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2018 Resolution of the Nutritional Requirements and Metabolic Profile of Clostridium scindens ATCC 35704, A Major Bile Acid-Dehydroxylating Anaerobe in the Human Gut Rachana Shrestha Eastern Illinois University
Recommended Citation Shrestha, Rachana, "Resolution of the Nutritional Requirements and Metabolic Profile of Clostridium scindens ATCC 35704, A Major Bile Acid-Dehydroxylating Anaerobe in the Human Gut" (2018). Masters Theses. 4420. https://thekeep.eiu.edu/theses/4420
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(TITLE)
By
Rachana Shrestha
THESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Master of Science
IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY
CHARLESTON, ILLINOIS
2018
YEAR
I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE
THESIS COMMITTEE CHAIR DATE DATE
/'/1/Zmt DATE
ii
------Resolution of the Nutritional Requirements and Metabolic Profile of Clostridium scindens ATCC 35704, A Major Bile Acid-Dehydroxylating Anaerobe in the Human Gut
(TITLE)
BY
Rachana Shrestha
THESIS
SUBMITIED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
Master of Science
IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY
CHARLESTON, ILLINOIS
2018
YEAR Abstract
Bile acids (cholate and chenodeoxycholate) are synthesized from cholesterol in the human liver, and secreted in small intestine for food digestion. However, a portion of these primary bile acids is converted in the colon to toxic secondary bile acids, deoxycholate and lithocholate. Secondary bile acid formation is the result of 7a dehydroxylating anaerobes, and one of the key players in this conversion is Clostridium scindens. Interestingly, little is known about the basic physiology and nutrition of C. scindens. Therefore, the goal of this research was to understand the nutritional requirement, and determine the 7a-dehydroxylation activity and end products from glucose fermentation in different growth conditions by C. scindens ATCC 35704. C. scindens was maintained in anaerobic defined medium (DM) containing 25 mM glucose, minerals, trace metals, amino acids, vitamins, bicarbonate, 100% C02 gas phase, and sodium sulfide. To determine the amino acid and vitamin requirements of C. scindens, the "leave one [amino acid group] out" technique and "leave one [vitamin] out" techniques were used to eliminate the non-essential amino acids and vitamins. With these techniques, it was found that an amino acid, tryptophan, and three vitamins
(riboflavin, pantothenate, and pyridoxal) were required for growth of C. scindens. C. scindens was able to convert cholate to deoxycholate along with some bile acid-derived products in defined as well as in minimal growth conditions. Also, C. scindens fermented glucose mainly to ethanol, acetate, formate, and Hi. The ratio of these end products varied with growth conditions. Understanding the metabolic profile of C. scindens ATCC
35704 may help in treatment of disease associated to secondary bile acids.
iii Acknowledgements
I would like to express my sincere appreciation to my advisor Dr. Steven Daniel for his constant support and mentorship during my graduate education. His unwavering enthusiasm and depth of knowledge on science kept me constantly focused on my research and his personal generosity helped me grow as a better person. I would also like to extend my appreciation to the members of my graduate committee, Dr. Gary Bulla and
Dr. Kai Hung for their valuable suggestions and motivations.
I would also like to thank my graduate coordinator Dr. Britto Nathan for his assistance throughout my study period. I owe great thanks to Dr. Thomas Can am for his help during my HPLC and GC work. I am grateful to Dr. Daniel and the Department of Biological
Sciences for the financial support to complete this project.
I really appreciate the help from my lab mates, Christopher Kalinka, Lina Sallam,
Nivedita Pareek, Kenji Ohseki, and Jade Mallaney. I would also like to thank Steven
Malehorn for his help during my entire research. Also, I want to thank my parents, my sister and my best friend Ranjana for their unceasing encouragement.
Lastly, I want to thank my fiance Pramod. You have always been my inspiration, and
I can't thank you enough for your love and support throughout this tough journey.
iv TABLE OF CONTENTS
Page
I. INTRODUCTION ...... 1
A. Human Gut Microbiome ...... 1
B. Bile Acid Biosynthesis and its Regulation in Humans ...... 2
C. Bile Acid Metabolism by Intestinal Microbiota ...... 4
1. Deconjugation of Bile Salts ...... 4
2. Oxidation/reduction and Epimerization of Bile Acids ...... 5
a. 3a- and 313-HSDHs...... 5
b. 7a- and 7j3-HSDHs ...... 6
c. 12a- and 1213-HSDHs...... 6
3. Bile Acid Dehydroxylation ...... 8
4. Genetics and Enzymology of 7a-/7j3-dehydroxylation ...... 12
a. baiA gene ...... 12
b. baiB gene ...... 12
c. baiCD gene ...... 12
d. baiH gene...... 13
e. baiE and bail genes ...... 13
f. baiF gene ...... 13
g. baiG gene...... 14
D. Bile Acids and Human Health ...... 15
E. Diseases Associated with Secondary Bile Acids ...... 16
v 1. Cholesterol Gallstones ...... 16
2. Colorectal Cancer ...... 16
3. Other Cancers...... 16
4. Clostridium difficile Infection ...... 17
F. Diversity of Gut Microbes with 7a/�-dehydroxylating Potential ...... 18
G. Characteristics and Metabolism of C. scindens ...... 18
II. Research Objectives ...... 21
Ill. Materials and Methods ...... 21
Bacterial Strain ...... 21
Stock Solutions ...... 21
Amino Acids...... 21
Vitamins...... 22
Mineral Solution ...... 24
Trace Metal Solution ...... 24
Resazurin Solution ...... 25
Culture Media ...... 25
Brain Heart Infusion ...... 25
Undefined Medium ...... 26
Defined Medium ...... 27
Minimal Medium ...... 28
Defined Medium with Phosphate Buffer ...... 29
Phosphate Buffer Media without Glucose...... 29
Inoculation and Maintenance of Culture...... 30
Determination of Amino Acid Requirements ...... 30
Group Determination ...... 30
vi Within Group Determination ...... 31
Determination of Vitamin Requirement ...... 32
Comparison of Two Forms of Pyridoxal...... 33
Thin Layer Chromatography (TLC) Analyses ...... 34
Bile Acids ...... 34
Bile Acid Standard in Culture Medium ...... 34
C. scindens ATCC 35704 in Culture Media with Bile Acids ...... 34
Bile Acid Extraction from Sterile Medium (Standards) and From Cultures ... .. 35
TLC Analysis ...... 35
High Performance liquid Chromatography (HPLC) ...... 36
Stock Solution for HPLC Standards...... 36
Preparation of HPLC Standards ...... 36
Glucose Stock Solution for use in culture media ...... 37
HPLC Conditions ...... 38
HPLC Analysis of Sterile Culture Media ...... 38
Resolving Ethanol Peak in Sterile Culture Media ...... 38
HPLC Analysis of Sterile DM with P04 Buffer ...... 39
Resolving Unknown Peak in DM with PQ4 Buffer ...... 39
Sampling and Processing of Culture for HPLC Analysis ...... 39
Gas Chromatography (GC) ...... 40
GC Conditions ...... 40
Preparations of GC Standards and Calibration ...... 40
GC Analysis from Cultures ...... 41
Genetic Information ...... 41
...... IV. Results ...... �······; ...... 42 :
Growth of C. scindens ATCC 35704 in Undefined and Defined Culture Media ..... 42
Determination of Amino Acid Requirements of C. scindens ATCC 35704 ...... 43
vii Determination of Amino Acid(s) Within Group Required for the Growth of C.
scindens ATCC 35704 ...... 45
Determination of the Vitamin Requirement of C. scindens ATCC 35704 ...... 50
Transformation of Bile Acids ...... 56
HPLC Analysis of Standards for Calibration ...... 62
HPLC Analysis of Sterile Culture Media ...... 64
Growth of C.scindens ATCC 35704 in Various Culture Media with Phosphate Buffer
····················································································································· 70
Substrate-end Product Profiles of C. scindens ATCC 3570 in Culture Media ...... 71
Information on Genes Associated in Metabolic Pathways of C. scindens ATCC
35704 ...... 82
V. Discussion ...... 87
VI. Conclusion ...... 101
VII. References ...... 102
viii LIST OF FIGURES
Figure Page
1. Enterohepatic circulation of bile acids in human ...... 3
2. Deconjugation and epimerization of primary bile acid ...... 7
3. Conversion of primary bile acids to secondary bile acids by 7a-/7f3-dehydroxylation 10
4. Proposed cholic acid 7a-dehydroxylation pathway in Clostridium scindens ...... 11
5. Gene arrangement in bile acid inducible (bai) operon in C. scindens ATCC 35704 ...... 14
6. Glucose fermentation with typical end products by Clostridium spp ...... 20
7. Growth of C. scindens ATCC 35704 in OM, UM, and BHI ...... 43
8. Determination of amino acid requirements of C. scindens ATCC 35704 using leave-one-
group out technique ...... 44
9. Determination of the histidine group requirement for C. scindens ATCC 35704 ...... 46
10. Determination of the aromatic group amino acids required for the growth C. scindens
ATCC 35704 ...... 47
11. Determination of the pyruvate group amino acids required for the growth of C.
scindens ATCC 35704 ...... 48
12. Determination of the serine group amino acids required for the growth of C. scindens
ATCC 35704 ...... 49
13. Determination of the vitamin requirements of C. scindens ATCC 35704 using the
leaving-one-out technique ...... 52
14. Determination of additional vitamin(s) required for the growth C. scindens ATCC
35704 ...... 53
ix 15. Growth of C. scindens ATCC 35704 in various defined medium (OM) and minimal
medium (MM) ...... 54
16. Comparison of two forms of pyridoxal (pyridoxal-HCI vs pyridoxal-P04) on growth of
C. scindens ATCC 35704 ...... 55
17. TLC analysis of bile acid standards in OM ...... 58
18. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 on defined and minimal
culture media ...... 59
19. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 on defined and minimal
culture media ...... 60
20. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 in BHI broth ...... 61
21. HPLC analysis of substances (20 mM) as standards ...... 63
22. HPLC analysis of sterile culture media using RI detection ...... 66
23. HPLC analysis of the OM component bicarbonate and a potential end product ethanol
using RI detection ...... 67
24. HPLC analysis of (A) OM with P04 buffer and (B) P04 buffer using refractive index (RI)
detection ...... 68
25. HPLC analysis of sterile culture medium and its component using UV (210 nm)
detection ...... 69
26. Growth of C. scindens ATCC 35704 in culture media with phosphate buffer...... 70
27. Glucose (growth substrate) and end products detected in OM with PQ4 buffer with C.
scindens ATCC 35704 at sampling time To ...... 74
x 28. Glucose (growth substrat�) and end products detected in OM with PQ4 buffer with C.
scindens ATCC 35704 at sampling time Tmax················ ············································ 75
29. Glucose (growth substrate) and end products detected in OM + Trp with P04 buffer
with C. scindens ATCC 35704 at sampling time To ...... 76
30. Glucose (growth substrate) and end products detected in OM + Trp with PQ4 buffer
...... with C. scindens ATCC 35704 at sampling time Tmax ...... 77
31. Glucose (growth substrate) and end products detected in MM with P04 buffer with C.
scindens ATCC 35704 at sampling time To ...... 78
32. Glucose (growth substrate) and end products detected in MM with P04 buffer with C.
...... scindens ATCC 35704 at sampling time Tmax ...... 79
33. KEGG map for biosynthesis of tryptophan in C. scindens ATCC 35704 ...... 90
34. KEGG map for biosynthesis of riboflavin in C. scindens ATCC 35704 ...... 92
35. KEGG map for biosynthesis of pantothenate in C. scindens ATCC 35704 ...... 93
36. KEGG map for biosynthesis of pyridoxal in C. scindens ATCC 35704 ...... 95
37. KEGG map for glucose fermentation with typical end products by C. scindens 35704
...... 99
38. Hypothetical pathway for coupling glucose fermentation to bile acid dehydroxylation
· by C. scindens ...... 101
xi LIST OF TABLES
Table Page
1. Bile acid standard in OM ...... 34
2. Culture preparation and inoculation ...... 35
3. Retention times of substances used as standards in high performance liquid
chromatography (HPLC) ...... 62
4. Substrate consumption and end products formation over time in defined and minimal
culture media by C. scindens ATCC 35704 ...... 80
5. Substrate-product profiles of C. scindens ATCC 35704 in defined and minimal culture
media ...... 81
6. Missing genes of C. scindens ATCC 35704 in amino acid anabolism using the Integrated
Microbial Genomes datasets ...... 84
7. Amino acid biosynthesis of C. scindens ATCC 35704 dependent on other intermediates
...... 85
8. Missing genes of C. scindens ATCC 35704 in vitamin anabolism using the Integrated
Microbial Genomes (IMG) datasets ...... 86
xii I. INTRODUCTION
Humans synthesize cholic acid (CA) and chenodeoxycholic acid (COCA) from
cholesterol in the liver. These two primary bile acids, conjugated to taurine or glycine,
are secreted in the small intestine and play vital roles in food digestion. However, a small
amount of these primary acids (unconjugated) are also converted to toxic secondary bile acids known as deoxycholic acid and lithocholic acid, respectively, in the human colon.
This reductant-consuming process is carried out via 7a-dehydroxylation by anaerobic bacteria present in the gut microbiome. Clostridium scindens is one of the major bacteria
in the microbiome responsible for 7a-dehydroxylation, and the toxic secondary bile acids
that result from this process have been linked to human diseases.
A. Human gut microbiome
The human gastrointestinal (GI) tract is inhabited by "'1013 microorganisms that are collectively termed as gut microbiota and comprises more than 1000 bacterial species (1-
3). Among the diverse gut microbiota, phyla Formicutes and Bacteriodetes comprises 90% of the total population (4). A large number of obligate anaerobes such as Bacteroides,
Bifidobacterium, Clostridium, Eubacterium, Fusobacterium, Peptococcus,
Peptostreptococcus, and Ruminococcus belongs to these groups (4). The gut microbiota
exhibit a symbiotic relationship with humans, and play important roles in host health (2,
S). Some common functions of gut microbes are: digestion of complex foods, synthesis of essential vitamins, and improving host defenses (3, 6). However, recent studies have revealed their association with various diseases (2, 7, 8).
