus HN001 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ABWJ00000000 Lactobacillus rhamnosus LMS2-1 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACIZ00000000 Lactobacillus ruminis ATCC 25644 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGS00000000 Lactobacillus salivarius ATCC 11741 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGT00000000 Lactobacillus salivarius UCC118 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_007929 Lactobacillus ultunensis DSM 16047 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACGU00000000 Leuconostoc mesenteroides subsp. cremoris ATCC 19254 Firmicutes Bacilli Lactobacillales Leuconostocaceae Leuconostoc NZ_ACKV00000000 Listeria grayi DSM 20601 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_ACCR00000000 Listeria monocytogenes F6900 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARU00000000 Listeria monocytogenes Finland 1988 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AART00000000 Listeria monocytogenes FSL J1-175 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARK00000000 Listeria monocytogenes FSL J1-194 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARJ00000000 Listeria monocytogenes FSL J2-003 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARM00000000 Listeria monocytogenes FSL J2-064 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARO00000000 Listeria monocytogenes FSL J2-071 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARN00000000 Listeria monocytogenes FSL N1-017 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARP00000000 Listeria monocytogenes FSL N3-165 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARQ00000000 Listeria monocytogenes FSL R2-503 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARR00000000 Listeria monocytogenes FSL R2-561 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARS00000000 Listeria monocytogenes HPB2262 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AATL00000000 Listeria monocytogenes J0161 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARW00000000 Listeria monocytogenes J2818 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARX00000000 Listeria monocytogenes LO28 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AARY00000000 Listeria monocytogenes serotype 4b str. F2365 Firmicutes Bacilli Bacillales Listeriaceae Listeria NC_002973 Listeria monocytogenes serotype 4b str. H7858 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AADR00000000 Paenibacillus sp. HGF5 Firmicutes Bacilli Bacillales Paenibacillaceae Paenibacillus NZ_AEXS00000000 Paenibacillus sp. HGF7 Firmicutes Bacilli Bacillales Paenibacillaceae Paenibacillus NZ_AFDH00000000 Pediococcus acidilactici 7_4 Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus NZ_ACXB00000000 Pediococcus acidilactici DSM 20284 Firmicutes Bacilli Lactobacillales Lactobacillaceae Pediococcus NZ_AEEG00000000 Streptococcus sp. 2_1_36FAA Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NZ_ACOI00000000 Streptococcus anginosus 1_2_62CV Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NZ_ADME00000000 Streptococcus equinus ATCC 9812 Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus NZ_AEVB00000000 Weissella paramesenteroides ATCC 33313 Firmicutes Bacilli Lactobacillales Leuconostocaceae Weissella NZ_ACKU00000000 Bacillus sp. 7_6_55CFAA_CT2 Firmicutes Bacilli Bacillales Bacillaceae Bacillus NZ_ACWE00000000 Bacillus smithii 7_3_47FAA Firmicutes Bacilli Bacillales Bacillaceae Bacillus NZ_ACWF00000000 Enterococcus faecalis 62 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NC_017312 Enterococcus faecalis OG1RF Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NC_017316 Enterococcus saccharolyticus 30_1 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_ADLY00000000 Lactobacillus sp. 7_1_47FAA Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_ACWR00000000 Lactobacillus delbrueckii subsp. bulgaricus 2038 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_017469 Lactobacillus fermentum CECT 5716 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_017465 Lactobacillus johnsonii DPC 6026 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_017477 Lactobacillus rhamnosus Lc 705 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_013199 Lactobacillus salivarius CECT 5713 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_017481 Listeria monocytogenes L99 Firmicutes Bacilli Bacillales Listeriaceae Listeria NC_017529 Enterococcus faecalis TX1302 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBK00000000 Enterococcus faecalis TX1341 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AECF00000000 Enterococcus faecalis TX1342 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBJ00000000 Enterococcus faecalis TX1346 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBI00000000 Enterococcus faecalis TX1467 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AFBS00000000 Enterococcus faecalis TX2137 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBQ00000000 Enterococcus faecalis TX4244 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NZ_AEBH00000000 Lactobacillus delbrueckii subsp. lactis DSM 20072 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_AEXU00000000 Lactobacillus rhamnosus ATCC 21052 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NZ_AFZY00000000 Listeria innocua ATCC 33091 Firmicutes Bacilli Bacillales Listeriaceae Listeria NZ_AGCN00000000 Lactobacillus rhamnosus GG Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus NC_013198 Enterococcus sp. 7L76 Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus NC_021023 Blautia hydrogenotrophica DSM 10507 Firmicutes Clostridiales IncertaeSedisXIV Blautia NZ_ACBZ00000000 Bryantella formatexigens DSM 14469 Firmicutes Clostridia Clostridiales Lachnospiraceae Bryantella NZ_ACCL00000000 Clostridium scindens ATCC 35704 Firmicutes Clostridia Clostridiales Clostridium NZ_ABFY00000000 Clostridium spiroforme DSM 1552 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABIK00000000 Dorea longicatena DSM 13814 Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea NZ_AAXB00000000 Eubacterium eligens ATCC 27750 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_012778 Eubacterium rectale ATCC 33656 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_012781 Faecalibacterium prausnitzii A2-165 Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium NZ_ACOP00000000 Faecalibacterium prausnitzii M21/2 Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium NZ_ABED00000000 Ruminococcus obeum ATCC 29174 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NZ_AAVO00000000 Ruminococcus torques ATCC 27756 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NZ_AAVP00000000 Clostridium bolteae ATCC BAA-613 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABCC00000000 Clostridium sporogenes ATCC 15579 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABKW00000000 Clostridium asparagiforme DSM 15981 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ACCJ00000000 Ruminococcus lactaris ATCC 29176 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NZ_ABOU00000000 Anaerococcus hydrogenalis DSM 7454 Firmicutes Clostridia Clostridiales IncertaeSedisXI Anaerococcus NZ_ABXA00000000 Pseudoflavonifractor capillosus ATCC 29799 Firmicutes Clostridia Clostridiales Pseudoflavonifractor NZ_AAXG00000000 Anaerotruncus colihominis DSM 17241 Firmicutes Clostridia Clostridiales Ruminococcaceae Anaerotruncus NZ_ABGD00000000 Coprococcus comes ATCC 27758 Firmicutes Clostridia Clostridiales Lachnospiraceae Coprococcus NZ_ABVR00000000 Blautia hansenii DSM 20583 Firmicutes Clostridia Clostridiales IncertaeSedisXIV Blautia NZ_ABYU00000000 Ruminococcus gnavus ATCC 29149 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NZ_AAYG00000000 Anaerofustis stercorihominis DSM 17244 Firmicutes Clostridia Clostridiales Eubacteriaceae Anaerofustis NZ_ABIL00000000 Anaerostipes caccae DSM 14662 Firmicutes Clostridia Clostridiales Lachnospiraceae Anaerostipes NZ_ABAX00000000 Butyrivibrio crossotus DSM 2876 Firmicutes Clostridia Clostridiales Lachnospiraceae Butyrivibrio NZ_ABWN00000000 Eubacterium ventriosum ATCC 27560 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_AAVL00000000

92

Table 2.6. (cont.)

Clostridium sp. L2-50 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_AAYW00000000 Eubacterium dolichum DSM 3991 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_ABAW00000000 Clostridium leptum DSM 753 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABCB00000000 Parvimonas micra ATCC 33270 Firmicutes Clostridia IncertaeSedisXI Parvimonas NZ_ABEE00000000 Coprococcus eutactus ATCC 27759 Firmicutes Clostridia Clostridiales Lachnospiraceae Coprococcus NZ_ABEY00000000 Clostridium bartlettii DSM 16795 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABEZ00000000 Clostridium ramosum DSM 1402 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABFX00000000 Clostridium sp. SS2/1 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABGC00000000 Dorea formicigenerans ATCC 27755 Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea NZ_AAXA00000000 Clostridium nexile DSM 1787 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABWO00000000 Clostridium hylemonae DSM 15053 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABYI00000000 Roseburia intestinalis L1-82 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia NZ_ABYJ00000000 Eubacterium biforme DSM 3989 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_ABYT00000000 Clostridium methylpentosum DSM 5476 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ACEC00000000 Eubacterium hallii DSM 3353 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_ACEP00000000 Clostridium hiranonis DSM 13275 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ABWP00000000 Subdoligranulum variabile DSM 15176 Firmicutes Clostridia Ruminococcaceae Subdoligranulum NZ_ACBY00000000 Clostridium sp. M62/1 Firmicutes Clostridia Clostridiales Lachnospiraceae Robinsoniella NZ_ACFX00000000 Clostridium symbiosum Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NC_Csymbiosum Eubacterium rectale DSM17629 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_Erectale_DSM17629 M23A Firmicutes Clostridia NC_M23A Ruminococcus bromiiL263 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NC_RbromiiL263 Anaerostipes sp. 3_2_56FAA Firmicutes Clostridia Clostridiales Lachnospiraceae Anaerostipes NZ_ACWB00000000 Bacteroides pectinophilus ATCC 43243 Firmicutes Clostridia Clostridiales NZ_ABVQ00000000 Clostridiales bacterium 1_7_47FAA Firmicutes Clostridia Clostridiales NZ_ABQR00000000 Clostridium sp. 7_2_43FAA Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ACDK00000000 Clostridium sp. D5 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADBG00000000 Clostridium difficile CD196 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NC_013315 Clostridium difficile NAP07 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADVM00000000 Clostridium difficile NAP08 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADNX00000000 Clostridium difficile R20291 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NC_013316 Clostridium hathewayi DSM 13479 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ACIO00000000 Clostridium sp. HGF2 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_AENW00000000 Clostridium sp. SY8519 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NC_015737 Clostridium symbiosum WAL-14673 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLR00000000 Eubacterium siraeum DSM 15702 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_ABCA00000000 Faecalibacterium cf. prausnitzii KLE1255 Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium NZ_AECU00000000 Finegoldia magna ATCC 29328 Firmicutes Clostridia Clostridiales IncertaeSedisXI Finegoldia NC_010376 Lachnospiraceae bacterium 1_1_57FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTM00000000 Lachnospiraceae bacterium 1_4_56FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTN00000000 Lachnospiraceae bacterium 2_1_46FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ADLB00000000 Lachnospiraceae bacterium 2_1_58FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTO00000000 Lachnospiraceae bacterium 3_1_46FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACWP00000000 Lachnospiraceae bacterium 3_1_57FAA_CT1 Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTP00000000 Lachnospiraceae bacterium 4_1_37FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ADCR00000000 Lachnospiraceae bacterium 5_1_57FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTR00000000 Lachnospiraceae bacterium 5_1_63FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTS00000000 Lachnospiraceae bacterium 6_1_63FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTV00000000 Lachnospiraceae bacterium 8_1_57FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACWQ00000000 Lachnospiraceae bacterium 9_1_43BFAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTX00000000 Roseburia inulinivorans DSM 16841 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia NZ_ACFY00000000 Ruminococcaceae bacterium D16 Firmicutes Clostridia Clostridiales Ruminococcaceae NZ_ADDX00000000 Ruminococcus sp. 5_1_39BFAA Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NZ_ACII00000000 Clostridium sp. 7_3_54FAA Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ACWK00000000 Clostridium citroniae WAL-17108 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLJ00000000 Clostridium clostridioforme 2_1_49FAA Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLL00000000 Clostridium hathewayi WAL-18680 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLN00000000 Dorea formicigenerans 4_6_53AFAA Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea NZ_ADLU00000000 Eubacterium sp. 3_1_31 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NZ_ACTL00000000 Flavonifractor plautii ATCC 29863 Firmicutes Clostridia Clostridiales Flavonifractor NZ_AGCK00000000 Lachnospiraceae bacterium 7_1_58FAA Firmicutes Clostridia Clostridiales Lachnospiraceae NZ_ACTW00000000 Roseburia hominis A2-183 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia NC_015977 Subdoligranulum sp. 4_3_54A2FAA Firmicutes Clostridia Ruminococcaceae Subdoligranulum NZ_ACWW00000000 Anaerostipes hadrus DSM 3319 Firmicutes Clostridia Clostridiales Lachnospiraceae Anaerostipes NZ_AMEY00000000 Clostridium celatum DSM 1785 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_AMEZ00000000 Clostridium difficile 70-100-2010 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_AGAC00000000 Clostridium perfringens WAL-14572 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLP00000000 Clostridium symbiosum WAL-14163 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NZ_ADLQ00000000 Desulfitobacterium hafniense DP7 Firmicutes Clostridia Clostridiales Peptococcaceae Desulfitobacterium NZ_AFZX00000000 Butyrivibrio fibrisolvens 16/4 Firmicutes Clostridia Clostridiales Lachnospiraceae Butyrivibrio NC_021031 [Clostridium] cf. saccharolyticum K10 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium NC_021047 Coprococcus catus GD/7 Firmicutes Clostridia Clostridiales Lachnospiraceae Coprococcus NC_021009 Coprococcus sp. ART55/1 Firmicutes Clostridia Clostridiales Lachnospiraceae Coprococcus NC_021018 [Eubacterium] cylindroides T2-87 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_021019 Eubacterium rectale DSM 17629 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_021010 Eubacterium rectale M104/1 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_021044 [Eubacterium] siraeum 70/3 Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_021011 Faecalibacterium prausnitzii L2-6 Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium NC_021042 Faecalibacterium prausnitzii SL3/3 Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium NC_021020 Roseburia intestinalis M50/1 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia NC_021040 Roseburia intestinalis XB6B4 Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia NC_021012 Ruminococcus champanellensis 18P13 = JCM 17042 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NC_021039 Ruminococcus sp. SR1/5 Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus NC_021014 [Eubacterium] siraeum V10Sc8a Firmicutes Clostridia Clostridiales Eubacteriaceae Eubacterium NC_021043 Catenibacterium mitsuokai DSM 15897 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Catenibacterium NZ_ACCK00000000 Holdemania filiformis DSM 12042 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Holdemania NZ_ACCF00000000 Coprobacillus sp. 29_1 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Coprobacillus NZ_ADKX00000000 Coprobacillus sp. D7 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Coprobacillus NZ_ACDT00000000 Erysipelotrichaceae bacterium 3_1_53 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae NZ_ACTJ00000000 Erysipelotrichaceae bacterium 5_2_54FAA Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae NZ_ACZW00000000 Turicibacter sp. HGF1 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Turicibacter NZ_AEXQ00000000 Turicibacter sanguinis PC909 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Turicibacter NZ_ADMN00000000 Coprobacillus sp. 3_3_56FAA Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Coprobacillus NZ_ACWL00000000 Coprobacillus sp. 8_2_54BFAA Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Coprobacillus NZ_ACTG00000000 Erysipelotrichaceae bacterium 2_2_44A Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae NZ_ADCZ00000000 Erysipelotrichaceae bacterium 21_3 Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae NZ_ACTI00000000 Mitsuokella multacida DSM 20544 Firmicutes Negativicutes Selenomonadales Veillonellaceae Mitsuokella NZ_ABWK00000000 Acidaminococcus sp. D21 Firmicutes Negativicutes Selenomonadales Acidaminococcaceae Acidaminococcus NZ_ACGB00000000 Phascolarctobacterium sp. YIT 12067 Firmicutes Negativicutes Selenomonadales Acidaminococcaceae Phascolarctobacterium NZ_AEVN00000000