1 B. Bile acid biosynthesis and its regulation in humans
Bile is a vital secretion of the human liver and play important role in digestion of food
(9). Bile acid is a saturated steroid synthesized in hepatocytes (10, 11). Cholic acid
(3a,7a, 12a-trihydroxy-5(3-cholan-24-oic acid) and chenodeoxycholic (3a, 7a-dihydroxy
SJ3-cholan-24-oic acid) undergo conjugation with either glycine or taurine to form bile
salts and are stored in the gallbladder (9, 11, 12).
The transformation of bile acids to bile salts restricts its passive absorption when
passed down the GI tract during food digestion (9). The release of cholecystokinin from
duodenum, after food consumption, stimulates secretion of bile salts from the gallbladder at a "'100 mM concentration (8, 11). Inside the small intestine, bile salts play
a crucial role in the solubilization and absorption of cholesterol, lipids and lipid soluble vitamins (A, D, E and K) (10, 11). With active re-absorption, 95% of the released bile salts
are recovered from the distal ileum (8, 10, 11). The bile salts absorbed by enterocytes of the terminal ileum are returned to the liver via portal blood (8, 10). The circulation of bile from liver to intestine and vice-versa is termed "enterohepatic circulation" (Fig. 1), and
this phenomenon occurs several times a day (10, 11). Nearly 5% of the bile salts escape
enterohepatic circulation as they enter the colon and are lost through feces (8, 10).
2 Deoxycholic acid ____ Bile Feces - .,..______Cholic acid .,_ • Lithocholic acid Chenodeoxycholic Bacterial acid dehydroxylation SMALL COLON INTESTINE
Fig. 1. Enterohepatic circulation of bile acids in human (12)
3 C. Bile acid metabolism by intestinal microbiota
The human gut microbiota have a crucial role in the metabolism of bile acids (11). Gut microbiota are capable of producing 15-20 different bile acid metabolites from primary bile acids (12). Some predominant species in the human bowel which are involved in bile acid metabolism are: Bacteroides, Bifidobacterium, Clostridium, Lactobaci//us,
Eubacterium, and Escherichia (11). Bile acid deconjugation and the oxidation of hydroxy groups are common transformations that occur in the small intestine (11). The bile acids
(500-600 mg/day) which escapes from enterohepatic circulation undergo further biotransformation into different metabolites by commensal anaerobes in the colon (8,
10, 13). The types of microbial transformations of bile acids in the bowel include: deconjugation, 7a-/7'3-dehydroxylation and oxidation/reduction, and epimerization of hydroxyl groups (14-16).
1. Deconjugation of bile salts. Deconjugation is a process where conjugated bile acids are hydrolyzed to free bile acids (Fig. 2) by the action of bile salt hydrolase (BSH) enzymes
(14, 17, 18). The enzymatic hydrolysis occurs at the C-24 N-acyl amide bond which is the bridge between bile acids and their amino acid conjugates (11). BSH is choloylglycine hydrolase (EC 3.5.1.24) which belongs to the super family N-terminal nucleophilic hydrolases (Ntn) (11, 14). Deconjugation activities have been observed in species like
Clostridium, Bifidobacterium, lactobacil/us, and Bacteroides (14, 15). The outcome of BSH shows both positive and negative consequences on the colonic environment (14).
Glycine-conjugated bile salts have been found as antimicrobial agent for gut microbes, therefore, BSH is considered as detoxifying agent in this case (14). On the other hand,
4 free bile acids are harmful to some beneficial species like Lactobacillus, and BSH is thus considered harmful (14).
2. Oxidation/reduction and epimerization of bile acids. Some species of gut microbiota can produce hydroxysteroid dehydrogenase (HSDH) enzymes which catalyze oxidation/reduction and epimerization reactions (Fig. 2) involving hydroxyl groups at C-3,
C-7, and C-12 positions of bile acids (19-22). Such microbes are capable of the oxidation/reduction of hydroxyl group of cholic acid producing 27 different oxo- and hydroxy-related metabolites (14, 21). HSDHs belong to the short-chain dehydrogenase/reductase family and are dependent on pyridine nucleotides (14, 19).
"Bacterial bile acids HSDHs differ in their pH optima, pyridine nucleotide specificity
NAD(H) or NADP(H) or both, subunit molecular weight, and gene regulation (14)."
Epimerization is a reversible process which occurs by action of two stereochemically different HSDHs on the hydroxyl groups of bile acids generating stable oxo-bile acid intermediates (11, 20, 23). Epimerization can be carried out either by intraspecies or by interspecies processes (12). For example, intraspecies epimerization occurs if a single bacterial species possessing both and (3-HSDHs act on the bile acid (11, 20). On the a- other hand, interspecies epimerization occurs when two different bacterial species, one possessing a a-HSDH and the other with a (3-HSDH,act on the bile acid (11, 20).
a. 3a- and 3J3-HSDHs. Oxidation/reduction reactions between 3-oxo-bile acids and
3a- or 3�-hydroxy bile acids are catalyzed by 3a/�-HSDHs (11, 24). 3a-HSDH activity has been found in species like C. scindens, C/ostridium perfringens, and Clostridium hiranonis.
3�- HSDHs have been detected in species of Clostridium and Ruminococcus (11).
5 b. 7a- and 7P-HSDHs. Oxidation/reduction reactions involving the 7a-and 7� hydroxyl groups of bile acids are catalyzed by 7a/�-HSDHs (11, 24). 7a/�-HSDHs are expressed by species of Bacteroides, Clostridium, and Ruminococcus (11). In addition, both 7a- and 7�-HSDH activities are present in several intestinal clostridial species, for example Clostridium absonum (25).
12a- and 12P-HSDHs. The 12a-and 12�-hydroxyl groups of bile acids undergo c. reversible oxidation/reductions by the catalytic action of 12a/�-HSDHs (11, 24). 12a/�
HSDH activity is found mainly in species of Clostridium; C. perfringens, Clostridium /eptum,
Clostridium tertium, Clostridium difficile, and Clostridium paraputrificum (11).
6 cholylglyclne l BSH
HSOH ~ 7a- ~ OH O,,..H lt
H H H 7-oxodeoxycholic acid .JJX3-dehydrocholic acid �·ct( oo'12-o:cochenodeoxycholicc& acid
HSOH t 3P- l 7Jl-HSDH l t 12P-HSOH OH OH i OH !
H H H 7-epicholic acid �4£isocholic acid ...cte ..,cb;:12-epicholic acid
Fig. 2. Deconjugation and epimerization of primary bile acid (11, 14).
Abbreviations: BSH, bile salt hydrolase; HSDH, hydroxysteroid dehydrogenase.
7 3. Bile acid dehydroxylation. In the human bowel, some of the predominant
microbial transformations are 7a-/7J3-dehydroxylationof free bile acids (11, 26). The 7a
/7�-dehydroxylation is a multi-step pathway carried out by colonic bacteria which
removes the 7a-hydroxy or 7�-hydroxy group from the primary bile acids, cholate and
chenodeoxycholate (Fig.3), forming secondary bile acids, deoxycholate and lithocholate
respectively (27-29). The deoxycholate is absorbed from the colon by passive non-ionic
diffusion and is returned to the bile acid pool (26). Compared to deoxycholate,
lithocholate is absorbed in very lesser amount and is usually lost in feces (11).
Deoxycholate in the bile acid pool can range from 0 to over 40% of the total pool (30).
High levels of deoxycholate in blood, bile and feces have been linked with a high risk for
cholesterol gallstone disease and colon cancer (26, 31). The human gut microbes that
dehydroxylate primary bile acid belong to the genus C/ostridium (11, 26). The
deconjugation of bile salts with BSH enzyme is required as the action 7a-/7�
dehydroxylation is limited to free bile acids (11). Some colonic bacteria show both 7a
/7�-dehydroxylation activities whereas some do either 7a- or 7�-dehydroxylation (11).
A proposed multi-step bile acid 7a-dehydroxylating pathway in C. scindens shown in
Figure 4 is based on a bile acid inducible (bai) operon (11, 14). The pathway begins with transport of free primary bile acid into the bacterial cell by an enzyme encoded by the baiG gene termed bile acids transporter (11, 14). The unconjugated bile acid inside the
bacterial cell is ligated with coenzyme A (CoA) where the reaction is catalyzed by an enzyme encoded by the baiB gene. After CoA ligation, the baiA gene product oxidizes 3a
hydroxy group with either NAO+ or NADP+ as an electron acceptor (11, 14).
8 The baiCD gene encodes a NADH:flavin dependent-oxidoreductase which introduces a double bond in the C4=CS position of CoA conjugates of 3-dehydro-4-cholenoic acid
(14). The hydrolysis of 3-dehydro-4-cholenoic acid-CoA is catalyzed by bile acid CoA hydrolase encoded by the baiF gene (11). The baiE gene encodes enzyme bile acid 7a dehydratase which catalyzes 7a-dehydration of 3-dehydro-4-cholenoic acid and forms an intermediate; 3-dehydro-4,6-deoxycholdienoic acid {11, 14). The gene encoding
EDS08212.1, a flavin dependent enzyme, is responsible in catalyzing two consecutive reduction reactions; from 3-dehydro-4,6-deoxycholdienoic acid to 3-dehydro-4- deoxycholenoic acid and then to 3-dehydro-deoxycholic acid (32).
Furthermore, 3-dehydro-deoxycholic acid is reduced to deoxycholic acid, and deoxycholic acid is removed from bacterium with bile acid exporter (11). The genes involved in these two last steps have not been identified (11, 14).
9 0
7a-dehydroxylation
',, HO'' H Deoxycholic acid Cholic acid
H DH - S 7P-dehydroxylation 7a I ll H�
P HSDH 7 -
H I!
7-oxo-cholic acid Ursocholic acid
7a-dehydroxylation
II Chenodeoxycholic acid lithocholic acid
Fig. 3. Conversion of primary bile acids to secondary bile acids by 7a-/7�- dehydroxylation (11, 12, 14).
10 CA
r I CA l I CoASH L ___J
NAD(P)+ ----.. r -- - 3 �dehydr-�-4- =A l _ _ -� J
AMP+PPi ___ _ ( NAD(P)H l CA"'CoA 3-dehydro-4-CA"'CoA - -- --· � ---_J [------] NAD(P) + - NAD(P)H + dehydro- -DCA NAD(P) r�--.,-:-- --- 1 -- ? NAD(P)
NAD(P) + t..- : · NAD(P) DCA� lI ' -'
Fig. 4. Proposed cholic acid 7a-dehydroxylation pathway in Clostridium scindens (14,
32). Abbreviations: CA, cholic acid; DCA, deoxycholic acid; baiG, primary bile acid
transporter; baiB, bile acid-coenzyme A ligase; baiA, 3a-hydroxysteroid
dehydrogenase; baiCD, 7a-hydroxy-3-oxo-64-cholenoic acid oxidoreductase; baiF, bile
acid coenzyme A transferase/hydrolase; baiE, bile acid 7a-dehydratase; EDS08212.1,
flavin dependent enzyme.
11 4. Genetics and enzymology of 7a-/7P-dehydroxylation. The 7a-/7f>- dehydroxylating intestinal bacteria have genetically distinct bai genes (31). It has been found that C. scindens encodes at least 10 open reading frames from the bai operon (11).
The bai operon (�12 kb) has been cloned in Escherichia coli and the functions of the proteins encoded by bai genes have been studied (11, 12, 30). Figure 5 shows a model of the bai operon in C. scindens American Type Culture Collection (ATCC) 35704.
a. baiA gene. A 27-kDa polypeptides containing 249 amino acids is encoded by the baiA gene which significantly resembles the short-chain alcohol/ polyol dehydrogenase gene family (11). Three different baiA genes are found in C. scindens; baiAl, baiA2, and baiA3 (11, 12). The baiAl and baiA3 are identical and monocistronic whereas the baiA2 gene is a portion of polycistronic bai operon (11, 12). The baiA gene encodes a 3a-HSDH when cloned and expressed in E.coli (11). The enzyme uses either NAO+ or NADP+ as an electron acceptor and catalyzes the oxidation or reduction of CoA conjugates of bile acids
(11, 12).
b. baiB gene. The baiB gene encodes a bile acid CoA ligase which is a 58 kDa polypeptide with 521 amino acids (11, 12, 30). The bile acid CoA ligase is dependent on
ATP, CoA, and Mg + and catalyzes the synthesis of CoA conjugated bile acids (11). 2
c. baiCD gene. The baiCD gene encodes a steroid oxidoreductase enzyme, a �10 kDa polypeptide (11). The enzyme is specific for 3-dehydro-4-cholenoic acid-CoA and 3- dehydro-4-chenodeoxycholenoic acid-CoA and requires FMN, NAO+, or NADP+ for its activity (11). The baiCD polypeptide shows significant similarity with various 2,4-dienoyl
12 CoA reductases and baiH ·from C. scindens as well as with NADH oxidase from Listeria monocytogenes (11).
d. baiH gene. A "'72 kDa polypeptide with 661 amino acids is encoded by the baiH gene (11, 12). The encoded protein shows NADH:flavin oxidoreductase activity and occurs as homotrimer (11). When cloned in E. coli, one mole of polypeptide subunit of baiH gene contains 1 mol of FAD, 2 mol of iron, and 1 mol of copper (11, 12). The baiH gene product shows similarity in its amino acid sequence with NADH oxidase from
Thermoanaerobium brockii and old yellow enzyme from Saccharomyces carlbergensis
(12). The steroid oxidoreductase encoded by baiH gene in C. scindens is specific for 3- dehydro-4-ursodeoxycholenoic acid and 3-dehydro-4-epicholenoic acid conjugated with
CoA (11).
e. baiE and bail genes. A "'19.5 kDa polypeptides with 166 amino acids is encoded by baiE gene (11, 12). The baiE gene product expressed in £. coli occurs as a dimer and shows 7a-dehydratase activity (12). The enzyme acts on the C-7 hydroxyl group of 3- dehydro-4-cholenoic acid and 3-dehydro-4-chenodeoxycholenoic acid and forms the stable intermediates, 3-dehydro-4,6-deoxycholdienoic acid and 3-dehydro-4,6- lithocholdienoic acid respectively (11). Since bail gene product shows amino acid sequence homologies with baiE gene, it is hypothesized to encode a bile acid 7� dehydratase (11, 14).
f. baiF gene. A 47.5-kDa polypeptide comprising 426 amino acids is encoded by baiF gene (12). The gene product of baiF gene shows bile acid CoA hydrolase activity and is similar with the type Ill family of CoA transferases (11).