93

Table 2.6. (cont.)

Veillonella sp. 3_1_44 Firmicutes Negativicutes Selenomonadales Veillonellaceae Veillonella NZ_ADCV00000000 Veillonella sp. 6_1_27 Firmicutes Negativicutes Selenomonadales Veillonellaceae Veillonella NZ_ADCW00000000 Veillonella parvula DSM 2008 Firmicutes Negativicutes Selenomonadales Veillonellaceae Veillonella NC_013520 Acidaminococcus intestini RyC-MR95 Firmicutes Negativicutes Selenomonadales Acidaminococcaceae Acidaminococcus NC_016077 Dialister succinatiphilus YIT 11850 Firmicutes Negativicutes Selenomonadales Veillonellaceae Dialister NZ_ADLT00000000 Megamonas funiformis YIT 11815 Firmicutes Negativicutes Selenomonadales Veillonellaceae Megamonas NZ_ADMB00000000 Megamonas hypermegale ART12/1 Firmicutes Negativicutes Selenomonadales Veillonellaceae Megamonas NC_021041 Fusobacterium sp. 4_1_13 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDE00000000 Fusobacterium varium ATCC 27725 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACIE00000000 Fusobacterium sp. 1_1_41FAA Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ADGG00000000 Fusobacterium sp. 11_3_2 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACUO00000000 Fusobacterium sp. 2_1_31 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDC00000000 Fusobacterium sp. 21_1A Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ADEE00000000 Fusobacterium sp. 3_1_27 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ADGF00000000 Fusobacterium sp. 3_1_33 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACQE00000000 Fusobacterium sp. 3_1_36A2 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACPU00000000 Fusobacterium sp. 3_1_5R Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDD00000000 Fusobacterium sp. 7_1 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDF00000000 Fusobacterium sp. D11 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDS00000000 Fusobacterium sp. D12 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDG00000000 Fusobacterium gonidiaformans ATCC 25563 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACET00000000 Fusobacterium mortiferum ATCC 9817 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDB00000000 Fusobacterium ulcerans ATCC 49185 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ACDH00000000 Fusobacterium sp. 12_1B Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_AGWJ00000000 Fusobacterium necrophorum subsp. funduliforme 1_1_36S Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_ADLZ00000000 Fusobacterium nucleatum subsp. animalis ATCC 51191 Fusobacteria Fusobacteria Fusobacteriales Fusobacteriaceae Fusobacterium NZ_AFQD00000000 Burkholderia oklahomensis EO147 Proteobacteria Betaproteobacteria Burkholderiaceae Burkholderia NZ_ABBF00000000 Burkholderiales bacterium 1_1_47 Proteobacteria Betaproteobacteria Burkholderiales NZ_ADCQ00000000 Laribacter hongkongensis HLHK9 Proteobacteria Betaproteobacteria Neisseriales Laribacter NC_012559 Neisseria macacae ATCC 33926 Proteobacteria Betaproteobacteria Neisseriales Neisseriaceae Neisseria NZ_AFQE00000000 Oxalobacter formigenes HOxBLS Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Oxalobacter NZ_ACDP00000000 Oxalobacter formigenes OXCC13 Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Oxalobacter NZ_ACDQ00000000 Parasutterella excrementihominis YIT 11859 Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Parasutterella NZ_AFBP00000000 Ralstonia sp. 5_7_47FAA Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Ralstonia NZ_ACUF00000000 Sutterella wadsworthensis 3_1_45B Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Sutterella NZ_ADMF00000000 Ralstonia sp. 5_2_56FAA Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Ralstonia NZ_ACTT00000000 Sutterella parvirubra YIT 11816 Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Sutterella NZ_AFBQ00000000 Sutterella wadsworthensis 2_1_59BFAA Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Sutterella NZ_ADMG00000000 Desulfovibrio piger ATCC 29098 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio NZ_ABXU00000000 Desulfovibrio piger GOR1 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio NC_DpigerGOR1 Bilophila wadsworthia 3_1_6 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Bilophila NZ_ADCP00000000 Desulfovibrio sp. 3_1_syn3 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio NZ_ADDR00000000 Bilophila sp. 4_1_30 Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Bilophila NZ_ADCO00000000 Desulfovibrio sp. 6_1_46AFAA Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio NZ_ACWM00000000 HPAG1 Proteobacteria Epsilonproteobacteria Helicobacteraceae Helicobacter NC_008086 Arcobacter butzleri JV22 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Arcobacter NZ_AEPT00000000 Campylobacter coli JV20 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AEER00000000 Campylobacter concisus 13826 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_009802 Campylobacter curvus 525.92 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_009715 Campylobacter hominis ATCC BAA-381 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_009714 subsp. jejuni 81-176 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AASL00000000 Campylobacter jejuni subsp. jejuni CG8486 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AASY00000000 Campylobacter jejuni subsp. jejuni HB93-13 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AANQ00000000 Campylobacter jejuni subsp. jejuni ICDCCJ07001 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_014802 Campylobacter jejuni subsp. jejuni NCTC 11168 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_002163 Campylobacter upsaliensis JV21 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AEPU00000000 Campylobacter upsaliensis RM3195 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_AAFJ00000000 Helicobacter bilis ATCC 43879 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ACDN00000000 Helicobacter bizzozeronii CIII-1 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_015674 Helicobacter canadensis MIT 98-5491 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABQS00000000 CCUG 18818 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABQT00000000 Helicobacter pullorum MIT 98-5489 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABQU00000000 Helicobacter pylori 26695 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_000915 Helicobacter pylori 98-10 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABSX00000000 Helicobacter pylori B128 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABSY00000000 Helicobacter pylori B38 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_012973 Helicobacter pylori B8 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_014256 Helicobacter pylori G27 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_011333 Helicobacter pylori J99 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_000921 Helicobacter pylori P12 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_011498 Helicobacter pylori PeCan4 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_014555 Helicobacter pylori Shi470 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_010698 Helicobacter pylori SJM180 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_014560 Helicobacter winghamensis ATCC BAA-430 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ACDO00000000 Campylobacter sp. 10_1_50 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NZ_ACWJ00000000 Campylobacter jejuni subsp. jejuni M1 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter NC_017280 Helicobacter pylori 2017 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017374 Helicobacter pylori 2018 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017381 Helicobacter pylori 35A Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017360 Helicobacter pylori 51 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017382 Helicobacter pylori 83 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017375 Helicobacter pylori 908 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017357 Helicobacter pylori Cuz20 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017358 Helicobacter pylori F16 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017368 Helicobacter pylori F30 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017365 Helicobacter pylori F32 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017366 Helicobacter pylori F57 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017367 Helicobacter pylori Gambia94/24 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017364 Helicobacter pylori HPKX_438_AG0C1 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ABJO00000000 Helicobacter pylori India7 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017372 Helicobacter pylori Lithuania75 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017362 Helicobacter pylori Sat464 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017356 Helicobacter pylori SouthAfrica7 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017361 Helicobacter pylori v225d Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017355 Helicobacter pylori GAM100Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_ANFP00000000 Helicobacter pylori GAM101Biv Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APCW00000000 Helicobacter pylori GAM103Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APCX00000000 Helicobacter pylori GAM105Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APCY00000000 Helicobacter pylori GAM112Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APCZ00000000

94

Table 2.6. (cont.)

Helicobacter pylori GAM114Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDA00000000 Helicobacter pylori GAM115Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDB00000000 Helicobacter pylori GAM118Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDD00000000 Helicobacter pylori GAM119Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDE00000000 Helicobacter pylori GAM120Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDF00000000 Helicobacter pylori GAM121Aii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDG00000000 Helicobacter pylori GAM201Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDC00000000 Helicobacter pylori GAM210Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDH00000000 Helicobacter pylori GAM231Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDI00000000 Helicobacter pylori GAM239Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDJ00000000 Helicobacter pylori GAM244Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDK00000000 Helicobacter pylori GAM245Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDL00000000 Helicobacter pylori GAM246Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDM00000000 Helicobacter pylori GAM249T Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDN00000000 Helicobacter pylori GAM250AFi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDO00000000 Helicobacter pylori GAM250T Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDP00000000 Helicobacter pylori GAM252Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDQ00000000 Helicobacter pylori GAM252T Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDR00000000 Helicobacter pylori GAM254Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDS00000000 Helicobacter pylori GAM260ASi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDT00000000 Helicobacter pylori GAM260Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDU00000000 Helicobacter pylori GAM260BSi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDV00000000 Helicobacter pylori GAM263BFi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDW00000000 Helicobacter pylori GAM264Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDX00000000 Helicobacter pylori GAM265BSii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDY00000000 Helicobacter pylori GAM268Bii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APDZ00000000 Helicobacter pylori GAM270ASi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEA00000000 Helicobacter pylori GAM42Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEB00000000 Helicobacter pylori GAM71Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEC00000000 Helicobacter pylori GAM80Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APED00000000 Helicobacter pylori GAM83Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEE00000000 Helicobacter pylori GAM83T Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEF00000000 Helicobacter pylori GAM93Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEG00000000 Helicobacter pylori GAM96Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEH00000000 Helicobacter pylori GAMchJs106B Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEI00000000 Helicobacter pylori GAMchJs114i Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEJ00000000 Helicobacter pylori GAMchJs117Ai Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEK00000000 Helicobacter pylori GAMchJs124i Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEL00000000 Helicobacter pylori GAMchJs136i Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEM00000000 Helicobacter pylori HP116Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEN00000000 Helicobacter pylori HP250AFii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEO00000000 Helicobacter pylori HP250AFiii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEP00000000 Helicobacter pylori HP250AFiV Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEQ00000000 Helicobacter pylori HP250ASi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APER00000000 Helicobacter pylori HP250ASii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APES00000000 Helicobacter pylori HP250BFi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APET00000000 Helicobacter pylori HP250BFii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEU00000000 Helicobacter pylori HP250BFiii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEV00000000 Helicobacter pylori HP250BFiV Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEW00000000 Helicobacter pylori HP250BSi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEX00000000 Helicobacter pylori HP260AFi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEY00000000 Helicobacter pylori HP260AFii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APEZ00000000 Helicobacter pylori HP260ASii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APFA00000000 Helicobacter pylori HP260BFii Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APFB00000000 Helicobacter pylori HP260Bi Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NZ_APFC00000000 Helicobacter pylori 35A Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017360 Helicobacter pylori 83 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter NC_017375 Escherichia coli str. K-12 substr. MG1655 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_000913 Escherichia fergusonii ATCC 35469 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_011740 O1 biovar eltor str. N16961 I Proteobacteria Gammaproteobacteria Vibrionales Vibrio NC_002505 Citrobacter youngae ATCC 29220 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter NZ_ABWL00000000 Providencia alcalifaciens DSM 30120 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Providencia NZ_ABXW00000000 Providencia stuartii ATCC 25827 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Providencia NZ_ABJD00000000 Providencia rustigianii DSM 4541 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Providencia NZ_ABXV00000000 Proteus penneri ATCC 35198 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Proteus NZ_ABVP00000000 Providencia rettgeri DSM 1131 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Providencia NZ_ACCI00000000 Shigella sp. D9 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ACDL00000000 Enterobacter cancerogenus ATCC 35316 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Enterobacter NC_Ecancerogenus Acinetobacter baumannii AB900 Proteobacteria Gammaproteobacteria Moraxellaceae Acinetobacter NZ_ABXK00000000 Acinetobacter junii SH205 Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Acinetobacter NZ_ACPM00000000 Citrobacter sp. 30_2 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter NZ_ACDJ00000000 ATCC BAA-895 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter NC_009792 Edwardsiella tarda ATCC 23685 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Edwardsiella NZ_ADGK00000000 Enterobacteriaceae bacterium 9_2_54FAA Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae NZ_ADCU00000000 Escherichia sp. 3_2_53FAA Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ACAC00000000 Escherichia sp. 4_1_40B Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ACDM00000000 Escherichia albertii TW07627 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ABKX00000000 Escherichia coli ATCC 8739 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_010468 Escherichia coli str. K-12 substr. DH10B Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_010473 Escherichia coli MS 107-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADWV00000000 Escherichia coli MS 115-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTL00000000 Escherichia coli MS 116-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTZ00000000 Escherichia coli MS 119-7 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADWU00000000 Escherichia coli MS 124-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADWT00000000 Escherichia coli MS 145-7 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADWS00000000 Escherichia coli MS 146-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTN00000000 Escherichia coli MS 175-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUB00000000 Escherichia coli MS 182-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTM00000000 Escherichia coli MS 185-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUE00000000 Escherichia coli MS 187-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTQ00000000 Escherichia coli MS 196-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUD00000000 Escherichia coli MS 198-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTJ00000000 Escherichia coli MS 200-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUC00000000 Escherichia coli MS 21-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTR00000000 Escherichia coli MS 45-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTO00000000 Escherichia coli MS 69-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTP00000000 Escherichia coli MS 78-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTY00000000 Escherichia coli MS 84-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTK00000000 Escherichia coli O157_H7 str. EDL933 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_002655

95 Table 2.6. (cont.)

Escherichia coli O157_H7 str. Sakai Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_002695 Escherichia coli O26_H11 str. 11368 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_013361 Escherichia coli O55_H7 str. CB9615 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_013941 Escherichia coli SE11 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_011415 Escherichia coli UTI89 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_007946 Grimontia hollisae CIP 101886 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Grimontia NZ_ADAQ00000000 Klebsiella sp. 1_1_55 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella NZ_ACXA00000000 Klebsiella sp. MS 92-3 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella NZ_AFBO00000000 Succinatimonas hippei YIT 12066 Proteobacteria Gammaproteobacteria Succinivibrionaceae Succinatimonas NZ_AEVO00000000 Vibrio cholerae MZO-2 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NZ_AAWF00000000 Vibrio cholerae V52 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NZ_AAKJ00000000 Vibrio furnissii CIP 102972 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NZ_ACZP00000000 Vibrio mimicus MB451 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NZ_ADAF00000000 Vibrio sp. RC341 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NZ_ACZT00000000 bercovieri ATCC 43970 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Yersinia NZ_AALC00000000 Yersinia pseudotuberculosis IP 31758 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Yersinia NC_009708 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Salmonella NC_003197 Acinetobacter radioresistens SH164 Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Acinetobacter NZ_ACPO00000000 4_7_47CFAA Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter NZ_ADLG00000000 Edwardsiella tarda FL6-60 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Edwardsiella NC_017309 Escherichia sp. 1_1_43 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ACID00000000 Escherichia coli 042 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_017626 Escherichia coli 536 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_008253 Escherichia coli B7A Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_AAJT00000000 Escherichia coli DH1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_017625 Escherichia coli E110019 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_AAJW00000000 Escherichia coli E24377A Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_009786 Escherichia coli ED1a Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_011745 Escherichia coli F11 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_AAJU00000000 Escherichia coli HS Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_009800 Escherichia coli IHE3034 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_017628 Escherichia coli LF82 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_011993 Escherichia coli O157:H7 str. EC508 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ABHW00000000 Escherichia coli SE15 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_013654 Escherichia coli UM146 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NC_017630 Hafnia alvei ATCC 51873 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Hafnia NZ_AGCI00000000 Klebsiella sp. 4_1_44FAA Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella NZ_ACWO00000000 Pseudomonas sp. 2_1_26 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas NZ_ACWU00000000 Vibrio furnissii NCTC 11218 chromosome 1 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio NC_016602 subsp. palearctica Y11 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Yersinia NC_017564 Yokenella regensburgei ATCC 43003 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Yokonella NZ_AGCL00000000 Escherichia coli MS 110-3 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTW00000000 Escherichia coli MS 117-3 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTS00000000 Escherichia coli MS 153-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADTX00000000 Escherichia coli MS 16-3 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUA00000000 Escherichia coli MS 57-2 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUG00000000 Escherichia coli MS 60-1 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella NZ_ADUF00000000 subsp. pneumoniae WGLW3 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella NZ_AMLN00000000 Klebsiella pneumoniae subsp. pneumoniae WGLW5 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella NZ_AMLO00000000 WGLW6 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Proteus NZ_AMGT00000000 subsp. cloacae NCTC 9394 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Enterobacter NC_021046

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100

CHAPTER 3: TRANSCRIPTIONAL AND TRANSLATIONAL ACTING TPP

RIBOSWITCHES IDENTIFIED AMONG BACTEROIDES SPECIES

Abstract:

Thiamine (vitamin B1) and its phosphorylated moieties are essential for all living organisms. Unfortunately, little is understood about how this cofactor impacts and shapes the human microbiome. Previous work has shown that both thiamine biosynthesis and transport are critical to the fitness of Bacteroides species. Recent studies have led to a better understanding of thiamine biosynthesis and transport genes in the gut. As of yet, little work has been done to characterize the regulation of these critical thiamine biosynthesis and transport operons among the prominent gut phylum Bacteroidetes. To this end, we have selected three representative and abundant species from the phylum

Bacteroidetes: Bacteroides thetaiotaomicron, Bacteroides uniformis, and Bacteroides vulgatus in which to study thiamine based regulatory mechanisms. These regulatory features are highly conserved cis-acting thiamine pyrophosphate (TPP) riboswitches. TPP riboswitches impact the expression of thiamine biosynthesis and transport genes under their control. In the three selected organisms we have identified distinct transcriptomic profiles in response to exogenous thiamine. These profiles include distinct responses across thiamine biosynthesis and transport operons. We also constructed and tested translational fusions of the TPP riboswitches found in B. thetaiotaomicron, B. uniformis, and B. vulgatus in which we found pronounced regulation of transport and biosynthetic operons. Together these data show that Bacteroides species employ both transcriptional and translational acting TPP riboswitches. B. thetaiotaomicron is novel in the utilization

101 of both transcriptional and translational acting TPP riboswitches. In addition, B. uniformis and B. vulgatus exclusively leverage transcriptional acting TPP riboswitches. The mechanism of TPP riboswitch regulation appears to be linked to the distance from the first start codon in the regulon of the TPP riboswitch. Consequently, this trend between distance and mechanism of regulation may be conserved among the

Bacteroidetes.

Importance:

Characterization of the regulatory mechanisms of TPP among gut microbes may help inform how gut microbiomes are formed and give insight into relative abundances of certain organisms in the gastrointestinal tract. Our study confirms there is regulation of thiamine biosynthesis and transport among Bacteroides species. In addition, our work shows how different organisms respond to thiamine starvation. Our data also implies

Bacteroides species are novel in employing both transcriptional and translational acting

TPP riboswitches in order to rapidly respond to the shifting environmental availability of thiamine in the gut. This work can inform how gut microbes will respond to and cope to changes in the availability of this essential cofactor and may help in therapeutic approaches to altering dysbiotic microbiomes.

102 Introduction:

Thiamine pyrophosphate (TPP) is the active, cofactor form of thiamine (vitamin

B1). Thiamine is required by all living organisms to perform metabolic activities.

Thiamine requiring pathways include central metabolism, branched chain amino acid biosynthesis, and nucleotide synthesis (11, 21, 32, 42). Humans primarily acquire thiamine from their diet and convert it to the cofactor form, TPP (17, 37). In addition to dietary thiamine, it is believed microbial communities in the gastrointestinal tract also provide thiamine to the host (38, 48). This thiamine is likely supplied by the indigenous gut microbes that possess ability to biosynthesize thiamine and TPP de novo (8,36). In contrast, studies have show that microbes common to the gastrointestinal tract sequester thiamine (1). The conflicting reports imply that more investigation is required to fully understand the role of thiamine in the gut. In addition, thiamine availability has been correlatively linked to the structure of microbial communities (51). Irregular host thiamine levels have also been linked to dysbiotic states such as Crohn’s Disease,

Irritable Bowel Disease, and Dementia (17, 19, 33). These studies provide insight into how thiamine and microbial species impacts human health. However, little is known about how thiamine impact the prominent gut phylum Bacteroidetes.

Work to characterize and understand thiamine biosynthesis and transport among many microbes such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and

Bacteroides thetaiotaomicron and the role it plays in their fitness continues (4, 8). A complex suite of enzymes performs thiamine biosynthesis. Despite this diversity, thiamine biosynthesis is performed through a conserved bifurcated pathway. TPP is biosynthesized by first synthesizing 4-amino-2-methyl-5-diphosphomethylpyrimidine

103 (HMP-PP) and 2-(2-carboxy-4-methylthiazol- 5-yl) ethyl phosphate (cTHz-P). HMP-PP and cTHz-P are condensed into thiamine monophosphate (TMP) and then phosphorylated to the active TPP (4). While the steps of TPP biosynthesis are conserved. The impact thiamine biosynthesis has on the transcriptome and metabolome of an organism is remarkably diverse (2, 3, 8, 30). In addition to thiamine biosynthesis, there are many thiamine, thiamine monophosphate (TMP), TPP, and thiamine precursor transporters found across all domains of life (8, 14, 34, 35, 39, 49). These diverse thiamine transport and biosynthesis pathways are regulated by highly conserved TPP riboswitches.