13 g. baiG gene. The baiG gene encodes a 50-kDa polypeptide containing 477 amino acids (11, 12). The baiG polypeptide consists of 14 membrane-spanning domains (11).
The baiG gene product expressed in E. coli shows W-dependent bile acid transporter activity and the sequence homology with a major facilitator super family (11, 12). The transportation of free primary bile acids is facilitated by the baiG gene product whereas the secondary bile acids are not transported (11).
Bile acid CoA transferase
H+ Putative Bile acid dependent 3-dehydro-4- transcriptional CoA li2ase 7a-dehydratase bile acid UDCA/7-epiCA transporter oxidoreductase
barA
3-dehydro-4- CDCA/CA 3a-HSDH 713-dehydratase
Fig. 5. Gene arrangement in bile acid inducible (bai) operon in C. scindens ATCC 35704
(11, 14).
14 D. Bile acids and human health
Bile acids are ordinary components present in the luminal content of human GI tract which helps in the metabolism of fats and lipids (33). Bile acids exhibit a broad range of biological activities in humans and are vital signaling molecules which regulate several metabolic pathways in the host (34, 35). Studies shows that the secondary bile acids synthesized by colonic bacteria are found in various human tissues (e.g., heart, kidney, and liver) and significantly influence the homeostasis process in humans (24, 34).
Secondary bile acids have both beneficial as well as detrimental effects on human health
(7, 34). Some secondary bile acids prevent the growth of pathogens, whereas some are used in treatment of disease (7, 34). For example, ursodeoxycholic acid, an epimerized product of chenodeoxycholic acid is used in the treatment of cholesterol gallstones and as such is considered to be a chemopreventive agent (34). In contrast, high concentration of secondary bile acids have been linked to human diseases like cholesterol gall stones and colon cancer (24, 33, 36, 37).
Studies have found that lithocholate is associated with colon carcinogenesis and is harmful to liver cells (34). Recent studies have shown that alterations in the bile acid pool affect gut microbes and vice-versa (38, 39). It has been found that microbial biotransformation of bile acids modifies the gut microbiota causing dysbiosis and health issues (34). The altered bile acid pool results in such disease states as C. difficile infection, inflammatory bowel diseases, irritable bowel syndrome, metabolic syndrome, and cancers (38).
15 E. Diseases associated with secondary bile acids
1. Cholesterol gallstones. The two main reasons for the development of cholesterol
gallstone disease (CGD) are the hypersecretion of cholesterol and the supersaturation of the bile with cholesterol (24). High levels of secondary bile acids as well as a high number of 7a-dehydroxylating fecal bacteria have been associated with a high risk of CGD (24).
Studies have found that the level of deoxycholate and 7a-dehydroxylating bacteria were significantly reduced when patients with CGD were treated with antibiotics (24).
2. Colorectal cancer. Many genetic and environmental factors are involved in colorectal cancer in humans (8). A positive correlation has been found in colorectal cancer and a high fat diet (8). Individuals on Western diets which contain a high fat content tend to exhibit higher levels of deoxycholate in their bile acid pool (40). The secondary bile acids formed by bacterial conversions act as carcinogens or cocarcinogens
(38). Studies have found that patients with colorectal cancer have higher amounts of bile acids and higher numbers of 7a-dehydroxylating clostridia in their feces (38). The accumulation of deoxycholate appears to damage mucosa! membranes and cellular DNA and elevates the level of reactive oxygen species (7, 8). The subsequent inflammatory response followed by hyperproliferation of undifferentiated cells for cell repair is likely to enhance tumor growth (41).
3. Other cancers. Apart from colon cancer, high levels of bile acids have also been associated with cancers at other sites in human body: laryngopharyngeal tract, esophagus, stomach, pancreas, small intestine, and liver (8, 38). Bile acids can act as carcinogens all along the human GI tract (8).
16 4. Clostridium difficile infection. C. difficile is a spore-forming organism and a potential pathogen of the human gut which causes severe diarrhea and life-threatening conditions in humans (6, 42). The antibiotics used in the treatment of C. difficile infections (COi) kill vegetative cells but do not eliminate the spores of C. difficile (43).
Moreover, the antibiotics alter the gut microbiota and increases the susceptibility to COi
(44-46). Bile acids present in the human GI tract have a huge impact in the pathogenesis of COi (6, 39). For example, the conjugated bile salt taurocholic acid (TCA) found in the human GI tract activates the germination of C. difficile spores in the small intestine (39).
In contrast secondary bile acids, specifically OCA,inhibit the growth of vegetative cells of
C. difficile which ultimately inhibits COi (39, 43). The growth of C. difficile is suppressed by C. scindens (47). However, broad spectrum antibiotics kills C. scindens and increase the risk of COi (39, 43). Therefore, narrow-spectrum antibiotics which could spare C. scindens or fecal microbial transplantation (FMT) are considered as possible treatments for COi (39, 43, 48).
17 F. Diversity of gut microbes with 7a/�-dehydroxylating potential
A small group of intestinal bacteria are capable of 7a/J3-dehydroxylation of primary bile acids to secondary bile acids (40, 49). These bacterial species belong to the genus
Clostridium, a group of Gram-positive and spore-forming anaerobes (SO, 51). The 7a/J3- dehydroxylating Clostridium species include: C. scindens, C. hiranonis, and Clostridium hylemonae which belong to Clostridium cluster XIVa, Clostridium sordellii and Clostridium bifermentans which belongs to Clostridium cluster XI, and C. leptum which belongs to
Clostridium cluster I (40, 49). The 7a/J3-dehydroxylation activity is considered lower in C. hylemonae, C. sordellii, C. bifermentans and C. leptum, and thus these organisms are placed in "low activity group" whereas higher dehydroxylating activities are observed in
C. scindens and C. hiranonis and thus are placed in a "high activity group" (49). As an example, C. scindens has 100-fold higher bile acid 7a-dehydroxylating activity than C. sordellii when analyzed in vitro (40).
G. Characteristics and metabolism of C. scindens
C.scindens, scindens meaning splitting, is named as it produces desmolase, an enzyme which cleaves the side chain from cholesterol (52, 53). C. scindens is a chemoheterotrophic, endospore forming obligate anaerobe (53). The cells are straight to
slightly curved rods with 0.5--0.7 µm width x 1.0-2.5 µm length for cell dimensions (53).
Cells are non-motile, occurring singly or in chains, and spores are terminal and wider than the vegetative cells (53). On blood agar, colonies are circular to slightly irregular, grayish white, convex, smooth, and glistening with a diameter of 0.3-lmm (53).
18 Carbohydrates like 0-glucose, 0-lactose, 0-fructose, 0-mannose, 0-ribose, and 0- xylose are fermented by C. scindens; however, amygdalin, L-arabinose, 0-cellobiose, meso-erythritol, esculin, glycogen, myo-inositol, maltose, 0-mannitol, 0-melezitose, 0- melibiose, 0-raffinose, L-rhamnose, salicin, 0-sorbitol, starch, sucrose, and 0-trehalose are not fermented (53). C. scindens does not produce enzymes like lecithinase, lipase, or catalase and is unable to digest gelatin, milk, and meat (53). This organism is also incapable of reducing nitrate or hydrolyzing starch and esculin; however, hydrogen sulfide is produced in sulfide-indole-motility medium (53). Overall, C. scindens is considered primarily a saccharolytic organism capable of metabolizing only simple sugars as a source of carbon and energy. C. scindens ATCC 35704 is usually cultivated in undefined medium which is very nutritive and growth supportive. Interestingly, very little research has been performed to understand the nutritional (amino acid and vitamin) requirements of this gut bacterium.
C. scindens produces acetic acid, ethanol, and hydrogen (H2) gas as fermentation end products in peptone-yeast extract glucose broth (53). However, a complete metabolic profile of sugar-derived metabolic end products has yet to be performed. A typical glucose fermentation scheme for Clostridium spp. is shown in Figure 6. Whether similar pathways exist in C. scindens is currently unknown. By understanding the types of catabolic pathways that are present in C. scindens, we will begin to resolve how sugar derived reductant (electrons and protons) and bile acid-dehydroxylation are coupled in this important gut bacterium.
19 1 mol Glucose + AOP • • t NAO EMP Pathway ')t ATP NAOH Lactate t Dehydrogenase ..------t C -. 1 Lactate . _ 2 mol Pyruvate , .1 • 7\ Fd I • ox N'AO NAOH HS-CoA NAOH
Ferredoxin: NAO+ Formate Oxidoreductase NAO+ Pi NAOH
Phosphotransacetylase HS-CoA NAO+
Acetyl Phosphate Acetaldehyde NAOH
NAO + Acetate Eth nol a
Fig. 6. Glucose fermentation with typical end products by Clostridium spp. (82).
20 II. RESEARCH OBJECTIVES
The objectives of this study were to determine:
1. The amino acid requirements of C. scindens ATCC 35704.
2. The vitamin requirements of C. scindens ATCC 35704.
3. If C. scindens ATCC 35704 can undergo bile acid dehydroxylation process under
minimal conditions.
4. The fermentation end products from glucose metabolism by C. scindens ATCC
35704.
Ill. MATERIALS AND METHODS
Bacterial strain
C. scindens ATCC 35704, originally obtained from the American Type Culture
Collection (ATCC; Manassas, VA), was used exclusively in this study and was routinely grown at 37°C in butyl rubber-stoppered crimp-sealed Belko tubes (18 by 150 mm; series
2048 [Bellco Glass, Inc., Vineland, N.J.]; 27.2-ml approximate stoppered volume at 1 atm flOl.29 kPa)) containing an anaerobic defined medium (DM).
Stock solutions
Amino acids. L-alanine, L-arginine-HCI, L-asparagine monohydrate, L-aspartic acid, L cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L lysine, L-phenylalanine, L-proline, L-methionine, L-serine, L-threonine, L-tryptophan, L tyrosine, and L-valine were used for this study. For the complete amino acids solution
21 (CAAS), 0.4 g of each amino acid (final concentration 2 g/I) was added to 200 ml reverse osmosis (RO) water and mixed properly. For amino acids that were insoluble in RO water,
10 N NaOH was added dropwise to dissolve them in 150 ml of RO water. After the solution was clear, the final volume was adjusted to 200 ml with RO water. The CAAS was stored at 4°C when it was not in use. To make 200 ml of OM, 4 ml of CAAS was added so that the final concentration of each amino acid in the culture medium was 40 mg/I.
For determination of amino acid requirements by C. scindens ATCC 35704, stock solutions (40 g/I) of amino acids were prepared individually. An amino acid (2 gm) was added to 50 ml of RO water and mixed properly. For those amino acids that were insoluble in RO water, lON NaOH was added dropwise to dissolve them in 40ml of RO water. After the solution was clear, the final volume was adjusted to a final volume of SO ml with RO water. The prepared stock solutions were diluted 1:20 to make various combinations of amino acid solutions. For instance, to make the tryptophan solution
(final concentration 2 g/I), 1 ml of tryptophan stock solution was added to 19 ml of RO water. The amino acid solutions were stored at 4°C when not in use. To make 200 ml of defined medium, 4 ml of an amino acid solution was added so that the final concentration of each amino acid in the culture medium was 40 mg/I.
Vitamins. In this study, d-biotin, folic acid, pyridoxal-HCI, lipoic acid, nicotinic acid, d pantothenic acid, p-aminobenzoic acid, riboflavin, thiamine, and cyanocobalamin were used. The complete vitamin solution (CVS) consisted of the following (mg/I): d-biotin,10; folic acid, 10; pyridoxal-HCI, 10; lipoic acid, 25; nicotinic acid, 25; d-pantothenic acid, 25; p-aminobenzoic acid, 25; riboflavin, 25; thiamine, 25; and cyanocobalamin, 25. Vitamins
22 were mixed properly and stored at 4 °C prior to use. To make of 200 ml OM, 4 ml of CVS was added so that the final concentration in the culture medium of each vitamin was
(µg/I): d-biotin, 200; folic acid, 200; pyridoxal-HCI, 200; lipoic acid, 500; nicotinic acid, 500; d-pantothenic acid, 500; p-aminobenzoic acid, 500; riboflavin, 500; thiamine, 500; and cyanocobalamin, 500.
In determination of vitamin requirements by C. scindens ATCC 35704, autoclaved vitamin solutions were added aseptically to the base medium. For autoclaved vitamin solution, individual stock solutions for each of the 10 vitamins were prepared: 0.212 g/I for d-biotin, folic acid, and pyridoxal-HCI and 0.530 g/I for lipoic acid, nicotinic acid, d pantothenic acid, p-aminobenzoic acid, riboflavin, thiamine, and cyanocobalamin. The prepared stock solutions were diluted 1:10 to make various vitamin solutions (CVS minus one vitamin). For instance, to prepare CVS minus biotin, 1 ml from each of the 9 individual vitamin stock solutions, except biotin, and 1 ml of RO water were added to a 18 x 150- mm Belko culture tube and sealed with a gray butyl rubber stopper and an aluminum crimp seal. In addition, individual vitamin solutions were prepared by diluting 1:10 the vitamin stock solutions. For example, to prepare a biotin solution, 1 ml of the stock biotin
solution and 9 ml of RO water were added to a 18 x 150-mm Belko culture tube and sealed with a gray butyl rubber stopper and aluminum crimp seal. All tubes containing vitamin solutions were flushed and pressurized to 15 pounds per square inch (PSI) over atmospheric pressure with argon, sterilized in autoclave at 121°C for 15 min at fast exhaust and stored at room temperature until utilized.
23 In some experiments, filter sterilized vitamin solutions were added aseptically to the base medium. Vitamin solutions were prepared as described above and sterilized by filtering with 0.2-µm pore size nylon syringe filters into sterile 18 150-mm Belko culture x tubes which were subsequently aseptically sealed with sterile gray butyl rubber stoppers and sterile aluminum crimp seals. Tubes were then flushed with sterile argon, pressurized to 15 PSI over atmospheric pressure with sterile argon and stored at room temperature until utilized.