Riboswitches are a highly conserved cis-encoded, cis-acting regulatory RNAs found in the untranslated region of the messenger RNA. These riboswitches control expression by binding the TPP aptamer and negatively regulating the genes under their control (7, 43, 50). TPP riboswitches are unique because they are the only riboswitch that can be found in all three domains of life (43). TPP riboswitches are so highly conserved, they have become key in identifying thiamine biosynthesis and transport genes (36). In bacteria, TPP riboswitches are known to regulate biosynthesis and transport genes at either the transcriptional or translational level. Transcriptional acting TPP riboswitches change the secondary structure of the mRNA creating a premature terminator stem-loop stopping transcription. TPP riboswitches that block translation of their regulon do so by occluding ribosomal binding sites (50). Transcriptional or translational acting TPP riboswitches are canonically believed to be split between Gram positive and Gram- negative bacteria respectively (28). It should be noted; previously characterized translational acting TPP riboswitches in E. coli also impact mRNA levels when significant portions of the coding region are added to transcriptional fusions (6). While

104 TPP riboswitch structures and functions have been studied and characterized in model organism (e.g. E. coli and B. subtilis) there has been surprisingly little characterization of these conserved regulatory elements among the abundant and prominent gut phylum,

Bacteroidetes.

Initial computational analysis predicts that TPP riboswitches are present and regulate predicted thiamin biosynthesis and transport pathways across the Bacteroidetes

(34, 36). TPP riboswitch controlled genes in Bacteroides thetaiotaomicron, a prominent member of the Bacteroidetes, have recently been characterized (8, 14). These data have shown that both thiamine transport and biosynthesis are critical to the fitness of B. thetaiotaomicron. Despite understanding that thiamine acquisition is important among

Bacteroidetes species; little work has been done to characterize how the predicted TPP riboswitches impact these genes. Here, we have attempted to study how TPP riboswitches are utilized by the Bacteroidetes. We have (i) leveraged transcriptomic data to observe how exogenous thiamine impacts the whole transcriptome in addition to TPP riboswitch controlled genes. In addition, we (ii) leveraged translational fusions on individual TPP riboswitches from representative and abundant Bacteroides species. Lastly, we (iii) carried out in silico analysis and identification of TPP riboswitches across the

Bacteroidetes to better understand the regulation of thiamine acquisition in the gut.

105 Results:

Global transcriptional responses vary between Bacteroides species

We have previously shown B. thetaiotaomicron differentially expresses certain metabolic pathways when thiamine is scarce (8). To determine if this trend is consistent across Bacteroides species we replicated an RNA sequencing experiment that we performed previously on B. thetaiotaomicron in Costliow and Degnan 2017 (8). To this end, strand-specific RNA sequencing of cells grown in minimal medium that was thiamine replete (15 µM) or deplete was carried out for Bacteroides uniformis and

Bacteroides vulgatus in comparison to our previously published RNA sequencing data

(FIG 3.1). Samples of each species were sequenced in duplicate for both thiamine replete and deplete conditions. In total, 173,849,709 reads were generated (ranging between 8 and 20 million reads per sample), quality trimmed, filtered, and analyzed utilizing

Rockhopper (Table 3.1) (8, 23, 45).

Whole transcriptomic responses to exogenous thiamine were markedly different between B. thetaiotaomicron, B. uniformis, and B. vulgatus. As previously shown, B. thetaiotaomicron had 151 genes significantly (false-discovery rate [FDR], q ≤ 0.05) differentially expressed by ≥ 2-fold in excess thiamine (FIG 3.1A). Significantly down regulated genes are representative of thiamine biosynthesis, amino acid biosynthesis,

TCA cycle enzymes, purine, and pyruvate metabolism (8). In contrast to B. thetaiotaomicron, transcriptomic responses observed in B. uniformis and B. vulgatus are more subdued in response to excess thiamine (FIG 3.1B and C). Between B. vulgatus and

B. uniformis, only B. uniformis showed a significant response to exogenous thiamine

(FIG 3.1B). B. uniformis down regulated 6 genes significantly, all of which are readily

106 identifiable as TPP riboswitch controlled thiamine biosynthesis genes. In addition, the predicted thiamine transport operon B. vulgatus did not show a transcriptional response to exogenous thiamine (FIG 3.1C). This lack of a response may be due in part to the unique genetic context of B. vulgatus and other closely related Bacteroides species. B. vulgatus does not have a readily identifiable outer membrane transporter to import thiamine into the cell. Lack of the outer membrane transporter suggests that thiamine may not be imported into the cell. In addition, B. vulgatus does not have a TPP riboswitch preceding the computationally predicted inner membrane transporter, pnuT. The absence of a TPP riboswitch preceding pnuT may suggest that the inner membrane transporter does not have specificity for thiamine. Both factors imply the lack of a significant transcriptomic response in B. vulgatus to exogenous thiamine is due in part to an absence of thiamine transport (8).

The different transcriptomic profiles of B. thetaiotaomicron, B. uniformis, and B. vulgatus in response to thiamine were unexpected. All three species are closely related with similar thiamine acquisition and biosynthesis genes. The divergent transcriptomic responses of closely related organisms that a complex level of diversity and regulation exist in the gut and that thiamine may play a key role in the structure of the gut microbiome.

Thiamine biosynthesis and transport genes have divergent transcriptional responses

The impact of the predicted TPP riboswitches regulation on thiamine biosynthesis and transport genes is not understood. To better understand the role of TPP riboswitches we investigated the riboswitch-controlled operons mentioned above. All together 6 TPP riboswitch controlled operons were investigated in B. thetaiotaomicron (3), B. uniformis

107 (2), and B. vulgatus (1) (FIG 3.2). B. thetaiotaomicron has three TPP riboswitch controlled operons (1 biosynthesis and 2 transport operons). Of these three operons only the biosynthetic operon had genes that were significantly (false-discovery rate [FDR], q ≤

0.05) down regulated in excess exogenous thiamine. Transport genes in B. thetaiotaomicron did not show significant transcriptional regulation, but that does not rule out that the TPP riboswitches are acting at the level of translation, the canonical

Gram-negative TPP riboswitch mechanism (FIG 3.2A). The TPP riboswitch controlled operons in B. uniformis show transcriptional response to exogenous thiamine. The thiamine biosynthesis operon regulated by a TPP riboswitch had a 3-fold decrease in expression in which 6 of the 8 genes were deemed significantly so (false-discovery rate

[FDR], q ≤ 0.05). The single three-gene thiamine transport operon also showed ~2.5 fold change in expression, but the changes in transcript level were not considered significant

(q > 0.05) (FIG 3.2B). Again, B. vulgatus does not show a response to exogenous thiamine even at the TPP riboswitch controlled thiamine biosynthesis operon (FIG 3.2C).

It is worth noting B. vulgatus does not have a riboswitch controlled pnu-like (inner membrane) or tonB (outer membrane) transporters, making it likely that there is not a specific thiamine transport system (8).

To confirm the RNA sequencing profiles observed in B. thetaiotaomicron, B. uniformis, and B. vulgatus, RT-qPCR was performed. Probes for RT-qPCR were designed for a biosynthesis gene (thiC), the outer membrane tonB dependent transporter

(OMthi), and the predicted inner membrane transporter (pnuT) for each organism they are present in. Among the RT-qPCR probes, the biosynthesis gene, thiC, is regulated by a

TPP riboswitch in all three organisms tested. Consequently, OMthi and pnuT are only

108 under the control of a TPP riboswitch in B. thetaiotaomicron and B. uniformis. In addition, despite lacking a TPP riboswitch only the inner membrane transporter can be identified in B. vulgatus (FIG 3.3). The trends observed in all three organisms for RNA sequencing were replicated in the RT-qPCR experiments. B. thetaiotaomicron and B. uniformis both showed a strong transcriptional response at thiC. Respectively, thiC is expressed 60-fold and 2-fold higher in 0 nM thiamine as compared to its expression in 10

µM thiamine in both B. thetaiotaomicron and B. uniformis (FIG 3.3A and B). The expression of the riboswitch regulated inner (pnuT) and outer (OMthi) membrane thiamine transporter in B. thetaiotaomicron showed a minimal change in expression between high and low thiamine conditions (FIG 3.3A). In contrast to B. thetaiotaomicron,

B. uniformis shows a strong transcriptional response across the TPP riboswitch controlled operon that contains both OMthi and pnuT. Respectively, OMthi and pnuT are significantly up regulated (p < 0.05) 2.8-fold and 2.7-fold in the absence of thiamine (FIG

3.3B). RT-qPCR also validated that B. vulgatus appears to be transcriptionally blind to exogenous thiamine. The expression of the riboswitch controlled thiC gene shows no significant response. In addition, the predicted inner membrane thiamine transporter also shows no response. It is worth noting, the predicted pnuT lacks a TPP riboswitch. The lack of a transport regulating riboswitch suggests that the predicted pnuT may be in active or is not specific for thiamine.

These transcriptional data suggest thiamine biosynthesis can be dramatically altered by exogenous thiamine when a readily identifiable thiamine transport system is present. All regulation observed is likely performed by the predicted TPP riboswitches as proteins that regulate these pathways have not been identified among bacteria (28).

109 Bacteroides utilize transcriptional and translational acting TPP riboswitches

In addition to RT-qPCR, we investigated if TPP riboswitch controlled operons that did not show a transcriptional response to exogenous thiamine were possibly regulating at the level of translation. To this end, we built translational fusions of the nanoluciferase gene in frame to the first three codons of genes under the control of a TPP riboswitch. All nanoluciferase fusions are under control of their native promoters in addition to the cis-acting TPP riboswitches (20, 24). Fusions for all six predicted TPP riboswitches in B. thetaiotaomicron (3), B. uniformis (2), and B. vulgatus (1) were tested across various exogenous thiamine concentrations (0 - 10,000 nM). Control vectors of the nanoluciferase gene without a promoter were tested in each of the bacterial backgrounds at 10,000 and 0 nM concentrations. These controls showed that thiamine alone did not significantly alter luminescence (RLU/OD630: 750-956). These fusions along with additional RT-qPCR analysis provided insight into the mechanism of TPP riboswitch regulation (FIG 3.4). In addition, the nanoluciferase fusions provided the concentrations of thiamine required for TPP riboswitch regulation among Bacteroides species (FIG

3.4A-D).

Unsurprisingly, strong regulatory responses in line with our RNAseq and RT- qPCR results (FIG 3.2 and 3) were observed. The nanoluciferase gene under the control of the thiamine biosynthesis riboswitch of B. thetaiotaomicron (Bt pTPP1-NanoLuc) showed significant down regulation as the concentration of thiamine increased. In addition, the Bt pTPP1-NanoLuc fusion showed that an exogenous concentration of thiamine greater than 1 nM was required significantly decrease expression (p ≤0.05, student's t-test) of thiamine biosynthesis in B. thetaiotaomicron (FIG 3.4A). In contrast to

110 transcriptomic data that showed no response (FIG 3.2A and 3A); significant (p ≤0.05, student's t-test) down regulation of transport regulating riboswitches in B. thetaiotaomicron (Bt pTPP2/3-NanoLuc) are reached when exogenous concentrations of thiamine are above 10 nM (FIG 3.4A). The above data in coordination with the nanoluciferase data suggest that B. thetaiotaomicron is novel in utilizing both transcriptional (Bt pBtTPP1-NanoLuc) and translational (Bt pTPP2/3-NanoLuc) acting

TPP riboswitches.

In agreement with the aforementioned transcriptional data (FIG 3.2B and 3B), both TPP riboswitches in B. uniformis significantly (p ≤0.05, student's t-test) regulate the genes under their control (FIG 3.4B). These data suggest that both TPP riboswitches in B. uniformis act at the level of transcription. Consistent with trends observed in B. thetaiotaomicron, the TPP riboswitch that controls expression of biosynthesis (Bu pBuTPP1-NanoLuc) is significantly down regulated when external thiamine concentrations exceed 1 nM. In addition, the transport regulating riboswitch of B. uniformis (Bu pBuTPP2-NanoLuc) are significantly down regulated at thiamine concentrations above 10 nM (FIG 3.4B).