Prior to inoculation, 0.1 ml of autoclaved or filter-sterilized vitamin solution was added to 10 ml of sterile base medium.
Mineral solution. The mineral stock solution contained: sodium chloride (10 g), ammonium sulfate (10 g), potassium chloride (5 g), monopotassium phosphate (5 g), and magnesium sulfate heptahydrate (0.5 g) in 1000 ml of RO water. The solution was mixed with stirring bar and stored at 4°C. The mineral solution was diluted 1:20 in culture medium. To make a 200 ml OM, 10 ml of mineral stock solution was used so the final concentration of minerals was as follows (mg/I): sodium chloride, 500; ammonium sulfate,500; potassium chloride, 250; monopotassium phosphate,250; and magnesium sulfate heptahydrate, 25.
Trace metal solution. Trace metal stock solution contained: trisodium nitrilotriacetate (1.5 g), manganese(ll) sulfate monohydrate (0.5 g), iron(ll) sulfate heptahydrate (0.1 g), cobalt(ll) nitrate hexahydrate (0.1 g), zinc chloride (0.1 g), nickel chloride heptahydrate (0.05 g), selenous acid (0.05 g), copper (II) sulfate pentahydrate
(0.01 g), aluminum potassium sulfate dodecahydrate (0.01 g), boric acid (0.01 g), sodium
24 molybdate dihydrate (0.01 g), and sodium tungstate dihydrate (0.01 g) in 1000 ml of RO
water. The solution was mixed with stirring bar and stored at 4°C. The trace metal solution was diluted 1:500 in culture media. To make 200 ml of DM, 0.4 ml of trace metal stock solution was added so the final concentration of trace metals was as follows (mg/I): trisodium nitrilotriacetate (chelating agent), 3; manganese(ll) sulfate monohydrate, 1; iron(ll) sulfate heptahydrate, 0.2; cobalt(ll) nitrate hexahydrate, 0.2; zinc chloride, 0.2; nickel chloride heptahydrate, 0.1; selenous acid, 0.1; copper {II) sulfate pentahydrate,
0.02; aluminum potassium sulfate dodecahydrate, 0.02; boric acid, 0.02; sodium molybdate dihydrate, 0.02; and sodium tungstate dihydrate, 0.02.
Resazurin solution. 0.1% resazurin was used as stock solution for this experiment.
0.1 g resazurin was added to 100 ml of RO water and stored at room temperature. To
make 200 ml of DM, 0.2 ml of resazurin stock solution was added so that the final concentration was 1 mg/I.
Culture media
Brain HeartInfusion (BHI). BHI is a very nutritious and a standard growth medium for
C. scindens ATCC 35704 (52). To prepare 200 ml of BHI broth, 7.4 g of BHI (Oxoid Ltd,
Cambridge, UK), 1 g of yeast extract (Becton, Dickinson and Company, Sparks, MD), 0.4 g of glucose (final concentration 11 mM), 1.5 g of sodium bicarbonate (final concentration
7500 mg/I), and 0.2 ml of resazurin solution were added to a 500 ml Erlenmeyer flask containing 220 ml RO water. Since the medium was boiled and bubbled with C02 to generate an anaerobic environment, it was likely to lose some amount of water so
25 additional water (20 ml) was added. After boiling for a few minutes, the medium was
cooled below room temperature in an ice bath and 0.1 g sodium sulfide nonahydrate
(final concentration 500 mg/I)) was added and mixed properly. The bubbling process was
switched to gently flushing the headspace with C02 for a few minutes. The culture
medium was dispensed (10 ml per tube) into 18 x 150 mm Bellco culture tubes which
were being flushed with C02. The tubes were sealed with gray-butyl stoppers and
aluminum crimp seals and autoclaved at 121°C for 15 min with fast exhaust. The pH of
the medium after autoclaving was 6.7.
Undefined medium (UM). UM was another nutritionally rich growth medium and
supported good growth of C. scindens ATCC 35704. To prepare 200 ml of UM, 0.9 g of
glucose (final concentration 25 mM), 10 ml of mineral solution, 0.4 ml of trace metal
solution, 4 ml of CMS, 4 ml of CVS, 0.2 g of yeast extract (Becton, Dickinson and Company,
Sparks, MD), and 0.2 ml of resazurin solution were added to a 500 ml Erlenmeyer flask containing 200 ml RO water and then stirred to dissolve all components. Prior to boiling, the total volume of the medium as approximated 218 ml with the additional volume
present to correct for water loss during gassing and boiling. The pH of medium was
adjusted to 7 by adding NaOH or HCI using a pH meter. After the pH was adjusted, 0.75 g sodium bicarbonate (final concentration 7,500 mg/I) was added to the medium; the
medium was mixed properly and then boiled. To the boiling medium, C02 was bubbled
until the medium turned to bright pink. After boiling, the medium was cooled below room temperature in an ice bath and 0.1 g sodium sulfide nonahydrate (final concentration 500
mg/I) was added and mixed properly. The bubbling process was switched to gently
26 flushing headspace with C02 until its color change from pink to light pink/colorless. UM was dispensed {10 ml per tube) into 18 150 mm Bellco test tubes which were being x flushed with C02. The test tubes were sealed with gray-butyl stoppers and aluminum crimp seals and autoclaved at 121°C for 15 min with fast exhaust. The pH of the medium after autoclaving was 6.8.
Defined medium {OM). To prepare 200 ml of OM, 0.9 g of glucose (final concentration
25 mM), 10 ml of mineral solution, 0.4 ml of trace metal solution, 4 ml of CAAS, 4 ml of
CVS, and 0.2 ml of resazurin solution were added into a 500 ml Erlenmeyer flask containing 200 ml RO water and then stirred to dissolve all components. Prior to boiling, the total volume of the medium as approximated 218 ml with the additional volume present to correct for water loss during gassing and boiling. The pH of medium was adjusted to 7 by adding NaOH or HCI using pH meter. After the pH was adjusted, 0.75 g sodium bicarbonate (final concentration 7,500 mg/I) was added to the medium; the medium was mixed properly and then boiled. To the boiling medium, C02 was bubbled until the medium turned to bright pink. After boiling, the medium was cooled below room temperature in an ice bath and 0.1 g sodium sulfide nonahydrate (final concentration 500 mg/I) was added and mixed properly. The bubbling process was switched to gently flushing headspace with C02 until its color change from pink to light pink/colorless. OM was dispensed (10 ml per tube) into 18 150 mm Bellco culture tube which were being x flushed with C02. The test tubes were sealed with gray-butyl stoppers and aluminum crimp seals and autoclaved at 121°C for 15 min with fast exhaust. The pH of the medium
27 after autoclaving was 6.�. Unless otherwise stated, OM was the defined medium containing both CAAS and CVS.
Minimal medium (MM). In this study, minimal medium contained the amino acid tryptophan and three vitamins (pantothenic acid, riboflavin, and pyridoxal-HCI). To prepare 200 ml of MM, 0.9 of glucose (final concentration 25 mM), 10 ml of mineral solution, 0.4 ml of trace metal solution, 4 ml of tryptophan solution (final concentration
40 mg/I), 4 ml of vitamin solution (final concentration 200 µg/I for pyridoxal-HCI and 500
µg/I for pantothenic acid and riboflavin), and 0.2 ml of resazurin solution were added to a 500 ml Erlenmeyer flask containing 200 ml of RO water and then stirred to dissolve all components. The pH of medium was adjusted to 7 by adding NaOH or HCI using pH meter.
After the pH was adjusted, 0.75 g of sodium bicarbonate (final concentration 7,500 mg/I) was added to the medium, the medium was mixed properly and then boiled. To the boiling medium, C02 was bubbled until the medium turned to bright pink. After boiling, the medium was cooled below room temperature in an ice bath and 0.1 g sodium sulfide nonahydrate (final concentration 500 mg/I) was added and mixed properly. The bubbling process was switched to gently flushing headspace with C02 until its color change from
pink to light pink/colorless. MM was dispensed (10 ml per tube) into 18 x 150 mm Belko culture tubes which were being flushed with C02. The test tubes were sealed with gray butyl stoppers and aluminum crimp seals and autoclaved at 121°C for 15 min with fast exhaust. The pH of the medium after autoclaving was 6.7.
28 Defined medium with phosphate buffer (OM + P04). Separate stock solutions of 1 M monobasic potassium phosphate and 1 M dibasic potassium phosphate were prepared by adding 13.61 g of monobasic to 80 ml of RO water and 17.42 g of dibasic to 80 ml of
RO water. The final volume of each solution was made to 100 ml and stored at 4 °C. To prepare 200 ml of OM with phosphate buffer, 0.9 g of glucose (final concentration 25 mM), 10 ml of mineral solution, 0.4 ml of trace metal solution, 4 ml of CAAS, 4 of ml CVS,
3.85 ml of monobasic potassium phosphate (final concentration 50 mM), 6.15 ml of dibasic potassium phosphate (final concentration 50 mM), and 0.2 ml of resazurin solution were added to 500 ml Erlenmeyer flask containing 200 ml reverse osmosis RO water and then stirred to dissolve all components. The pH of medium was adjusted to 7 by adding NaOH or HCI using pH meter. After the pH was adjusted, the medium was boiled, and argon was bubbled until the medium turned to bright pink. The medium was cooled below room temperature in an ice bath and 0.1 g sodium sulfide nonahydrate
(final concentration 500 mg/I) was added and mixed properly. The bubbling process was switched to gently flushing headspace with argon until its color change from pink to light pink/colorless. DM + P04 medium was dispensed (10 ml per tube) was dispensed into 18
x 150 mm Belko culture tubes which were being flushed with argon. The test tubes were sealed with gray-butyl stoppers and aluminum crimp seals and autoclaved at 121°C for 15 min with fast exhaust. The pH of the medium after autoclaving was 6.8.
Phosphate buffered media without glucose. OM, OM containing tryptophan as sole amino acid, and MM were all prepared without glucose and containing PQ4 buffer. The process of preparation was the same as OM + P04 prepared above.
29 Inoculation and maintenance of culture
To start a culture in a new growth medium, 0.5 ml of a late log/stationary phase culture of C. scindens ATCC 35704 was transferred aseptically with a sterile 23-gauge BO needles and sterile 1-ml syringe to a tube of sterile growth medium. All inoculated tubes were incubated vertically and in the dark at 37°C and growth was measured over time as optical density (OD) at 600 nm using a spectrophotometer (Spectronic Instruments Inc.,
Rochester, NY; the optical path width [inner diameter of culture tubes] was 1.6 cm). The culture medium was considered growth supportive if the 00 of cultures after 3 sequential transfer was ;?; 0.05. Uninoculated culture media servesas references.
Determination of amino acid requirements
The amino acid determination for C. scindensATCC 35704 was performed exclusively in OM with variation in supplemented amino acids.
Group determination. Based on known pathways for amino acid interconversions, twenty amino acids were divided into six groups (54): (i) glutamate group (Glu grp; glutamine, glutamic acid, proline, and arginine), (ii) serine group (Ser grp; serine, glycine, and cystine), (iii) aspartate group (Asp grp; aspartate, asparagine, methionine, lysine, threonine, and iso-leucine), (iv) pyruvate group (Pyu grp; alanine, valine, and leucine), (v) aromatic group (Aro grp; tryptophan, tyrosine, and phenylalanine), and (vi) histidine group (His grp; histidine). For the leave-one-group-out technique, six different defined media, each without one of the above groups were prepared. Growth was initiated by transferring 0.5 ml of C. scindens ATCC 35704 from late-log/stationary phase of OM
30 aseptically with 23-gauge BO needles attached to 1-ml syringes and tubes were incubated at 37°C. Growth was measured over time and sequential transfers were made to respective sterile media from tubes showing good growth.
Within group determination. Within group determination of amino acids was performedfor amino acids from histidine, aromatic, pyruvate and serine groups. In group determination test, C. scindens ATCC 35704 supported growth without glutamate and aspartate groups so the two groups were not included in further tests.
To determine the His grp requirement, OM containing Aro, Pyu, and Ser grps was supplemented with and without histidine. The inoculum source for this test was OM with
Ser, Pyu, Aro, and His grps. The growth was initiated by transferring 0.5 ml of C. scindens
ATCC 35704 aseptically from late-log/stationary phase with 23-gauge BO needles attached to 1-ml syringes to sterile growth media with and without histidine and tubes were incubated at 37°C. Growth was measured over time, and sequential transfers were made to respective sterile media showing growth.
To determine the Aro grp requirement, OM containing Pyu and Ser grps was supplemented individually with amino acids of the Aro grp (tryptophan, tyrosine, and phenylalanine). The inoculum source for this test was OM with Ser, Pyu and Aro groups, and growth was initiated by transferring 0.5 ml of C.scindens ATCC 35704 aseptically from late-log/stationary phase with 23-gauge BO needles attached to 1-ml syringes to sterile growth media. Tubes were incubated at 37°C. Growth was measured over time and sequential transfers were made to respective sterile medium from tubes showing growth.
31 To determine the Pyu grp requirement, OM containing tryptophan and Ser grp was supplemented individually with amino acids of the Pyu group (alanine, valine, and leucine). The inoculum source for this test was OM with tryptophan, Ser, and Pyu grps, and growth was initiated by transferring 0.5 ml of C.scindens ATCC 35704 aseptically from late-log/stationary phase with 23-gauge BO needles attached to 1-ml syringes to sterile growth media. Tubes were incubated at 37°C. Growth was measured over time, and sequential transfers were made to respective sterile medium from tubes showing growth.
To determine the Ser grp requirement, OM containing tryptophan as the sole amino acid was supplemented individually with amino acids of the Ser group (serine, glycine, and cystine). The inoculum source for this test was OM with tryptophan and Ser grp, and growth was initiated by transferring 0.5 ml of C. scindens ATCC 35704 aseptically from late-log/stationary phase with 23-gauge BO needles attached to 1-ml syringes to sterile growth media. Tubes were incubated at 37°C. Growth was measured over time, and sequential transfers were made to respective sterile medium from tubes showing growth.