Also in agreement with our previous transcriptional data (FIG 3.2C and 3.3C), the thiamine biosynthesis regulating riboswitch in B. vulgatus (Bv pBvTPP-NanoLuc) does not show any change in expression across various concentrations of exogenous thiamine

(FIG 3.4C). The lack of a response in B. vulgatus is likely due to the lack of a defined thiamine transport system. To ensure the TPP riboswitch in B. vulgatus was actually active and functional we moved pBvTPP-NanoLuc into a B. thetaiotaomicron background (Bt pBvTPP-NanoLuc). B. thetaiotaomicron, unlike B. vulgatus, has an

111 active and characterized transport system (8, 14). In a B. thetaiotaomicron background the TPP riboswitch of B. vulgatus responds to exogenous thiamine. Bt pBvTPP-NanoLuc is significantly down regulated at exogenous concentrations of thiamine equal to or greater than 1 nM (FIG 3.4D).This response is similar to both the B. thetaiotaomicron and B. uniformis thiamine biosynthesis regulating TPP riboswitches (FIG 3.4A and B).

The response observed in Bt pBvTPP-NanoLuc shows that the biosynthesis regulating riboswitch of B. vulgatus is active. This does not however provide the mechanism of regulation for this riboswitch.

To characterize the regulatory mechanism implemented by BvTPP we leveraged

RT-qPCR of the nanoluciferase gene to rule out or confirm transcriptional regulation

(FIG 3.4E). Transcriptional assays were carried out in minimal medium with 10,000 nM

(+) or 0 nM (-) thiamine added. As a control we utilized the transcriptional acting TPP riboswitch in B. thetaiotaomicron (Bt pBtTPP1-NanoLuc). In addition, we utilized both

Bt pTPP2-NanoLuc and Bt pTPP3-NanoLuc as controls for riboswitches that act at the level of translation. The biosynthesis regulating TPP riboswitch in B. vulgatus (Bt pBvTPP-NanoLuc) is significantly down regulated in the presence of thiamine. Bt pBvTPP-NanoLuc responds similarly to the transcriptional acting B. thetaiotaomicron

TPP riboswitch (Bt pTPP1-NanoLuc) suggesting that the TPP riboswitch in B. vulgatus operates at the level of transcription (FIG 3.4E).

These data suggest Bacteroides species utilize both transcriptional and translational acting TPP riboswitches to maintain homeostasis of thiamine. The nanoluciferase assays also show that thiamine biosynthesis is down regulated at levels of thiamine above 1 nM of thiamine. Thiamine transport is regulated at a 10-fold higher

112 concentration of thiamine (> 10 nM) in all three of the species tested. These data define a clear regulatory hierarchy between thiamine biosynthesis and transport.

Distances of TPP riboswitches predict regulon type and mechanism

The transcriptional and translational data from B. thetaiotaomicron, B. uniformis, and B. vulgatus pointed to a trend in how riboswitch distance from the regulon relates to the mechanism of regulation. To determine if this trend is conserved across the

Bacteroidetes; we leveraged in silico analysis of 114 Bacteroidetes genomes. Among these 114 genomes, 219 TPP riboswitches were predicted and then categorized by the genes that they regulate (FIG 3.5A, Table 3.2). Among the 219 riboswitches, 122 of them were predicted to regulate putative thiamine biosynthetic operons. All aspects of thiamine biosynthesis (e.g. HMP-PP, cThz-P, and condensation) are tightly controlled by TPP riboswitches across Bacteroidetes species. A closer look riboswitch controlled operons show that closely related Bacteroides species have similarly ordered biosynthetic operons. More distantly related Bacteroidetes (e.g. Parabacteroides and Paraprevotella) species appear to have split biosynthesis operons, but have acquired a second TPP riboswitch in order to fully regulate the process. The other 97 TPP riboswitches regulate predicted thiamine transport genes, primarily operons that start with OMthi (85/97) or pnuT (12/97)(FIG 3.5A,Table 3.2). In contrast to the diversity observed among TPP riboswitch controlled thiamine biosynthesis operons; thiamine transport operon organization is highly conserved. TPP riboswitches regulate both the inner and outer membrane transporters in addition to a thiamine pyrophosphokinase (tnr3).

113 Turning our attention to the mechanism of regulation, we identified the distance of the TPP riboswitches from the first gene in the regulon (Table 3.2). We then plotted the distances of each riboswitch from its regulon along with the predicted function of each operon (FIG 3.5B). The average distance of the 122 thiamine biosynthesis regulating are approximately 60 nucleotides (± 34 nucleotides)(Table 3.2) suggesting that most biosynthesis operons are regulated at the level of transcription (FIG 3.5B).

Conversely, many TPP riboswitches regulating thiamine transport operons are closer to their regulon (21 ±20 nucleotides)(Table 3.2) suggesting that they might be regulating through ribosomal occlusion (translation) (FIG 3.5B).

The presence and trends observed throughout the Bacteroidetes show a clear trend that Bacteroidetes species are novel in utilizing both transcriptional and translational acting TPP riboswitches. Further characterization of this observed trend would help predict how this prominent class of gut microbes will respond to the availability of thiamine in the gastrointestinal tract.

Discussion:

Thiamine plays an essential role in host and microbial metabolism, as such the acquisition of thiamine is key for host health and microbial abundance. It is believed that most of thiamine in the gastrointestinal tract is made available by the host diet (37, 39), and that microbial prominence may be linked to gaining required levels of thiamine (8).

Here, we have shown that Bacteroides species are novel in employing both transcriptional and translational acting TPP riboswitches. To this end, we utilized

114 expression analysis in the prominent and abundant gut microbes B. thetaiotaomicron, B. uniformis, and B. vulgatus, characterizing the mechanism employed by thiamine biosynthesis and transport regulating TPP riboswitches in these species. In addition, we utilized in silico approaches to identify if TPP riboswitches across the Bacteroidetes. The identification of these TPP riboswitches also helped us predict the mechanism of regulation used by these riboswitches. We also identified a clearly conserved regulatory hierarchy between biosynthesis and transport regulating TPP riboswitches.

Our data suggest that the Bacteroides may also be utilizing different types of riboswitch regulation to carry out the regulation of thiamine biosynthesis and transport

(FIG 3.2, 3.3, and 3.4). Leveraging both transcriptional and translational assays we determined that riboswitches that are close to their regulon (<20 bp) regulate by blocking translation, while riboswitches shown to be further upstream (>50 bp) are acting through transcriptional attenuation. These mechanisms have been shown to be canonical mechanisms of regulation for TPP riboswitches. However, the mechanisms of regulation were thought to be divided between Gram negatives (translational blocking) and Gram positives (transcriptional attenuation) (16, 28, 44). In B. thetaiotaomicron, B. uniformis, and B. vulgatus transcriptional assays have shown that thiamine biosynthesis regulating riboswitches affect a strong transcriptional response (FIG 3.2A, 3.2B, 3.3A, 3.3B, and

FIG 3.4E). In addition, the transport regulating riboswitch in B. uniformis illicts a strong transcriptional response in response to thiamine (FIG 3.2B and FIG 3.3B). These riboswitches are greater than 40 nucleotides away from the start codon of their regulon, indicating that they may be acting through transcriptional termination by forming a stem- loop structure. In addition to these transcriptional responses, we saw no significant

115 transcriptional regulation from riboswitches under 40 nucleotides from their start codon

(transport regulating riboswitches) (FIG 3.2 and FIG 3.3). We then leveraged translational fusions (FIG 3.4) and saw regulation from all riboswitches tested. This suggests that riboswitches that did not show a significant transcriptional response operate at the level of translation. Further analysis of these riboswitches and the untranslated regions preceding thiamine biosynthesis and transport genes across the Bacteroidetes are required to characterize the mechanism regulation. These initial data imply that

Bacteroidetes are unique in the fact that they utilize both transcriptional and translational acting riboswitches.

Building on the data that the Bacteroides species tested appear to have both transcriptional and translational acting riboswitches and that the mechanism is linked to the distance from its regulon. We identified the TPP riboswitches across the

Bacteroidetes confirming and expanding on the present data available we identified 219

TPP riboswitches (FIG 3.5A)(8, 36). Of these 219 TPP riboswitches 122 of them are predicted to regulate biosynthesis genes and 97 regulated putative thiamine transport genes (Table 3.2). Manual inspection of the genomes in which these predicted riboswitches shows that they only regulate thiamine biosynthesis or transport. We then identified the distances of these TPP riboswitches from their regulons. Predicted distances of TPP riboswitches that regulate biosynthesis genes are much farther from the presumed regulon (FIG 3.5B) suggesting that they regulate through transcriptional termination. This prediction is based on data from B. thetaiotaomicron (biosynthesis), B. uniformis (biosynthesis and transport), and B. vulgatus (biosynthesis) that regulate through transcriptional termination (FIG 3.2, FIG3, and FIG 3.4E). In contrast, many

116 transport regulating riboswitches are much more proximal to the genes they regulate.

This suggests that proximal transport regulating TPP riboswitches occlude the ribosomal binding site (FIG 3.5B). This data is based off of characterized translational acting riboswitches identified in B. thetaiotaomicron (FIG 3.3A and FIG 3.4A). Further characterization of these trends is warranted. Inspecting RNA stability, mapping individual riboswitch structure, and a better understanding of Bacteroidetes ribosomal binding sites would help in clarifying the precise mechanism of regulation. In addition, further transcriptional assays may help in narrowing down the transition from transcriptional to translational acting riboswitches and determine if distance is the sole factor in the mechanism of TPP riboswitch regulation.

In addition our results clearly show TPP riboswitches respond to different magnitudes of exogenous thiamine. In B. thetaiotaomicron and B. uniformis, the transport regulating riboswitches decrease the expression of the genes when the concentrations of thiamine are greater than 10 nM (FIG 3.4A,B). Comparatively, the biosynthetic regulation riboswitches in B. thetaiotaomicron, B. uniformis, and B. vulgatus are an order of magnitude more sensitive to exogenous thiamine. Biosynthesis regulating riboswitches block transcription at concentrations below 1 nM of thiamine (FIG 3.4A,B,and D). Our studies showed that in the wildtype background we get no response from the TPP riboswitch in B. vulgatus (FIG 3.1C, FIG 3.2C, and FIG 3.3C). We hypothesized that B. vulgatus may not respond to exogenous thiamine because it lacks a dedicated thiamine transport system. This hypothesis became more plausible upon further investigation of the genomic context of thiamine acquisition in B. vulgatus. Through in silico analysis we could not identify TPP riboswitch controlled inner (pnuT) or outer (OMthi) membrane

117 transporters (FIG 3.5A). As TPP riboswitches are highly conserved and appear to regulate thiamine biosynthesis and transport across the Bacteroidetes we suggest B. vulgatus relies solely on the biosynthesis of thiamine and that the predicted pnu-type transporter is likely a pnuC homolog that transports vitamin B3 (18). Further characterization of the pnu-type transport homologs across the Bacteroidetes is warranted. To better understand the regulation of the B. vulgatus biosynthesis TPP riboswitch we moved its reporter fusion into B. thetaiotaomicron that has a partially characterized thiamine transport system (8, 14). Under this system we see the B. vulgatus riboswitch now regulates similarly to the other thiamine biosynthesis controlling TPP riboswitches (FIG 3.4D). We then confirmed through RT-qPCR that the B. vulgatus TPP riboswitch acts at the level of transcription (FIG 3.4E). Remarkably, the levels at which transport and biosynthesis regulating riboswitches change expression are consistent in the

Bacteroides species tested (FIG 3.4A-D).

TPP riboswitches appear to have conserved mechanisms and regulatory concentrations of thiamine. In contrast, the global transcriptome of B. thetaiotaomicron,

B. uniformis, and B. vulgatus are markedly different from one another (FIG 3.1). As shown previously, B. thetaiotaomicron down regulates multiple metabolic pathways upon the addition of thiamine to the environment (8). However, B. uniformis only down regulates its thiamine biosynthesis genes to any significant degree and B. vulgatus as stated above shows no response to exogenous thiamine (FIG 3.1B,C). These different expression profiles show each organism may have vastly different thiamine requirements and utilizations strategies in order to cope with variable thiamine availability in the human gastrointestinal tract. These differences may allow for different species to become

118 prominent at disparate thiamine concentrations. To fully understand these differences a meta-transcriptomic profile of a gut community in various concentrations of thiamine in addition to analysis of the prominence of each microbe under these conditions is needed to clarify the importance of thiamine in the gut.