Determination of vitamin requirement
The vitamin determination for C. scindens ATCC 35704 was performed exclusively in
OM with variations in supplemented vitamins. The leave-one-vitamin-out method was used in this study. OM without CVS was supplemented (0.1 ml) of one of ten different autoclaved vitamin solutions (i.e., each solution consisted of CVS minus a vitamin).
Growth was initiat�d by transferring 0.5 ml of C. scindens ATCC 35704 aseptically from late-log/stationary phase of OM with 23-gauge BO needles attached to 1-ml syringes, and tubes were incubated at 37°C. Growth was measured over time, and sequential transfer
32 was made to respective sterile medium from tubes showing growth. The culture medium which supported growth through the 3rd transfer was considered growth supportive.
OM with riboflavin and pantothenic acid as sole vitamins was prepared, and growth was initiated by transferring 0.5 ml of C. scindens ATCC 35704 aseptically from late log/stationary phase of OM. On 2nd and 3rd transfers, an autoclaved vitamin solution consisting of (biotin, folic acid, pyridoxal-HCI, lipoic acid, nicotinic acid, p-aminobenzoic acid, thiamine, or cyanocobalamin) were added (0.1 ml) to OM with riboflavin and pantothenic acid, and the growth measured over time. Supplemented vitamins which enhanced growth on 2nd and 3rd transfers were considered growth-stimulatory.
Comparison of two forms of pyridoxal. Filter-sterilized vitamin solutions with two different forms of pyridoxal were prepared. One solution contained riboflavin, pantothenic acid and pyridoxal-HCI; the other solution contained riboflavin, pantothenic acid and pyridoxal-P04. OM with tryptophan as the sole amino acid was supplemented with 0.1 ml of the filter-sterilized vitamin solutions separately, and growth was initiated by transferring 0.5 ml of C. scindens ATCC 35704 aseptically from late-log/stationary phase of OM. Growth was measured over time, and sequential transfers were made to respective sterile media from cultures showing growth.
33 Thin Layer Chromatography (TLC) analyses
Bile acids. 10.6 mM stock solution of bile salts (sodium cholate and sodium deoxycholate) were prepared. 0.0456 g of sodium cholate and 0.0439 g of deoxycholate were individually dissolved in 8 ml RO water and final volume was made to lOml. The solution was filter sterilized with 0.2 µm pore size nylon syringe filters into dry and sterile
18 x 150-mm culture tube and aseptically sealed with sterile gray butyl rubber stoppers and sterile aluminum crimp. The tube was then flushed and pressurized to 15 PSI over atmospheric pressure with argon aseptically and stored at room temperature.
Bile acid standard in culture medium. OM with 0.1 mM cholate (CA) or 0.1 mM deoxycholate (OCA) was prepared as bile acid standards as shown in Table 1.
Table 1. Bile acid standard in OM Control Sterile DM RO water CA (10.6 mM) OCA (10.6 mM) OM 1050 µI 10 µI 0 0 DM +CA (0.1 mM) 1050 µI 0 10 µI 0 OM + OCA (O.l mM) 1050 µI 0 0 10 µI
C. scindens ATCC 35704 in culture media with bile acids. OM was aseptically supplemented with sterile cholate and inoculated with C. scindens ATCC 35704 with 23- gauge BD needles attached to 1-ml syringes as shown in Table 2. The tubes were incubated at 37°C, and growth was measured over time. Once the growth reached stationary phase, 1 ml of culture was removed aseptically with 23-gauge BD needles attached to 1-ml syringes, and bile acids were extracted from the culture sample immediately or the culture sample was stored at -20°C until extracted later.
34 Table 2. Culture preparation and inoculation Step 2 Step 3 Step 1 Sterile anoxic RO Sterile cholate Step 4 Culture mediuma Broth water (10.6 mM) lnoculum DM lO ml 0.1 ml 0 0.5 ml DM lO ml 0 O.l ml O.S ml DM + Trp lO ml 0 0.1 ml O.S ml DM + MVS 10 ml 0 0.1 ml 0.5 ml MM lO ml 0 0.1 ml 0.5 ml aAll cultures were maintained in duplicate tubes. For this experiment, DM + Trp was DM with tryptophan as sole amino acid, and DM + MVS was DM with riboflavin, pantothenic acid and pyridoxal-HCI as sole vitamins.
Bile acid extraction from sterile medium (standards) and from cultures. The bile acid standards (1060 µI) shown in Table 1 and cultures (1000 µI) shown in Table 2 were divided equally into two microcentrifuge tubes. Each sample was acidified with SO µI of 3 N HCI, and SOO µI of ethyl acetate was added to extract the bile acids. The tubes were capped, vortexed, and spun for 1 min at 14,000 rpm. The organic phase collected at the top of the microcentrifuge was collected in a wide-mouth vial. The steps from addition of ethyl acetate to collection of organic phase was repeated for a second time from each acidified sample. The collected (pooled) organic phase was dried at room temperature under stream of nitrogen. Methanol (100 µI) was added to the vial, and the dried extract was resuspended. TLC was then performed as soon as possible.
TLC analysis. All of the next steps of TLC analysis were performed in a fume hood.
Samples (SO µI) of the dried, resuspended extracts from standards and from cultures were spotted onto 20 x 20 cm AL SIL G, 2SO µm thick, non-UV TLC plates (Macherey-Nagel,
Duren, Germany) with an Eppendorf pipettor (Volume 10-100 µI). To minimize big spots, a small amount of sample was applied at a time, the spot was allowed to dry, and then
35 more sample was applied until all of the 50 µI sample had been applied to the plate. To evaporate the methanol and dry the spot quickly, a hair dryer was used at low setting.
After the spotted samples were completely dry, the TLC plate was placed in a TLC tank containing 50 ml of developing solvent; the solvent was placed in the tank 15 min prior to adding the TLC plate in order to saturate the tank atmosphere with the solvent. The solvent system contained ethyl acetate, cyclohexane, chloroform, and glacial acetic acid in a ratio of 11:4:3:2. A second solvent system of ethyl acetate, cyclohexane, and glacial acetic acid in a ratio of 12:12:1 was also used to analyze bile acids. Once the solvent was nearly to the top of the TLC plate, the plate was removed from the tank and allowed to completely dry at room temperature. Charring agent solution was then sprayed over the entire surface of the dry TLC plate. The charring agent contained: methanol (150 ml), RO water (150 ml), concentrated HiS04 (10 ml), and MnCli.6H20 (1 g). The plate was placed in an oven at 6S°C for 10 min and then observed under long UV light (365 nm).
High performance liquid chromatography (HPLC)
Stock solution for HPLC standards. Stock solutions (400 mM) of standards (glucose, sodium acetate trihydrate, sodium formate, sodium lactate, succinic acid, ethanol, propionate, isobutyrate, butyrate, isovalerate, and valerate) were prepared individually.
All solutions were stored at 4 °C.
Preparation of HPLC standards. The HPLC external standardization method was used to calibrate the system. Standards either as individual substances or as mixtures were prepared with glucose, ethanol, acetate, formate, lactate, succinate, propionate,
36 isobutyrate, butyrate, isovalerate and valerate. A standard mix containing glucose,
ethanol, acetate, formate, lactate, and succinate was prepared with each substance in the mix was at a final concentration of 40 mM. To prepare standard mix, 100 µI of the individual stock solutions (400 mM) and 400 µI of RO water were combined. A second standard mix containing propionate, isobutyrate, butyrate, isovalerate, and valerate was also prepared with each substance in the mix at a final concentration of 40 mM. To prepare this standard mix, 100 µI of the individual stock solutions (400 mM) and 500 µI of
RO water were combined. Next, 2-fold serial dilutions were performed from both 40 mM standard mix solutions to achieve the following final concentrations (mM): 20, 10, 5 and
2.5.
Since the peak for phosphate buffer in OM + P04 showed up before glucose, calibration with lower concentrations of glucose standard were carried out for accurate glucose analysis. For this, 100 µI of glucose (400 mM) was added to 900 µI of RO water to achieve a 40 mM glucose solution. A 2-fold serial dilution was performed from 40 mM glucose solution to achieve the following final concentrations (mM): 201 10, 5, 2.5, 1.25,
0.625, 0.3125 and 0.15625. To determine glucose consumption by C. scindens 35704 precisely, the samples were diluted 1:10 in O.OlN H2S04 (HPLC solvent) and analyzed with this calibration system.
Glucose stock solution for use in culture media. A glucose stock solution (1.177 M) was prepared by dissolving 2.12 g of glucose in 8 ml of RO water and final volume brought to 10 ml. The glucose solution was filter sterilized with 0.2 µm pore size nylon filter into a dry and sterile 18 150-mm Bellco culture tube and aseptically sealed with sterile gray x
37 butyl rubber stoppers and sterile aluminum crimp seals. The tube was then flushed and
pressurized to 15 PSI over atmospheric pressure with argon aseptically and stored at
room temperature. The filter- sterilized glucose stock solution (0.2 ml) was added
aseptically to 10 ml of sterile culture medium without glucose so that the final
concentration of glucose in culture media was 22 mM.
HPLC conditions. The following HPLC equipment and conditions were used for
analysis of standards and cultures: Beckman Gold System (125 Solvent Module, model
166 variable wave length detector, Jasco Rl-1530 refractive index [RI] detector, Alltech
530 Column Heater, model 508 autosampler, and 32 Karat software [version 5]), Aminex
HPX-87H (300-mm long) analytical column (BioRad); solvent, O.OlN HiS04 (0.6 ml of
concentrated HiS04 in 2 liters RO water)solvent; solvent flow rate, 0.6 ml/min; column
temperature, SS°C; injection size, 20 µI; detection, UV (210 nm) or refractive index (Jasco
Rl-1530 detector); run time, 45 min per sample.
HPLC analysis of sterile culture media. Samples (0.7 ml) of sterile culture media
(OM, OM with tryptophan as sole amino acid, and MM) were removed with 23-gauge BO
needles attached to 1-ml syringes and dispensed into HPLC vials. Samples were analyzed
by HPLC using RI and UV detection (210 nm).
Resolving ethanol peak in sterile culture media. In order to discriminate between
ethanol and interfering substances (i.e., similar retention as ethanol) in OM, all components of OM (mineral solution, trace metal solution, complete vitamin solution,
complete amino acid solution, resazurin, sodium bicarbonate, and sodium sulfide) were
prepared individually with concentrations equal to their final concentrations in OM.
38 Samples (0.7 ml) of each component were added to separate HPLC vials and then subjected to HPLC analysis.
In addition, a mixture of 89.3 mM sodium bicarbonate and 20 mM ethanol and a mixture of 89.3 mM sodium bicarbonate and 40 mM ethanol were prepared. Samples
(0.7 ml) of both mixtures were added to separate HPLC vials and analyzed by HPLC.
HPLC analysis of sterile OM with P04 buffer. Samples (0.7 ml) of sterile OM with PQ4 buffer was removed with 23-gauge BO needles attached to 1-ml syringes and dispensed into HPLC vials. Samples were analyzed by HPLC using RI and UV detection (210 nm).
Resolving unknown peak in OM with P04 buffer. To resolve an unknown peak
(substance) interfering with glucose analysis, a mixture of monobasic and dibasic potassium phosphate (SO mM} was prepared. Sample (0.7 ml} of a mixture was added to separate HPLC vials and analyzed on HPLC.
Sampling and processing of culture for HPLC analysis. Six tubes each of OM, OM containing tryptophan as sole amino acid, and MM prepared in PQ4 buffer without glucose were used. Prior to inoculation, all tubes were supplemented with 0.2 ml of 1.177
M filter-sterilized stock glucose solution to achieve a 22mM final glucose concentration. lnocula (O.S ml) from OM with P04 buffer cultures were used, and nine tubes (three from each growth medium) were incubated at 37°C. Growth was measured over time, and 1 ml of culture was removed aseptically with 23-gauge BO needles attached to 1-ml syringes
once the cells reached maximum OD (Tmax ) in stationary phase. From the remaining inoculated tubes that were not incubated, 1 ml of culture was removed immediately to obtain a sample at time zero (To) with sterile 23-gauge BO needles attached to 1-ml
39 syringes. The samples from both periods (Tmax, To) were centrifuged at high speed for 2
min, and samples (0.7 ml) of supernatant fluid were removed with a sterile 1-ml syringe; a 0.2-µm pore size nylon filter was attached to the syringe and the supernatant fluid was
filtered into an HPLC vial and capped for subsequent analysis. The samples (Tmax and To) were also diluted 1:10 in 0.01 H2SQ4 (mobile phase) and analyzed for glucose on HPLC.
The samples were stored at -20°C until analyzed by HPLC.
Gas Chromatography (GC)
GC conditions. The following GC conditions were used with a Thermo Fischer Trace
1310 gas chromatograph, thermal conductivity detector (TCD), and Oionex chromeleon
(version 7.1.2.1478) to quantitate gas standards and headspace gas composition of cultures: column; stainless steel (2 mm by 2 m) containing Molecular Sieve (13 60/80; x
Restek, Bellefonte, PA); carrier gas, 100% argon; carrier gas flow rate, 20 ml/min; injection port, 150°C; column oven, 60°C; detector, 175°C; injection size, 100 µI; run time: 2 min per sample.
Preparation of GC standards and calibration. A syngas mix containing H2, CO, C02,
CH4, and N2 was used as a standard for GC analysis. The 100% syngas was diluted to 41.4% and 26.11% in argon containing serum bottles, and the three concentrations were used for GC calibration. The Hi concentrations in the 100, 41.4 and 26.11% syngas mixes were
25, 10.35 and 6.53%, respectively. Samples (100 µI) of gas were removed with 27-gauge
BD needles attached to 1-ml syringes and injected into the GC. The standards were analyzed by GC, and the system was calibrated.
40 GC analysis from cultures. Before chromatographic analysis, t�e volume of headspace gas was measured (in millibar) with a TensioCheck (Tensio-Technik,
Geisenheim, Germany) needle manometer. Hydrogen (H2) solubility (0.017 ml of Hi per ml of water at standard temperature and pressure) was calculated from a standard solubility table (SS), and the amount of gas produced was calculated by taking in account both gas and liquid phases. Samples (100 µI) of headspace gas were removed from
cultures with 27-gauge BO needles attached to 1-ml syringes from Tmax tubes (three tubes each from OM, OM containing tryptophan as sole amino acid and MM in PQ4 buffer), and analyzed using the above GC conditions.