The regulatory mechanisms of thiamine acquisition play an important role in the

Bacteroidetes. The study of how thiamine impacts microbial metabolism and global expression is necessary for understanding how certain organisms rise to prominence in the gastrointestinal tract. By further characterization of thiamine acquisition and regulation we can begin to design therapies through utilizing compounds that target TPP riboswitches (8, 31) and model how to change microbial communities mitigating diseases such as diabetes, obesity, and many other diseases.

Methods:

Bacterial culturing and genetic manipulation

Culturing of Bacteroides strains B. thetaiotaomicron VPI-5482, B. uniformis

ATCC 8492, and B. vulgatus ATCC 8482 occurred anaerobically at 37°C in liquid tryptone yeast extract glucose (TYG) medium (25) or a modified minimal medium with 0 to 10 µM of thiamine hydrochloride supplemented depending on the assay and strain (8,

40, 47) or Difco brain heart infusion (BHI) agar with the addition of 10% defibrinated horse blood (QuadFive, Ryegate, MT, USA). Cultures were grown anaerobically within a vinyl anaerobic chamber with an input gas mix consisting of 70% nitrogen, 20% carbon dioxide, and 10% hydrogen (Coy Laboratory Products, Grass Lake, MI, USA). E. coli

119 S17-1 λ pir strains used for cloning and conjugation of reporter vectors were grown in

LB medium at 37°C aerobically. Antibiotics were added to the medium when appropriate in the concentrations as follows: ampicillin 100 µg/mL, gentamicin 200 µg/mL, and erythromycin 25 µg/mL.

Nanoluciferase reporter vectors were constructed via traditional cloning of the nanoluciferase gene (20,24) into the vector pNBU2 (9, 10, 22). TPP riboswitches were then amplified from genomic DNA by PCR using HiFi Taq MasterMix (KAPA

Biosystems, Wilmington, MA, USA) and inserted in frame preceding the nanoluciferase gene in the pNBU2 vector. These vectors were then transformed into donor E. coli S17-1

λ pir. Reporter vectors were then confirmed via PCR amplicon size as well as sequencing. After confirmation vectors were then conjugated into B. thetaiotaomicron

VPI-5482, B. uniformis ATCC 8492, and B. vulgatus ATCC 8482 making a single insertion into the genome of each strain (9, 10, 22). A complete list of primers and vectors used for this study are provided (Table 3.3).

Expression analysis of TPP riboswitch controlled operons

Replicate cultures of wildtype B. uniformis and B. vulgatus were grown overnight in 5 mL minimal medium supplemented with a concentration of 10,000 nM thiamine HCl

(THI, > 99% pure) (Sigma Aldrich, St. Louis, MO, USA). Aliquots of each culture were pelleted by centrifugation (1 min at 13,300 x g), culture supernatants were decanted and the cells were washed 4 times in minimal medium with 0 nM thiamine. Cells were inoculated into 10 mL of minimal medium with either 0 or 15 µM thiamine HCl at a final dilution of 1:2,000 in biological duplicate. Cell growth was monitored and cells were

120 harvested between OD600 0.4 and 0.6 on a UV spectrometer. Total RNA was extracted using the Qiagen RNeasy kit and stored at -80°C. Total RNA was DNAse treated with

DNase I (Thermo Fisher, Waltham, MA, USA) and NEB DNase treatment protocol.

DNAse treated RNAs were re-cleaned using the Qiagen RNeasy kit (Hilden, Germany) and quantitated using a Qubit 2.0 (Life Technologies, Carlsbad, CA, USA) and stored at -

80°C. This was the same method used for a previously published RNA sequencing experiment for B. thetaiotaomicron in Costliow and Degnan 2017 (8).

RNA was submitted for integrity analysis, ribosomal RNA depletion, and library construction at the W.M. Keck Center for Comparative and Functional Genomics at the

University of Illinois at Urbana-Champaign.

For targeted gene expression analysis, total RNA was purified as above and used to generate cDNA libraries using the first strand cDNA synthesis kit (Thermo Fisher,

Waltham, MA, USA). RT-qPCR was performed on a Biorad CFX connect instrument

(Biorad, Hercules, CA, USA) and SYBR Fast MasterMix 2x Universal (Kapa

Biosystems, Wilmington, MA, USA) following the manufacturer’s instructions for triplicate biological samples in technical triplicate. Five genes in three strains were amplified 16 ribosomal RNA, thiC, thiS, OMthi, and pnuT. 16S rRNA primers were used as the control gene (9) and novel primers for thiC, thiS, OMthi, and pnuT for B. thetaiotaomicron (8), B. uniformis, and B. vulgatus were designed using Primer3 (46).

Standard curves were used to evaluate the efficiency of the amplification, all 5 genes had

R2 ≥ 0.98 and slope between -3.30 and -3.40. Relative expression changes were calculated using the ∆∆Cq method (5).

121 Nanoluciferase reporter fusion assays

Reporter assays were performed through a modified and combined protocol of

Lim et al. 2017 and Mimee et al. 2015 (20, 24). Bacteroides species with an integrated pNBU2 TPP riboswitch nanoluciferase reporter were grown in a modified minimal medium and then washed and back diluted to an OD600 of 0.004 in 1.5 mL Axygen deep

96-well plates (Corning, Inc., Corning, NY) over a gradient of thiamine HCl (Sigma-

Aldrich, St. Louis, MO). Growth was monitored by via a BioTek Synergy HTX Multi-

Mode microplate reader (BioTek, Winooski, VT) (8).

When cells reached mid-log phase growth (0.35-0.58 OD600) growth was stopped.

1 mL of mid log culture was spun down in the Axygen deep well plate at 3,000-x g for 10 minutes and medium was poured off of pellets. Cell pellets were then resuspended in

50uL of 10x BugBuster protein extraction reagent (MilliporeSigma, Darmstadt,

Germany). Pellets-BugBuster suspension was then incubated at room temperature while gently shaking for 10 minutes. Nanoluciferase extracts were then mixed 1:1 (10 µL:10

µL) with Nano-Glo Luciferase mixture. Reporter mixture was then allowed to incubate for 5 minute at room temperature. Nanoluciferase luminescence was then read on BioTek

Synergy HTX Multi-Mode microplate reader (BioTek, Winooski, VT).

Relative Luminescence Units were then normalized to the OD600 of each respective pellet. Comparisons were then determined across concentrations for each TPP riboswitch Nanoluciferase reporter utilizing Pairwise Student’s t tests in GraphPad Prism v6, and P values of ≤0.05 were considered significant.

122 In silico identification of TPP riboswitches in Bacteroides species

Detection of TPP riboswitches among Bacteroidetes was done in the same manner as Costliow and Degnan 2017 (8). Infernal 2.0 was used to identify these riboswitches using the covariance model of TPP riboswitches in RFAM (RF00059)(15, 26) in 114

Bacteroidetes genomes available of RefSeq from the Human Microbiome Project (27,

29). In an attempt confirm that Infernal predicted riboswitches were TIGRFAM, PFAM, and Hidden Markov Models with HMMR were used to identify thiamine pyrophosphate biosynthesis and transport genes among the same 114 RefSeq Bacteroidetes genomes (13,

29, 41).

To identify trend of distances between riboswitches and the genes they were predicted to regulated. Nucleotides distances between coordinated identified with Infernal

2.0 and TIGRFAM, PFAM, and HMMR were taken and subtracted from one another (12,

15, 26, 41). Annotations and genes were then manually inspected utilizing Artemis to confirm that genes and riboswitches were called correctly and that distances were accurate and agreed with previous predictions.

Acknowledgements:

We thank members of the Degnan and Vanderpool labs at the University of

Illinois at Urbana-Champaign for comments on the manuscript. In addition, we thank Dr.

Andrew Goodman and Dr. Bentley Lim from Yale University for protocols and plasmids associated with the NanoLuciferase reporter system.

123 We also thank Alvaro Hernandez from the W.M. Keck Center for Comparative and Functional and Genomics at the University of Illinois at Urbana-Champaign for their technical services.

124 Figures:

A B 5 105 B. thetaiotaomicron 105 B. uniformis C 10 B. vulgatus

m 4 104 104 10 e Mediu

3 103 103 10 hiamin T

Minimal

M in

μ 2 102 102 10 15 sion + s 1 1 xpre 101 10 10 E

0 0 100 10 10 2 3 4 5 2 3 4 5 0 1 2 3 4 5 100 101 10 10 10 10 100 101 10 10 10 10 10 10 10 10 10 10 Expression in Minimal Medium Expression in Minimal Medium Expression in Minimal Medium

Down-Regulated Up-Regulated Non-Differentially Regulated Fig. 1 FIG 3.1. Global transcriptional response to exogenous thiamine

RNA sequencing was carried out on (A) B. thetaiotaomicron (previously in Costliow and

Degnan 2017), (B) B. uniformis, and (C) B. vulgatus in a defined minimal medium that was thiamine deplete (0 nM thiamine added) and thiamine replete conditions (15 µM thiamine added). Grey circles indicated genes that are neither significantly up- nor down- regulated. Green circles indicate genes that are significantly up regulated. Red circles indicate genes that are significantly down regulated in the presence of excess exogenous thiamine. Significance was defined by a q-value ≤ 0.05 for samples analyzed in

Rockhopper RNA sequencing analysis tool and a change in expression of ≥ 2-fold between conditions. The experiment was carried out in biological duplicate.

125 A

2.1 1.3 1.6 3.2 3.7 2.5 1.3 0.9 1.8 0.7 thiS* thiE thiG thiC* thiH* thiF tenI OMthi pnuT tnr3 BT0653-BT0647 BT2390 BT2396-BT2397 804,000 802,400 800,800 799,200 797,600 2,978,400 2,980,000 2,992,000 2,993,600

B

3.1 3.3 3.6 3.6 3.7 3.7 3.2 3.6 2.8 2.4 2.4 thiS* thiE* thiG* thiC* thiH* thiF* tenI thiD OMthi pnuT tnr3 BACUNI00698-BACUNI00705 BACUNI02372-BACUNI02374 742,000 744,000 746,000 2,355,000 2,356,000 2,357,000

C Fold Change ≥ -4 1.5 1.0 1.0 1.2 1.0 1.0 0.2 0.0 -3 thiS thiE thiG thiC thiH thiF pnuT tnr3 -2 BVU3540-BVU3547 BVU3637-BVU3636 4,503,600 4,502,000 ≤ -1 4,396,000 4,398,000 4,400,000 Fig. 2 FIG 3.2. Transcriptional response of TPP riboswitch controlled operons

Operons controlled by TPP riboswitches in the RNA sequencing experiment were investigated. Expression changes across operons are visualized in a scale schematic of each operon in which asterisk (*) and bold outlines indicate significance (q-value ≤ 0.05 and a change in expression ≥ 2-fold). Each species investigated (A) B. thetaiotaomicron,

(B) B. uniformis, and (C) B. vulgatus has a unique structure to their riboswitch controlled regulons and a differential transcriptional response. The experiment was carried out in biological duplicate. All operons and distances are represented to horizontal scale.

126 A 100 p < 0.0001

10 ± SD) (

ns ns hange

C 1

d l o F

0.1 Thiamine - + - + - + thiC OMthi pnuT B. thetaiotaomicron B 10 p = 0.0047

p = 0.0005 p = 0.0008 ± SD) (

1 hange C

d l o F

0.1 Thiamine - + - + - + thiC OMthi pnuT B. uniformis

C 10

ns ± SD)

( ns

1 hange C

d l o F

0.1 Thiamine - + - + thiC pnuT B. vulgatus FIGFig. 3.3. 3 RT-qPCR confirmation of TPP transcriptional response

127 FIG 3.3. (cont.)

Expression of TPP riboswitch regulated biosynthesis and transport genes were confirmed through RT-qPCR. In (A) B. thetaiotaomicron, (B) B. uniformis, and (C) B. vulgatus biosynthesis expression was analyzed through the expression of the thiC gene and transport by the expression of the predicted OMthi and pnuT gene when present.

Expression of each gene was compared to the expression of the gene in 10,000 nM exogenous thiamine (+) as opposed to medium with no added thiamine (-). Error bars represent the standard deviation (S.D.) of each experiment carried out in biological triplicate. Statistical significance for the expression of genes was determined via a student's t-test. Changes in expression were deemed significant when the fold change was

≥ 2-fold and had a p-value ≤ 0.05.