Genetic Information. An Integrated Microbial Genome (IMG) system was used for genomic analysis of C. scindens ATCC 3S704. IMG is a genome browsing software
(https ://img.jgi.doe.go v) which provides information about microbial genomes using several tools like Kyoto encyclopedia of genes and genomes (KEGG) and MetaCyc
Metabolic Pathway Database (S6). First, the genome for C. scindens ATCC 35704 was searched on IMG and then added to the genome cart. Using search tool in KEGG, metabolic pathways were searched, and the maps with missing genes were reviewed.
The information based on missing genes was investigated to understand the nutritional requirements of C. scindens ATCC 3S704.
41 IV. RESULTS
Growth of C. sclndens ATCC 35704 in undefined and defined culture media. In previous work, culture media reduced with cysteine hydrochloride were used to grow C. scindens ATCC 35704 (57). In contrast, in the present study, culture media reduced with sodium sulfide were used. Sulfide-based media were importantfor this study as cysteine was a part of the complete amino acid solution (CAAS) used to determine the amino acid requirement(s) of C. scindens ATCC 35704. The presence of cysteine in culture media as a reducing agent would therefore interfere with determining if C. scindens ATCC 35704 required cysteine for growth.
All undefined and defined media reduced with sulfide supported growth of C.scindens
ATCC 35704 (Fig. 7). In highly nutritious undefined media like brain heart infusion (BHI) and undefined medium (UM), growth was faster and greater than in the defined medium
(OM). However, good growth in OM reduced with sodium sulfide was observed and thus suggested that sulfide-based medium was suitable for determining the amino acid requirement{s) of C. scindens ATCC 35704. It should be noted that OM contained the complete amino acid solution (CAAS) and complete vitamin solution (CVS) unless indicated otherwise.
42 - E 1 c 0 0 �OM {8) \D1U c -UM (3) 0 - (3) --i'.r-BHI
0.01 0 5 10 15 20 30 35 25 Time (h)
Fig. 7. Growth of C. scindens 35704 in OM, UM, and BHI. The values
were the mean of duplicate tubes. Parenthetical values indicate
passage number. Abbreviations: BHI, brain heart infusion; UM,
undefined medium; and OM, defined medium
Determination of amino acid requirements of C. scindens ATCC 35704. The primary goal of this study was to determine the amino acid(s) required for the growth of C. scindens ATCC 35704. First, the essential 20 amino acids were divided into six groups: glutamate (Glu group [grp]), serine (Ser grp), aspartate (Asp grp), pyruvate (Pyu grp), aromatic (Aro grp), and histidine (His grp). To identify which group(s) was required for growth, the leave-one group-out technique was applied where each group was deleted from the CAAS present in OM. C. scindens grew in media when glutamate and aspartate groups were left out (Fig. 8), suggesting that the amino acids in these two groups were
43 not required for growth. Therefore, the preliminary results on group determination
suggested that amino acid(s) in serine, pyruvate, aromatic, and histidine groups were
necessary for growth of C. scindens.
1.2 ....------�
-
� 1 Iii1st transfer 0 �0 0.8 O 2nd transfer IU 131 3rd transfer c 0 0.6
-
0 DM-Glu grp DM-Ser grp DM-Asp grp DM-Pyu grp DM-Aro grp DM-His DM
DM without (-) the indicated group of amino acid(s)
Fig. 8. Determination of amino acid requirements of C. scindens ATCC 35704
using the leave-one-group out technique. The values were mean overall
changes (�) in OD for duplicate tubes, except for the positive control (DM) where
only single tubes were used. Abbreviations: Glu grp, glutamate group; Ser grp,
serine group; Asp grp, aspartate group; Pyu grp, pyruvate group; Aro grp,
aromatic group, and His grp, histidine group.
44 Determination of amino acid(s) within group required for the growth of C. scindens
ATCC 35704. To determine which amino acid(s) from the remaining four groups
{histidine, aromatic, pyruvate, and serine) were required for the growth of C. scindens
ATCC 35704, each group was tested individually. First, the histidine group was tested
where OM with Aro, Pyu and Ser grps was supplemented with and without histidine.
Growth was observed in OM with and without histidine (Fig. 9). The growth in OM
without histidine suggested that histidine was not required for C. scindens ATCC 35704.
Interestingly, this contradicted the previous findings from the leave-one- group-out test.
Second, the requirement for amino acids from the aromatic group was tested. OM
with only Pyu and Ser grps was supplemented with and without each amino acid of the
aromatic group (tryptophan [Trp], tyrosine [Tyr] or phenylalanine [Phe]). Only OM supplemented with tryptophan supported growth (Fig. 10), suggesting that only tryptophan in the aromatic group was required for growth by C. scindens ATCC 35704.
Third, the requirement for amino acids from the pyruvate group was tested. OM with only Trp and the Ser grp was supplemented with and without each amino acid of the
pyruvate group (alanine [Ala], valine [Val] or leucine [Leu]). All culture conditions were
growth supportive (Fig. 11). The observed growth in the absence of amino acids from the
pyruvate group suggested that these amino acids were not required for C. scindens ATCC
35704 and contradicted previous results with the leave-one-group-out technique (Fig. 8).
Lastly, the requirement for amino acids from serine group was tested. OM with only
Trp was supplemented with and without each amino acid of the serine group (glycine
[Gly], serine [Ser] or cystine [Cys]). All culture conditions supported growth (Fig. 12).
45 Growth was also observed without serine grp suggesting that the amino acids were not required for C. scindens ATCC 35704. This result also contradicted the leave-one-group- out technique group test (Fig. 8).
The overall result for amino acid requirement of C. scindens ATCC 35704 showed that only tryptophan was required for it s growth. At this time, it is unclear as to why the leave- one-group-out technique yielded conflicting results when determining the amino acid requirements from the histidine, pyruvate, and serine groups.
1.2
- • 1st transfer E 1 G 2nd transfer c 0 3rd transfer 0 0.8 o \0.... cns 0 0.6
-... 0.2
0 DM+His OM-His DM OM +/- Histidine
Fig. 9. Determination of the histidine group requirement for C. scindens
ATCC 35704. Organism was grown in OM with amino acids of the
aromatic, pyruvate, and serine groups with or without histidine. The
values are mean overall changes (6) in OD for duplicate tubes, except for
the positive control (DM) where only single tubes were used.
Abbreviations: His, histidine.
46 1.2
- 1 E
c ii1st transfer 0 0.8 0 O 2nd transfer '° ... �3rd transfer QCV 0.6 0
.s=... 03 0.2 �... 0 DM DM+Trp DM+Tyr DM+Phe -0.2 OM +/- Aro grp amino acids
Fig. 10. Determination of the aromatic group amino acids required
for the growth C. scindens ATCC 35704. The organism was grown in
OM with amino acids from serine and pyruvate groups and with or
without amino acids from the aromatic group. The values are mean
overall changes (6} in OD for duplicate tubes. Abbreviations: Trp,
tryptophan, Tyr, tyrosine and Phe, phenylalanine.
47 1.2
- El1st transfe E 1 r
c ffi12ndtransfer 0 0 0.8 �3rd transfer '° ....ns Q 0 0.6 �
.s::..... 0.4 0� .... C> 0.2
0 OM DM+Ala DM+Val DM+Leu OM +/- Pyu group amino acids
Fig. 11. Determination of the pyruvate group amino acids required
for the growth of C. scindens ATCC 35704. The organism was grown
in DM medium with amino acids from the serine group, with
tryptophan, and with or without amino acids from the pyruvate
group (Pyu grp). The values are mean overall changes (fl) in OD for
duplicate tubes. Abbreviations: Ala, alanine; Va l, valine and Leu,
leucine.
48 1.2 • 1st transfer 1 - E 0 2nd transfer c 3rd transfer 0 0.8 D 0 \0... CV : : : I c 0.6 ; <:J 0 0 :������\i=ii�rn OM DM+Gly DM+Ser DM+Cys OM +/- Ser grp amino acids
Fig. 12. Determination of the serine group amino acids required for the
growth of C.scindens ATCC 35704. The organism was grown in OM with
tryptophan and with or without amino acids from the serine group (Ser
grp). The values are mean overall changes (�) in OD for duplicate tubes.
Abbreviations: Gly, glycine; Ser, serine and Cys, cystine.
49 Determination of the vitamin requirement of C. scindens ATCC 35704. The second goal of this study was to determine vitamin(s) required for the growth of C. scindens ATCC
35704 and thus to ultimately resolve the minimal nutritional requirements of this important gut bacterium. To determine the vitamin requirements, the leave-one vitamin-out technique was applied where each vitamin was deleted from complete vitamin solution (CVS) used to prepare OM. When the media were prepared without pantothenic acid or riboflavin, growth did not occur (Fig. 13) which suggested that these two vitamins were essential for the growth of C. scindens ATCC 35704. To confirm this finding, growth was tested in OM with pantothenic acid and riboflavin only. Interestingly, this medium supported growth but only on initial transfers from OM cultures; successive transfers in OM containing only pantothenic acids and riboflavin as vitamins did not yield maintainable cultures (Fig. 14). Overall, this finding suggested that one or more additional vitamin(s) were necessary for the growth of C. scindens ATCC 35704.
In order to find out which additional vitamin(s} were required for growth, the remainder of the eight vitamins (biotin, folic acid, pyridoxal-HCI, lipoic acid, nicotinic acid, p-aminobenzoic acid, thiamine and cyanocobalamin) were added individually to the OM with riboflavin and pantothenic acid on second and third transfers. Only in OM with added pyridoxal-HCI did good growth occur (Fig. 14). Based on these findings, riboflavin, pantothenic acid and pyridoxal-HCI was required for growth of C. scindens ATCC 35704.
Overall, the minimal nutritional requirements for C. scindens ATCC 35704 were resolved with riboflavin, pantothenic acid and pyridoxal-HCI as the sole vitamins and with tryptophan as the sole amino acid required for growth.
so Consequently, minimal media (MM; OM with riboflavin, pantothenic acid and pyridoxal-HCI as sole vitamins and tryptophan as sole amino acid) was prepared, and growth of C. scindens was compared between MM and the other defined media. All MM and OM culture conditions were growth supportive (Fig. 15); however, as expected, the amount of growth was greater in OM compared to MM. Also, C. scindens struggled to grow after the third transfer in MM. This may be associated with the instability of vitamins during the autoclaving process or with the chemical form of the vitamin itself.
Pyridoxal-HCI is an inactive form which must be converted to the active form pyridoxal
PQ4 for use in cellular reactions (58, 59). Thus, using the active form of pyridoxal might allow for good, sustainable growth.
To resolve this issue, filter-sterilized vitamin solutions (riboflavin, pantothenic acid and pyridoxal) containing two different-forms of pyridoxal, pyridoxal-HCI or pyridoxal
P04, were used. C. scindens grew equally in both media on first and second transfers, however, by third transfer, growth was supported only in OM with vitamin solution containing pyridoxal-HCI (Fig. 16). This suggested that pyridoxal-HCI was more effective than pyridoxal P04; filter sterilized vitamin solution did not support better growth than that observed with the autoclaved version of vitamin solution (Fig. 14). Overall, the reason for unmaintainable cultures after the third transfer in MM remains unclear.
51 OM without (-) indicated vitamin •2nd transfer 3rd transfer B
Fig. 13. Determination of the vitamin requirements of C. scindens ATCC 35704 using the leaving-one-out technique. The values were mean overall changes (�) in
OD for duplicate tubes. Abbreviations: DM, defined medium; OM-CVS, DM without any vitamins added.
52 . 1.2 -E c 1 0 0 U) 0.8 ... "' Q 0 0.6
0� 0.2 CJ
0 e �o(\
1.§1lst transfer OM with (+) indicated vitamin 2nd transfer • G 3rd transfer
Fig. 14. Determination of additional vitamin(s) required for the growth C.
scindens ATCC 35704. The values were mean overall changes (D.) in OD for
duplicate tubes. For this experiment, OM represented OM with riboflavin
and pantothenic acid as sole vitamins.
53 - 1 E c 8 \0 ... "' Q 0 0.1 -+-DM (8) - -0-DM Trp (3) .s:; + i -<>-OM+MVS (4) 0� -MM (3) C> 0.01 0 10 20 30 40 60 70 80 90 100 110 120 130 so Time (h)
Fig. 15. Growth of C. scindens ATCC 35704 in various defined
medium (OM) and minimal medium (MM). The values were the
means of duplicate tubes. Parenthetical values indicate passage
number. For this experiment, OM + Trp was OM with tryptophan
as sole amino acid, and OM + MVS was OM with riboflavin,
pantothenic acid and pyridoxal-HCI as sole vitamins.
54 1.2
- E 1 • 1st transfer c 0 OD2nd transfer 0 0.8 3rd transfer '°.... O ta Q 0.6 0
- OM with (+) indicated vitamin solution Fig. 16. Comparison of two forms of pyridoxal {pyridoxal-HCI vs pyridoxal-P04) on growth of C. scindens ATCC 35704. The values were mean overall changes (ti)i n OD for duplicate tubes. For this experiment: OM + ORO was OM with tryptophan as sole amino acid but without vitamins; OM + MVS with p-HCI was OM with tryptophan as sole amino acid and filter-sterilized riboflavin, pantothenic acid and pyridoxal-HCI; OM + MVS with p-P04 was OM with tryptophan as sole amino acid and filter-sterilized riboflavin, pantothenic acid and pyridoxal-P04; and DM + CVS was OM with tryptophan as sole amino acid and filter-sterilized complete vitamin solution. 55 Transformation of bile acids. In previous studies, 7a-dehydroxylation activity has been observed in nutritious media like BHI (27, 29); therefore, this experiment was conducted to determine if C. scindens ATCC 35704 could undergo 7a-dehydroxylation activity under minimal growth conditions. The first step was to standardize the system with standard bile salts, sodium cholate and sodium deoxycholate. For this, stock solutions (10.6 mM) of standard bile salts were diluted to 0.1 mM in DM and examined on thin layer chromatography (TLC) plates using solvent system of ethyl acetate, cyclohexane, chloroform and glacial acetic acid in the ratio of 11:4:3:2. Results showed that the separation of cholate and deoxycholate was good in this solvent system (Fig. 17). After standardization, 0.1 mM cholate was supplemented into OM, OM containing tryptophan as sole amino acid, DM containing riboflavin, pantothenic acid and pyridoxal HCI as sole vitamins and MM and the 7a-dehydroxylation activity of C. scindens ATCC 35704 in minimal media was tested. TLC analysis showed that C. scindens converted chelate to deoxycholate in all defined and minimal culture media, suggesting that the 7a dehydroxylation activity was possible under minimal conditions (Fig. 18). However, the level of conversion from cholate to deoxycholate varied under different culture conditions; based on band intensity, less deoxycholate was formed in OM. Apart from cholate and deoxycholate, there were also other bands on TLC plates, suggesting that additional bile acid-derived products may have been formed during the bioconversion process; in addition, some products appeared to migrate quickly (bands above deoxycholate) and were not captured with this solvent system. 56 A new solvent system of ethyl acetate, cyclohexane and glacial acetic acid in a ratio of 12:12:1 was tested to allow better resolution of cholate and deoxycholate as well as other potential products on one TLC plate. The new solvent system worked well; products as well as cholate and deoxycholate were well resolved on a single TLC plate (Fig. 19). A product between cholate and deoxycholate appeared in all culture conditions, and its concentration appeared greater in OM. Similarly, a second product which appeared above deoxycholate was also observed under all culture conditions. Additional products appeared in OM containing tryptophan as sole amino acid, OM containing riboflavin, pantothenic acid and pyridoxal-HCI as sole vitamins. However, the identities of these are currently unknown. Overall, these results suggested that products formed during 7a dehydroxylation process changed with nutritional conditions provided to C. scindens ATCC 35704. To further evaluate the effect of culture conditions on the differential formation of bile acid-derived products, C. scindens grown in different defined or minimal media was transferred separately to tubes of BHI with 0.1 mM chelate, incubated, and the cultures then analyzed by bile acid-derived products. The product profiles derived from cholate in BHI cultures were essentially the same (Fig. 20); there was a faint band above deoxycholate on OM containing tryptophan as sole amino acid (lane 5; Fig 20). This was in contrast with OM and MM cultures where product profiles differed (Fig. 19). Overall, these results suggested that culture conditions had the potential to impact the 7a dehydroxylation process by C. scindens ATCC 35704. 57 Fig. 17. TLC analysis of bile acid standards in OM. From left to right: (1) OM; (2) cholate (CA; 0.1 mM CA in OM); and (3) deoxycholate (OCA; 0.1 mM OCA in OM). The solvent system was ethyl acetate, cyclohexane, chloroform and glacial acetic acid in the ratio of 11:4:3:2. 58 Fig. 18. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 on defined and minimal culture media. From left to right: (1) chelate (CA); (2) deoxycholate (OCA); (3) OM + C. scindens 35704; (4) OM + CA + C. scindens 35704; (5) OM with tryptophan as sole amino acid + CA + C. scindens 35704; (6) OM with riboflavin, pantothenic acid and pyridoxal HCI as sole vitamins + CA + C. scindens 35704; and (7) MM + CA + C. scindens 35704. The solvent system was ethyl acetate, cyclohexane, chloroform and glacial acetic acid in ratio of 11:4:3:2. The initial concentration of CA and OCA was 0.1 mM. 59 '") '") �:_- ? / DCA -I .. .. -� ? CA • • 0 1 5 6 Fig. 19. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 on defined and minimal culture media. From left to right: (1) cholate (CA); (2) deoxycholate (OCA); (3) OM + C. scindens 35704; (4) OM + CA + C. scindens 35704; (5) OM with tryptophan as sole amino acid + CA + C. scindens 35704; (6) OM with riboflavin, pantothenic acid and pyridoxal-HCI as sole vitamins + CA + C. scindens 35704; and (7) MM +CA+ C. scindens 35704. The solvent system used ethyl acetate, cyclohexane and glacial acetic acid in ratio of 12:12:1. The initial concentration of CAand OCA was 0.1 mM. 60 Fig. 20. TLC analysis of bile acid metabolism by C. scindens ATCC 35704 in BHI broth. From left to right: (1) cholate (CA}; (2) deoxycholate (OCA}; {3} BHI + C. scindens 35704; (4) BHI +CA+ C. scindens 35704 with inoculum from a culture grown in OM; (5) BHI +CA+ C. scindens 35704 with inoculum from a culture grown in OM with tryptophan as sole amino acid; (6} BHI + CA + C. scindens 35704 with inoculum from a culture grown in OM with riboflavin, pantothenic acid and pyridoxal-HCI as sole vitamins; and (7) BHI + CA + C. scindens 35704 with inoculum from a culture grown in MM. The solvent system used ethyl acetate, cyclohexane and glacial acetic acid in ratio of 12:12:1. The initial concentration of CA and OCA was 0.1 mM. 61 HPLC analysis of standards for calibration. The fi�st step of HPLC analysis was to calibrate the system 1.vith standards. The HPLC chromatogram for the substances used as standard (glucose, ethanol, acetate, formate, lactate, succinate, propionate, isobutyrate, butyrate, isovalerate and valerate) showed good peaks on both refractive index (RI) detection and UV detection (210 nm) (Fig. 21). Different retention times were detected for all substances which suggested a good separation between all components (Table 3). Table 3. Retention times of substances used as standards in high performance liquid chromatography (HPLC) . Standard Detection system Retention time (min) used Glucose Refractive index (RI) 9.20 Ethanol 21.92 Succinate Ultra violet (UV @ 11.72 Lactate 210 nm) 12.52 Formate 13.77 Acetate 15.00 Propionate 17.62 lsobutyrate 19.93 Butyrate 21.75 lsovalerate 25.13 Vale rate 30.67 62 Refractive Index (RI) Detector A � � N � �! ! � � ..... � cigN � �� ;:;;� � .., 0 0 5 10 1S 20 2S 30 3S Minutes Detector (21p nm) B UV 100000 J I ! ; ..,... � I .. ! 75000 I ' I i I ' � ! I Ii 8 I §8 C!C> I Odd· ;:i ci d 8881 I "' OW')N I N �� .,..., .., � i 0 vi cO � � ,...:� ... �� \ I . ·, 1::� I�,\ I I I 1 1 I I I ' I iI I 0 s 10 15 20 2S 30 3S Minutes Detector (210 nm) c UV I ' aC!C> 8c> � §8ci ci 8ci 80 ! 80 N I C> ..... � .... vi ,..; � .., 0 s 10 1 S 20 25 30 35 40 4SI Minutes Fig. 21. HPLC analysis of substances (20 mM) as standards. Panels: (A) glucose and ethanol with refractive index (RI) detection; (B) succinate, lactate, formate and acetate with UV detection; and (C) propionate, isobutyrate, butyrate, isovalerate and valerate with UV detection. 63 HPLC analysis of sterile culture media. After HPLC calibration, the next step was to subject sterile culture media used in the cultivation of C. scindens ATCC 35704 to HPLC analysis. Glucose and ethanol from OM, OM with tryptophan as sole amino acid, and MM was observed along with some additional small positive and negative peaks (Fig. 22). Other than that, the baseline of chromatogram looked clear. The concentration of glucose in OM, OM with tryptophan as sole amino acid, and MM were 23.4, 20.2 and 19.13 mM, respectively, which were lower than the actual (calculated) amount of glucose (25 mM). This could be due to glucose breakdown molecule during the autoclaving process. Though culture media were not supplemented with ethanol, the RI detected ethanol ("'20 mM) in all culture media, which may be attributed components in culture media with a similar retention time as ethanol. This would account for false positive. Therefore, the next step was to find out which component(s) in culture media showed similar retention time as ethanol. None of the components of DM (mineral solution, trace metal solution, complete vitamin solution, complete amino acid solution, resazurin, sodium bicarbonate, and sodium sulfide) gave a peak with an identical retention time of 21.9 min (retention time for ethanol); however, sodium bicarbonate showed a peak at 23.23 min (Fig. 23 A) which was just after ethanol. From a mixture of 89.3 mM sodium bicarbonate and 20 mM ethanol and a mixture of 89.3 mM sodium bicarbonate and 40 mM ethanol, 41.6 mM and 60.6 mM of ethanol were detected respectively (Fig. 23 B,C). Both mixtures showed "'20 mM additional ethanol concentration which confirmed that sodium bicarbonate was being detected as ethanol in culture media. 64 DM with P04 buffer (SO mM) showed a flat baseline with no ethanol detected and suggested culture medium with phosphate buffer (P04) could be used for substrate-end products detection. However, a large peak just before glucose at 8.43 min was also observed in DM with P04 buffer (Fig. 24 A) and this could be due to the phosphate buffer itself. A mixture of monobasic and dibasic potassium phosphate (SO mM) was detected as a peak at 8.43 min (Fig. 24 B) which ultimately verified that the unknown peak was due to the phosphate buffer. Based on retention times and UV detection (210 nm), trace amounts (< O.S mM) of the substances which appear to be succinate, acetate, formate, isobutyrate and butyrate and a moderate amount (6 mM) of propionate were present in OM (Fig. 2S A). However, since none of these organic acids were actually added to DM, their presence may be the result of the following: (i) succinate, acetate, formate, isobutyrate, butyrate, and propionate were present as contaminants in components used to make DM or were generated as a breakdown products during autoclaving and (ii) substances that produced similar retention times as those for succinate, acetate, formate, isobutyrate, butyrate, and propionate were present as contaminants in components used to make OM or were generated from breakdown of components during autoclaving. Interestingly, the peak with the same retention time as propionate was detected in all media except culture media which contained only added tryptophan, for example, DM with tryptophan as sole amino acid (not shown in figure) and MM (Fig. 2S B). Since, propionate was detected only in medium with CAAS, it suggested that an amino acid from CAAS yielded a substance (a 65 peak) with same retention time {19.5 mins) as propionate (Fig. 25 C). Thus, media containing CAAS could not be used to detect or quantify propionate. A Refractive R Detector Index ( I) 6000 § ! C> ,_ .. 4000 N .... co § ::: § § § § C> c::; C> C> C> C> ,_ ,_ ,_ � 2000 «> .... "" "' "" N ;:; c( .., � J.. lQ � � 0 -2000 0 s 1 0 I S 20 25 30 35 Mtnu1es Refractive Index (RI) Detector B 4000 C> ... "' § § § C>§§ C> C>§ C>§ .... C> C> d 2000 � ,_ � lil c; c( � $ � .. � CD"' � � � e .. � � ;;; 0 - --- J__I..-- -- -2000 0 10 I S 20 25 30 35 Mtnutes Refractive Index (RI) Detector c ,_ 4000 ;i.., § 2 C> § § § § § § i::: 2000 C> C> C> ! !! ! � c( l::'.!:! f::! :::. � � le � ;s;gg :s � !! � I :e Ila � ;::;;;; ;s;I 0 - _ _ 1 ·2000 0 5 35 1 0 1 5 20 25 30 Minute:s. Fig. 22. HPLC analysis of sterile culture media using RI detection. Panels: (A) DM; (B) DM with tryptophan as sole amino acid; (C) MM. 66 RefTactlve Index (RI) Detector A § § §§ o§ § o § Oo c:> c:> c:> ..,,_ 500 '° ::> c( 0 .500 ..,"' ..,"' 0 5 10 15 20 25 30 35 Minutes Refractive Index (RI) Detector B § § 8 § § c:> c:> c:> c:> 500 c:> c:> ..,s- - ;e ai .... "' --- ::> 0 --- c( I d I I I I --I "'! I L- L- CIO -1000 - �LJ 0 5 10 15 20 30 35 Minutes c Refractive Index (RI) Detector 1500 1000 § § 0,._ ... 