128 A 40 B 40 a,b h h a SEM) 30 SEM) d d ± ± ( 30 d

h ( h

630 b b h 630 d d c,d 20 c 20

x 1,000 c,d f f i x 1,000 d e i e e i a,b 10 10 a,b e a a e e g g RLU Normalized to OD g b,c c RLU Normalized to OD c c

0 0

1 0 1 0 1 0

0 1 0 1

10 10 10

10 10

0.1 0.1 0.1

0.1 0.1

100 100 100 100

[Thiamine] nM 100

0.01 0.01 0.01

0.01 0.01

1,000 1,000 1,000

1,000 1,000

10,000 10,000 10,000

10,000 10,000 Bt pBtTPP1-NanoLuc Bt pBtTPP2-NanoLuc Bt pBtTPP3-NanoLuc Bu pBuTPP1-NanoLuc Bu pBuTPP2-NanoLuc C D E 8 15 10 a p = 0.0001 a a a a p = 0.007 a a a,b a SEM) SEM) a ± ± 6 ( ( a 630 630 10 a

± SEM) ns ns 4 1 x 1,000 x 1,000 b 5 b b b

2 Fold Change ( RLU Normalized to OD RLU Normalized to OD

0 0 0.1

1 0

0 1 10

10 - + - + - + - + 0.1

0.1 Thiamine

100

100 0.01

[Thiamine] nM 0.01 Bt Bt Bt Bt

1,000

1,000 10,000 10,000 pBtTPP1- pBtTPP2- pBtTPP3- pBvTPP- Bv pBvTPP-NanoLuc Bt pBvTPP-NanoLuc NanoLuc NanoLuc NanoLuc NanoLuc

Fig. 4 FIG 3.4. Nanoluciferase assays of Bacteroides TPP riboswitches

Nanoluciferase translational fusions of all six TPP riboswitches found between (A) B. thetaiotaomicron, (B) B. uniformis, and (C) B. vulgatus were tested. The B. vulgatus riboswitch was also studied in a B. thetaiotaomicron background (D) to account for the lack of a response to exogenous thiamine seen by B. vulgatus. In addition RT-qPCR was carried out on Nanoluciferase fusions in B. thetaiotaomicron in thiamine replete (+) or thiamine deplete (-) conditions to confirm the mechanism of regulation by predicted riboswitches from B. thetaiotaomicron and B. vulgatus (E). Relative light units (RLU) were normalized to the OD630 of the bacterial culture before the lysis of cell and activation of the nanoluciferase reaction. Cells were harvested in mid-log for all samples

(OD630: 0.4-0.6).

129 FIG 3.4. (cont.)

The expression of each riboswitch-controlled fusion was represented as the averaged

RLU/OD630 x 1,000 from six independent biological representatives. Error bars represent the standard error of the mean (SEM). Letters (a, b, c, etc.) indicate groupings of that were significantly different from one another based off of a p-value ≤ 0.05 using a student’s t-test.

130 A

Biosynthesis Transport Bacteroides fragilis (16) B. nordii B. salyersiae B. thetaiotaomicron (5) B. faecis B. caccae (2) B. finegoldii (2) B. ovatus/xylanisolvens (15) B. oleiciplenus B. intestinalis B. cellulosilyticus (3) B. eggerthii (2) B. stercoris B. clarus B. uniformis (5) B. fluxus B. helcogenes B. coprophilus B. plebeius B. salanitronis B. coprocola B. vulgatus (5) B. dorei (7) Paraprevotella xylaniphila Pp. clara Prevotella salivae P. copri P. stercorea Parabacteroides merdae (3) Pb. johnsonii (2) Pb. goldsteinii Pb. distasonis (9) Tannerella forsythia Porphyromonas gingivalis (3) Tannerella. sp. Dysgonomonas mossii Odoribacter splanchnicus O. laneus Bacteroides sp. Alistipes indistinctus A. putredinis A. finegoldii (2) 0.2 a.a. substitutions Alistipes sp. per site A. shahii

Thiazole synthesis HMP synthesis Thiamine transport TPP riboswitch thiS thiC OMthi thiF thiD pnuT hypothetical gene thiH thi5 Thi pyrophosphokinase thiG tnr3 tenI TMP condensation thi4 thiE B 25 Biosynthesis Transport 20

15

10

5 No. of TPP riboswitches TPP No. of

0 Thi gene 240 200 160 120 80 40 0 Distance upstream of ATG (nt)

FIGFig. 3. 55. TPP riboswitches throughout the Bacteroidetes

131 FIG 3.5. (cont.)

Infernal and the RFAM 00059 covariance model were used to identify TPP riboswitches across the Bacteroidetes. (A) Predicted TPP riboswitches ORFs were then inspected to determine the operons directly preceded by putative TPP riboswitches. Function and syntenic context were assigned to all predicted riboswitch regulated operons. The distances of each TPP riboswitch from its predicted regulon was then recorded and plotted and divided into the subcategories of whether the riboswitch regulated a thiamine biosynthesis operon (red) or a thiamine transport operon (blue).

132 Tables:

Table 3.1. Reads of quality trimmed and mapped RNA sequencing Final SRA Processed Mapped % Species Treatment Replicate Accession Raw Reads Reads Reads Mapped

–Thi 1 SRP116143* 9,170,438 8,231,596 7,712,374 93.7%

B. 2 SRP116143* 8,258,740 7,454,637 6,830,124 91.6% thetaiota -omicron +Thi 1 SRP116143* 9,017,267 8,169,029 7,783,046 95.3%

2 SRP116143* 8,444,554 7,652,018 7,144,413 93.4%

–Thi 1 SRP148918 15,534,762 13,334,281 13,036,981 97.8%

B. 2 SRP148918 15,576,078 13,358,338 12,771,513 95.6% uniformi s +Thi 1 SRP148918 17,382,438 14,900,079 14,437,339 96.9%

2 SRP148918 15,505,061 13,290,981 12,756,005 96.0%

–Thi 1 SRP148917 19,391,595 13,334,281 13,036,981 97.8%

B. 2 SRP148917 18,821,066 13,358,338 12,771,513 95.6% vulgatus +Thi 1 SRP148917 18,447,248 14,901,073 14,437,549 96.9%

2 SRP148917 18,300,462 13,748,120 13,183,146 95.9% *denotes results previously published in Costliow and Degnan 2017

133 Table 3.2. Average distance distributions of TPP riboswitches from the first gene of their regulon

Gene TPP Riboswitch Distance Function Gene TIGRFAM / PFAM Riboswitches (Avg. ± St. Dev.)

thiS TIGR01683 / PF02597 77 64 ± 14 thiD TIGR00097 / PF08543 17 24 ± 34 thi4 TIGR00292 / PF01946 16 55 ± 19 thiE TIGR00693 / PF02581 9 106 ± 82 Biosynthesis thiC TIGR00190 / PF05690 3 132 ± 0 Total: 122 60 ± 34

N.A. / PF00593, OMthi PF07715, PF14905 85 23 ±23

Transport pnuT PF04973 / TIGR01528 12 3 ± 0 Total 97 21 ± 23

134 Table 3.3. Strains, vectors, and primers used Primer, Plasmid, or Strain name Oligonucleotide sequence (5'-3')/Notes1 Citation qPCR Primers Degnan et Bt_16s_F ggtagtccacacagtaaacgatgaa al. 2014 Degnan et Bt_16s_R cccgtcaattcctttgagtttc al. 2014 Costliow and Degnan Bt_thiC_F gagcagctccgatggcagag 2017 Costliow and Degnan Bt_thiC_R ggtccgggacacgtaccg 2017 Bt_pnuT_F tgtcggaatgtggatgctg This Study Bt_pnuT_R acaatgcggaggtgaaatagag This Study Bt_OMthi_F gttctttcaacacccacaagg This Study Bt_OMthi_R gcatcgaatgtccagtgattg This Study Bu_16s_F gcagaaagagtctatctgtc This Study Bu_16s_R tccgttttccacttataaga This Study Bu_thiC_F gcacagtatgacgttgccat This Study Bu_thiC_R cttcgatgaaagcctgcaca This Study Bu_pnuT_F ctggttggactccttcacca This Study Bu_pnuT_R catgcccaccattgctctac This Study Bu_OMthi_F aacaacgccacaaagaacga This Study Bu_OMthi_R ttgatgtggcggtattgcag This Study Bv_16s_F ggcttcttactttctctcttccg This Study Bv_16s_R cacaccgcccgtcaagccat This Study Bv_thiC_F atatcacttcggccatcggt This Study Bv_thiC_R tgggcaatcccagatgttct This Study Bv_pnuT_F cagggcaccgtactatctgt This Study Bv_pnuT_R caccgtctaagctgctgttg This Study NL_F tgtgaccccgaatatgattga This Study NL_R cgggttaatcagacgctc This Study

TPP riboswitch Primers Bt_TPP1_F atgggatccgcgggaatcgga agaaaaatg This Study ttgtctagatttcattgtcctatcagtattaataagt Bt_TPP1_R tg This Study Bt_TPP2_F tactctagagctgacttgtcatatttcaacaaactac This Study

135 Table 3.3. (cont.)

Bt_TPP2_R atcggatccggcgacggccatcat This Study Bt_TPP3_F gagtctagacacgacaaatgaggagaagag This Study Bt_TPP3_R tgcggatccctccagataattcatttccatatttgc This Study Bu_TPP1_F aaatctagacggttccaaacgtctcttattatc This Study aaaggatcctctgttcattaagaaataaaacaatgga Bu_TPP1_R agt This Study Bu_TPP2_F aaatctagacggcaataatcagcagaatgg This Study Bu_TPP2_R aaaggatcctctcttcatttgcaattgtttcgtttag This Study Bv_TPP1_F aaatctagagctgggtttcgatgtatgg This Study aaaggatcccagtttcatactatatcagtttatgaat Bv_TPP1_R ttacttgt This Study

NanoLuciferase Fusion Plasmids Plasmids with cloned fragments integrates at NBU2 Koropatkin pNBU2_erm att sites, ErmR, AmpR et al. 2008 pNBU2_erm plasmid with NanoLuciferase gene pNanoLuc inserted into the MCS This Study B. thetaiotaomicron TPP1 (biosynthesis) UTR and 3 pBtTPP1- codon thiS fusion added upstream and in frame of NanoLuc Nanoluciferase gene This Study B. thetaiotaomicron TPP2 (Transport) UTR and 3 pBtTPP2- codon OMthi fusion added upstream and in frame of NanoLuc Nanoluciferase gene This Study B. thetaiotaomicron TPP3 (Transport) UTR and 3 pBtTPP3- codon pnuT fusion added upstream and in frame of NanoLuc Nanoluciferase gene This Study B. uniformis TPP1 (biosynthesis) UTR and 3 codon pBuTPP1- thiS fusion added upstream and in frame of NanoLuc Nanoluciferase gene This Study B. uniformis TPP2 (Transport) UTR and 3 codon pBuTPP2- OMthi fusion added upstream and in frame of NanoLuc Nanoluciferase gene This Study B. vulgatus TPP (biosynthesis) UTR and 3 codon thiS pBvTPP1- fusion added upstream and in frame of Nanoluciferase NanoLuc gene This Study

136 Table 3.3. (cont.)

Parental Strains E. coli S17-1 λ thi pro hdsR hdsM+ recA, chromosomal insertion of Simon et al. pir RP4-2(Tc::Mu Km::Tn7) , AmpS 1983 B. thetaiotaomicron Koropatkin VPI-5482 ∆tdk 'wildtype' deletion background, BT2275–, FUDRR et al. 2008 B. uniformis 'wildtype' deletion background, BACUNI03774–, ATCC 8492 ∆tdk FUDRR This Study B. vulgatus ATCC 8482 ∆tdk 'wildtype' deletion background, BVU1857–, FUDRR This Study

Fusion Strains B. thetaiotaomicron VPI-5482 ∆tdk pBtTPP1-NanoLuc This Study B. thetaiotaomicron VPI-5482 ∆tdk pBtTPP2-NanoLuc This Study B. thetaiotaomicron VPI-5482 ∆tdk pBtTPP3-NanoLuc This Study B. uniformis ATCC 8492 ∆tdk pBuTPP1-NanoLuc This Study B. uniformis ATCC 8492 ∆tdk pBuTPP2-NanoLuc This Study B. vulgatus ATCC 8482 ∆tdk pBvTPP-NanoLuc This Study

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142 Chapter 4: SUMMARY AND FUTURE DIRECTIONS

Summary:

This thesis exams the impact thiamine has on the prominent gut phylum, the

Bacteroidetes. In addition, the mechanisms that regulate the level of thiamine in the

Bacteroidetes were also characterized. These studies showed that in Bacteroides species both thiamine biosynthesis and transport are required to remain competitive in the gut.

Consequently, the investigation of thiamine biosynthesis genes highlighted an unexpected diversity among these operons. This thesis also showed that Bacteroides thetaiotaomicron is novel in utilizing both transcriptional and translational acting TPP riboswitches. In addition, we viewed diverse transcriptional responses to thiamine across

Bacteroides species. These studies create a foundation for future studies that will attempt to alter dysbiotic states through the use of essential cofactors (e.g. thiamine).

Chapter 2 explains how gut microbes acquire thiamine. While thiamine biosynthesis has been well studied in model organisms, little work has been done to study these processes in the prominent gut phylum, the Bacteroidetes (1, 8, 12). Chapter 2 investigated the genomes of 641 gut microbes in an attempt to identify the thiamine biosynthesis and transport genes found in these organisms. Our results were in line with other studies, and showed that gut microbes primarily utilize previously characterized thiamine biosynthesis genes (10). This study also showed that, among gut bacteria, the

Bacteroidetes and γ-Proteobacteria are unique in possessing both complete biosynthesis and transport pathways (10). Thiamine biosynthesis operons among the gut Bacteroidetes however utilize a diverse set of genes to carry out this process (3, 10). In contrast, we confirmed that gut bacteria utilize a different thiamine transport system than the

143 previously characterized systems in Bacillus and Salmonella species (9, 16). This thiamine transport system, PnuT, is highly conserved throughout the Bacteroidetes (3, 5,

9). We also showed that PnuT only imports free thiamine. This is in contrast to other characterized bacterial thiamine transport systems that can import the phosphorylated moieties of thiamine in addition to the free-form (8, 9, 16).

In addition, Chapter 2 utilizes B. thetaiotaomicron as a model gut microbe to characterize the biosynthesis and transport of thiamine among Bacteroides species. To this end, we showed that both thiamine biosynthesis and transport systems are critical for

B. thetaiotaomicron. Deletion of either the biosynthesis or transport systems dramatically inhibit the growth of B. thetaiotaomicron. These data along with expression analyses, show that biosynthesis and transport systems are somehow interlinked. This connection is interesting and warrants further investigation into how they are linked. It would also be a worthwhile endeavor to characterize the flux that occurs from deleting these systems. The inhibited growth of thiamine biosynthesis or transport mutants in isolation directly correlates to a competitive defect in B. thetaiotaomicron thiamine acquisition mutants.

Considering that thiamine biosynthesis and transport systems are conserved across the

Bacteroidetes; we hypothesize that these results could be used to model the responses to thiamine for the entire phylum.

In Chapter 3, we focused on the TPP riboswitches that precede thiamine biosynthesis and transport genes among the Bacteroidetes. TPP riboswitches are universally conserved across all domains of life (7, 17). We found that TPP riboswitches universally precede the thiamine biosynthesis and transport operons in the Bacteroidetes investigated in Chapter 2.

144 While the TPP riboswitch structure is conserved, they have been shown to regulate through different mechanisms. In bacteria, TPP riboswitches can act by attenuating transcription or by blocking translation. The mechanism of regulation was canonically believed to be split between Gram negative (translation) and Gram positive

(transcription) bacteria (13, 14). Initial transcriptional analyses confirmed that some predicted TPP riboswitches were functional and attenuate transcription in Bacteroides species.

Building on the identification of TPP riboswitches, we characterized the function of these regulatory elements in B. thetaiotaomicron, B. uniformis, and B. vulgatus. The characterization of these riboswitches showed an explicit regulatory hierarchy between the riboswitches that regulate transport or biosynthesis of thiamine. We found that all biosynthetic riboswitches decreased the expression of biosynthesis at external concentrations of thiamine greater than 1 nM. In addition, we found that all the transport regulating riboswitches decreased the expression of the regulon at external concentrations of thiamine higher than 10 nM. We hypothesized that these riboswitches strategically regulate biosynthesis and transport operons in this manner. The strategic regulation of both acquisition pathways may be part of the reason that the Bacteroidetes are prominent members of the gut microbiome.

Consequently, our investigation of the TPP riboswitches identified that B. thetaiotaomicron is novel in employing both transcriptional (biosynthesis) and translational (transport) riboswitches. This was confirmed by leveraging translational fusions and transcriptional assays. Further study of the riboswitches in B. thetaiotaomicron suggested that the mechanism may be dictated by the distance of the

145 riboswitch from the first start codon of the regulon. Building on this trend we found that the distance phenomena is common across the Bacteroidetes. Commonly transport regulating riboswitches are much closer to their regulon. In contrast, biosynthesis regulating riboswitches are much farther upstream of the regulon. These data suggest that many of the Bacteroidetes may employ both transcriptional and translational acting riboswitches.

Together the data in this thesis increases our knowledge of how vitamins impact the gut microbiota. The impact thiamine has on the gut microbiota then informs how the two work together to affect human health. Our data may eventually lead to a better understanding of this dynamic biome. This increased understanding may then lead to novel therapies for the diseases linked to dysbiotic states of the gut microbiome.

Future Directions:

Are different thiamine biosynthetic strategies linked to species prominence?

Multiple bioinformatic studies have shown that divergent gene pathways carry out thiamine biosynthesis in the Bacteroidetes (3, 10). It is commonly seen that many

Bacteroides species possess both thi5 and thiC. Both of these genes biosynthesize HMP-

PP. Possessing both thiC and thi5, suggests a fitness advantage for using multiple precursors to synthesize the same molecule. In contrast, thiazole synthesis appears to have more constraints. Among the Bacteroidetes thi4 appears to displace the canonical bacterial synthesis genes (thiSFGH). This suggests that the loss of thiSFGH may be beneficial if thi4 has been acquired. This may be beneficial because thi4 requires fewer

146 precursors to synthesize thiazole than the thiSFGH pathway. In addition, the presence of different suites of thiamine biosynthesis genes may lead to the differential abundances we observe in the gut.

To further investigate this topic, specific deletions and complements could be made in a model species, such as B. thetaiotaomicron. Selectively deleting either thi5 or thiC in B. thetaiotaomicron may help give us insight into why both HMP-PP synthesis genes are retained. Initial tests would be to determine if either deletion impacts the ability of B. thetaiotaomicron to grow in thiamine limited minimal medium. In addition, we can confirm if these mutants are now auxotrophs for different thiamine precursors. To further characterize the mutants, we could compete them against one another in addition to wildtype strains. In these competitions, fitness defects or advantages may be sussed out.

Similar analyses can be performed for the synthesis of thiazole in B. thetaiotaomicron.

In contrast to thiC and thi5, thiSFGH and thi4 do not coexist in any Bacteroidetes genomes. To study these differences a thi4 gene will have to be cloned from a closely related Bacteroidetes member into a B. thetaiotaomicron mutant lacking thiSFGH. After these strains have been constructed, we can assess the precursors that each set of genes require to synthesize thiamine. These assays will help us confirm the current predictions for how each thiazole synthesis pathway works. We can then perform competition assays between B. thetaiotaomicron strains containing either thi4 or thiSFGH. These competition assays will help us ascertain if one set of thiazole synthesis genes is truly more efficient than the other in the gut.

Adding to the phenotypic and competitive assays, transcriptomic assays could be leveraged to see if relying on different thiamine synthesis genes impacts the global

147 transcriptome. We can then utilize metabolomic assays to investigate what the net metabolic flux of these different thiamine biosynthetic pathways are. Both assays would help inform why certain strains or pathways have higher or lower abundances in the gut.

What is the link between thiamine transport and biosynthesis?

In Chapter 2, we showed that both the thiamine biosynthesis and transport systems are critical to B. thetaiotaomicron (3). While this was an unexpected phenotype, it is not entirely novel. It has been shown that a similar phenomena occurs in Listeria monocytogenes (11). Both organisms encounter shifting thiamine concentrations in the environments they inhabit. This shifting level of availability may be why both systems have been maintained, but it does not explain why both systems are essential. In B. thetaiotaomicron, we showed that when the entire transport system is deleted there is a

10-100 fold down regulation of the biosynthetic operon (3). This dramatic down regulation suggests an uncharacterized link between the two systems.

In an attempt to study this link we must first determine why the biosynthetic operon is down regulated when transport is deleted. As there are no known protein regulators of thiamine biosynthesis, there must be another explanation for this down regulation (6, 15). One theory is that the deletion of transport creates excess free thiamine. Free thiamine could be increased if the tnr3 gene that recycles free thiamine to

TPP localizes to the deleted transport proteins. If tnr3 can no longer effectively localize, it may lose function preventing the conversion of free thiamine to TPP. Excess free thiamine could then bind and down regulate thiamine biosynthesis via the TPP riboswitch

(17). To test this theory we can use the thiamine biosynthesis riboswitch fusion utilized in

148 Chapter 3. The reporter system will conclusively tell us if the decreased expression of biosynthesis in the transport mutant is due to riboswitch dysregulation.

If this phenotype is not due to TPP riboswitch dysregulation, there may be a novel protein regulator of this system to consider. In order to identify this novel regulator, we may leverage a transposon mutagenesis library. This library would identify mutants that alleviate repression of the biosynthetic operon. Transposon insertions can then sequenced identifying what insertions improved the fitness of this mutant.

Together these studies may help explain the link between the two systems in the

Bacteroidetes. Learning how these systems are linked will in turn inform how thiamine impacts and changes the gut microbiome as a whole. In addition, characterizing the link between thiamine biosynthesis and transport may lead to the development of targeted antibiotics and therapies.

How does thiamine impact gut community structure?

Pairwise-competitions between B. thetaiotaomicron and thiamine acquisition mutants showed thiamine can dramatically impact fitness and competition (3).

Unfortunately, pairwise competitions do not represent the dynamic community of the gut microbiome. To fully understand how thiamine impacts abundances of gut microbes a community needs to be studied. As of yet, a defined medium that supports the growth of many species and allows for the control of thiamine levels does not exist. To circumvent this issue, mouse models with a representative humanized microbiome could be used.

Using this system we can control the thiamine intake of the mouse. We can then interpret how thiamine availability impacts the colonization of gut microbes. Utilizing mouse

149 models will also allow for the study of how thiamine and the microbiome impact disease states. These studies will lead to a better understanding of thiamine and how it impacts the human gut microbiome. In turn, this will lead to novel treatments of vitamin deficiencies and diseases in the future.

Do the Bacteroidetes possess transcriptional and translational acting TPP riboswitches?

As identified in Chapter 3, B. thetaiotaomicron is novel in employing both transcriptional and translational acting TPP riboswitches. This phenomena was linked to the distance that the TPP riboswitch was from the start codon of the first gene in the regulon. In addition, this phenomena was associated with previously characterized TPP riboswitches in microbes (2, 4, 17). Traditionally, transcriptional and translational acting riboswitches are different distances from their regulon, but this trend has never been characterized.

In Chapter 3, we identified that transport and biosynthesis acting riboswitches were typically at different distances. This suggests that across the Bacteroidetes transport and biosynthesis riboswitches may be operating through different regulatory mechanisms.

To build on this, we can leverage the nanoluciferase fusion system and transcriptional assays (e.g. RT-qPCR) to characterize the mechanism of TPP riboswitch regulation.

Leveraging both of these assay will help characterize how these riboswitches are working and determine if the trends observed in B. thetaiotaomicron are observed in other species.

150 Cumulatively, understanding the regulation of thiamine acquisition pathways can create strategies for modulating the gut microbiome. In addition, we will have a better understanding of how a prominent phylum, Bacteroidetes, responds too shifting metabolite availability.

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