500 N 0 ·500 0 5 10 1!1 30 35 Minutes Fig. 23. HPLC analysis of the OM component bicarbonate and a potential end product ethanol using RI detection. Panels: (A) sodium bicarbonate (89.3 mM); (B) sodium bicarbonate (89.3 mM) +ethanol (20 mM); and (C) sodium bicarbonate (89.3 mM) + ethanol (40 mM). 67 Refractive Index (RI) Detector A 0 5000 8 0 N N U> 0 8 8 8 0 8 § 0 0 0 ci� 0 2500 O> "' NO> • co li N :8 a) ::> � � � � < 0 I F; / � §J;0 .2500 9! �§ °' 0 5 10 15 20 25 30 35 Ml nut•• B Refr.:ietive Index (RI) Detector eCXIO _, g_, 5 _, g g l5 4000 o CD CD � I o 0§ 0§ 0§ 0§ � 0§ -.. -.;-- I (1 � ::> =s; ��p:-. �- ....N � .,, � E: .... � ti ""('.? N c-> � I N N � 0 _...... -...... -...... -� 0 5 10 15 zo 25 30 35 Mlnuies Fig. 24. HPLC analysis of (A) OM with P04 buffer and (B) P04 buffer using refractive index (RI) detection. 68 )> AU AU .. OJ n AU n H � ... l3 :::u 0 � 0 � � § 8 § § � � I �I I I I I I O 0 0 0 I' r·" �3II � Cl. 'Tl I 4"o 00 ; .....ro l ro �· 0.000 .....n N "l 2.42 U1 J I I I ( :::! I "'O U\ Vl1W nr- Q)"'O)" U\ ===- - �·I · s""f :::! Q) I - :::! 5�00-::--- =--- - ct> \ 0.000 ..v;- Q) -< 6.12 a.o VI �=--- =� o ); o - �o v;· h'_ _ oooo a98 0�.42 . � -0 0.000 - · 0 �--=::- 0 0.000 c 10.63 0.000 ' VI..... 0 t �. 43 - ct> 9.72 ';.,,•;:""' '" 0.3 I �ucunat� 0 _if. " 657 O OOO BDl �.72 - �. 0.000 � OJ ii) BDL 11OO:>o0 S7 BDL0000 � n Hi� 1Jr:!io.2s5 � 13 '� c OOOOBDL c: blnl!llC 0 388 . c 14. 90 acetate o 33 8 1 aee1�1e 0 c ..,;::; '" 0.000 < •• < c < 136S 526 I ;;. BOL 0 0... Q) 0 (D :::! ro i(D ; 14 62 ID 3 0.000 (').. -Cl. -(') 3: (')....ID ct> 3:: . 83 S':c - Cl. c:; 0., S'c 0... 0., n .. - s c:· .. i n • 'N..... I\).... � 18 1S propionate 6 481 'N 3 ... � 0 0 0 Q) � IJ (/) :::! :I :I ,, z. � :i Cl. 20 53 1sob1aym1e O 067 i ... ;:::::;: OOOOBDL VI � 0n I I 3 I , "' "' ,, "'C0 � .,. O.OOOBOL •�•oc :::! I I O 000 BDL .....ro:::! I ) 0000 c I "" VI I 0000 :::! I (1Q I 1 1,,., � I The above results indicated OM with bicarbonate-C02 buffer could be replaced with OM with phosphate buffer for better HPLC analysis of substrates and end products. However, in previous experiments, C. scindens ATCC 35704 has been grown exclusively with a bicarbonate-C02 buffer system (57). Therefore, the next step of this study was to determine if C. scindens could adapt and grow effectively in culture media with phosphate buffer instead of the bicarbonate-C02 buffer system. Good growth of C. scindens ATCC 35704 was observed after tertiary transfers in defined and minimal media with P04 buffer (Fig. 26), indicating that culture media with phosphate buffer was suitable for growth of C. scindens ATCC 35704 and thus the determination of substrate-end product profiles in cultures. 1 - E c 0 0 U> ....IV c 0.1 -+-DM (3) ci -DM +Trp(3) - -ii-MM (3) .c... 3: 0 �... 0.01 0 50 100 150 200 Time (h) Fig. 26. Growth of C. scindens ATCC 35704 in culture media with phosphate buffer. The values were the means of triplicate tubes. Parenthetical values indicate passage number. For this experiment, OM + Trp was OM with tryptophan as sole amino acid. 70 Substrate-end product profiles of C. scindens ATCC 35704 in culture media. After the growth of C. scindens ATCC 35704 was observed in culture media with phosphate buffer, the next goal of this study was to analyze by HPLC and GC the end products produced under various culture conditions and determine if there were any differences in the metabolite production when the nutritional requirements were changed. To carry out substrate-end product analysis, C. scindens ATCC 35704 was grown in culture media (OM with P04, OM containing tryptophan as sole amino acid with P04 and MM with P04) containing filter-sterilized glucose (final concentration approximated 20 mM). Samples were collected immediately after inoculation (time zero or To) and when cells reached stationary phase with maximum growth (Tmax ). HPLC analysis was performed on both samples. In OM + P04, chromatograms from all samples indicated a few random peaks (i.e., substances) upon RI and UV detection (Fig. 27 and 28); however, because of the CAAS in OM, propionate detection was not possible. Furthermore, since there was a phosphate associated peak which occurred before the actual glucose peak, samples were diluted 1:10 in O.OlN HiS04 (HPLC solvent) for accurate determination of glucose (Fig. 27 B and 28. B}. In Tmaxsamples, glucose was not detected indicating that C. scindens ATCC 35704 completely consumed the glucose (19.4 mM) during growth, converting it to ethanol, succinate, lactate, acetate, isobutyrate, and isovalerate (Table 4 and 5). The major end products formed were ethanol (24.6 mM), acetate (10.7 mM), formate (6.3 mM), and H2 (16.9 mM) whereas other products were produced in very negligible (< 1 mm) amounts (Table 4 and 5). Based on the substrate-product profile of glucose-grown cells in OM + 71 P04, carbon recovery was 72% and may be attributed to the fact that C02 and biomass carbon production were not determined. In OM with tryptophan as sole amino acid and P04 buffer, C. scindens ATCC 35704 completely consumed the glucose (19.4 mM), converting it to essentially the same products as cells grown in OM with P04 buffer (Table 4). However, ethanol (35.2 mM), acetate (12.9 mM), and H2 (18.9 mM) levels were increased compared to cells growing in OM + P04 (Table 5). In MM with P04 buffer, glucose was not completely consumed by C. scindens ATCC 35704 with 10.9 mM remaining at the end of growth (Table 4). The types of end products generated were same as those from cells grown in OM with P04 buffer and OM containing tryptophan as sole amino acid with P04 buffer. However, the concentrations for most of the end products were very low due to the decreased glucose consumption. Nonetheless, to compare cells grown under different conditions, the ratio of product formed (mM) per glucose (mM) consumed was used. On this basis, glucose-grown cells in different culture media exhibited similar substrate-product profiles (Table 5}: 72 OM + P04 buffer: 1 Glucose 7 1.27 ethanol + 0.04 succinate + 0.02 lactate + 0.32 formate + 0.55 acetate + 0.01 isobutyrate + 0.02 isovalerate + 0.87 H2 OM+Trp+P04 buffer: 1 Glucose 7 1.81 ethanol + 0.04 succinate + 0.03 lactate + 0.14 formate + 0.66 acetate + 0.05 isobutyrate + 0.03 butyrate + 0.05 isovalerate + 0.97 H2 MM + P04 buffer: 1 Glucose 7 2.02 ethanol + 0.03 succinate + 0.04 lactate + 0.05 formate + 0.39 acetate + 0.07 isobutyrate + 0.01 butyrate + 0.03 isovalerate + 0.37 H2 It is worth noting that as the culture conditions became more nutritionally demanding, ethanol production increased slightly while formate levels decreased. 73 A Refractlve Index (RJ) Detector 5000 i5 CD 0§ § N § 2500 N .... / "' -2500 / I 1 0 5 10 15 20 25 30 35 40 45 Minute$ B Refractive Index (RI) Detector 800 §0 600 "' .., co § §§ §§ §C> C> § c;j §C> Oo §0 C>C> §C> 00 ,_ N 00.., -too � O>M 200 0 10 IS 20 25 30 35 40 45 0 Minute$ UV Detector nm) c (210 15000 ...... 15 20 30 3 5 40 45 0 10 Mlnuttt Fig. 27. Glucose (growth substrate) and end products detected in DM with P04 buffer with C. scindens ATCC 35704 at sampling time T0• Panels: (A) T0 with RI detection; (B) T0 with 1:10 dilution of sample prior to RI detection; and (C) T0 on UV detection. 74 A Refractive Index (RI) Detector 2000 C> C>C> C> C> � C> C> §� ::> 0 /' < O> J c:; -1000 «>� U) c;;; -2000 §C> /' cs Q; c: U) jJ-/i 0 5 10 15 20 25 30 45 Minutes Refractive Index (RI) Detector B C> _, � 600 C> � "" .... a C> 200 0 0 5 10 15 20 25 30 40 45 Minutes Detector (21 nm) UV O c 80000 tiOOOO ::> <( 40000 20000 0 I I 0 5 10 15 20 25 30 35 40 45 Minutes Fig. 28. Glucose (growth substrate) and end products detected in OM with P04 buffer with C. scindens ATCC 35704 at sampling time Panels: (A) T with RI detection; Tmax · max (B) T with 1:10 dilution of sample prior to RI detection; and (C) T on UV detection. max max 75 A Refractive Index (RI) Detector 5000 "' "' "'! § "' 0 0 (") 0 § �0 �0 �0 �0 �0 � 5 10 15 20 25 30 35 4() d5 0 Minutes Refractive !ndex (RI) Detector B 000 0 GOO 8 dM C> o c::> 00 0 C>0 C>0 0 c:>0 200 0 10 1 5 2 C 30 40 0 5 UV et ctor (210 nm) c D e 10000 75j:> � ::> 5JOO 0 10 1 5 35 0 20 2: d C Minutes Fig. 29. Glucose (growth substrate) and end products detected in DM + Trp with P04 buffer with C. scindens ATCC 35704 at sampling time T0• Panels: (A) T0 with RI' detection; (B) T0 with 1:10 dilution of sample prior to RI detection; and (C) T0 on UV detection. For this experiment, DM + Trp was DM with tryptophan as sole amino acid. 76 buffer with C. scindens ATCC 35704 at sampling time T Panels: (A) T with RI m max ax· detection; (B) T with 1:10 dilution of sample prior to RI detection; and (C) T on UV max max detection. For this experiment OM + Trp was OM with tryptophan as sole amino acid. 77 A Index (FU) Detector Refractive 5000 � § C> C> N §§ C> � §§ §§§ C> § ;;;: C>O 8 OOC> g :a � C> <:> 2500 d d co 0 <:> <:> c::; <:> <:> 0 ;:: "" � � $;t; �8 � ..... � 112 IE! ..,.., .., � � �$ ...... ::>• � � 2? i �� 0 -2500 0 10 15 20 25 30 3 5 40 Minutes B Refractive Index (RI) Detector 75:) § 0 500 ::> < 25:1 0 0 5 10 25 35 ;:o Minutes UV Detector {21 nm) c O IOOCO _ ------,---� - 0 -- J D 0 25 30 Fig. 31. Glucose (growth substrate) and end products detected in MM with P04 buffer with C. scindens ATCC 35704 at sampling time T0. Panels: (A) T0 with RI detection; (B) T0 with 1:10 dilution of sample prior to RI detection; and (C) T0 on UV detection. 78 A Refractive Index (RI) Oetector 1000 0 1 ::> 1 / < .., -1000 I v � I � ....co -2000 § § c:> c:> ;:;; ....co �co !!? -3000 i 0 30 40 5 10 15 20 Minutes 25 45 Refractive Index (RI) Oetector B 800 § 0 co.... §§ § §§ §§ § § § § § § § co Oo 0 00 0 c:> c:> 0 c:> c:> 0 ..,.,. oO .... � c; 0 O> .... �� � � ...... �� $ � N �... � ... :8 � ii;;:; �� ;;; ;:; .., 200 0 5 1 0 15 25 0 20 30 40 Minutes Detector (210 nm) c UV 15000 10000 5000 0 - -- 0 10 30 5 15 20 35 Minutes Fig. 32. Glucose (growth substrate) and end products detected in MM with P04 buffer RI with C. scindens ATCC 35704 at sampling time Tmax· Panels: (A) Tmax with detection; (B) (C) T Tm;ix with 1:10 dilution of sample prior to RI detection; and max on UV detection. 79 Table 4. Substrate consumption and end products formation over time in defined and minimal culture media by C. scindens ATCC 35704° Substrate consumed or OM with P04 buffer OM + Trp with P04 MM with P04 buffer product formed (mM) buffer (mM) (mM) To Tmax To Tmax To Tmax Substrate consumed Glucose 19.4 ± 2.7 _b 19.4 ± 0.3 - 19.5 ± 3.2 8.6 ± 7.4 Product formed Ethanol 0.6 ± 0.6 25.2 ± 6.1 2.2± 0.3 37.4 ± 4.1 2.4 ± 0.4 24.4 8.4 ± Succinate - 0.8 ± 0.0 0.1 ± 0.1 0.8 ± 0.1 0.1 ± 0.0 0.4 ± 0.0 Lactate - 0.4 ± 0.0 - 0.5 ± 0.5 - 0.4 ± 0.0 Formate 0.3 ± 0.0 6.6 ± 0.4 0.2 0.0 3.1 ± 1.3 0.1 ± 0.0 0.6 ± 0.5 ± Acetate 0.5 ± 0.0 11.2 ± 0.9 0.5 ± 0.0 13.4 ±1.5 0.6 ±0.0 4.8 ± 1.5 I so butyrate - 0.1 ± 0.2 - 0.9 ± 0.2 - 0.8 ± 0.6 Butyrate - - - 0.5 ± 0.1 - 0.1 0.1 ± lsovalerate - 0.4 ± 0.1 - 0.9 ± 0.1 - 0.3 0.1 ± Valerate ------H2 Noc 16.9 ±2.0 ND 18.91 ±2.0 ND 7.21 ±1.7 0 All the values are mean of triplicate tubes ± S.D. To was concentration at time zero and Tmax was concentration at maximum growth. For this experiment, OM + Trp was OM with tryptophan as sole amino acid. b -. not detected c ND, not determined. For hydrogen (H2) concentrations at To, it was assumed that H2 carryover in inoculum would be minimal (not detectable) given the small inoculum size used and poor solubility of H2 in water. 80 Table 5. Substrate-product profiles of scindens ATCC 35704 in defined and minimal culture media0 C. Substrate consumed or product formedb DM with PQ4 buffer DM +Trp with MM with PQ4 [molecular formula, structural formula] (mM) PQ4 buffer (mM) buffer (mM) Substrate consumed Glucose [CsH120s, HCO(CHOH)sH] 19.4 ± 2.7 19.4 ± 0.3 10.9 ± 5.5 Product form ed Ethanol [C2HsO, CH CH20H] 24.6 ± 6.6 (1.27)C 35.2 ± 3.8 (1.81) 3 22.0 ± 8.4 (2.02) Succinate [C4H604, (CH2)i(C02H)i] 0.8 ± 0.0 (0.04) 0.7 ± 0.1 (0.04) 0.3 ± 0.0 (0.03) Lactate [ C3Hs03, CH3CH(OH)COOH] 0.4 ± 0.0 (0.02) 0.5 ± 0.5 (0.03) 0.4 ± 0.0 (0.04) Formate [CH202, HCOOH] 6.3 ± 0.4 (0.32) 2.8 ± 1.2 (0.14) 0.5 ± 0.5 (0.05) Acetate [C2H402, CH3CQOH] 10.7 ± 0.9 (0.55) 12.9 ± 1.5 (0.66) 4.2 ± 1.5 (0.39) lsobutyrate [C4Hs02, (CH3)iCHCOOH] 0.1 ± 0.2 (0.01) 0.9 ± 0.2 (0.05) 0.8 ± 0.6 (0.07) Butyrate [C4Hs02, CH3(CH2)zCOOH] -d 0.5 ± 0.1 (0.03) 0.1 ± 0.1 {0.01) lsovalerate [CsH1o02, (CH3)2CHCH2COOHJ 0.4 ± 0.1 (0.02) 0.9 ± 0.1 (0.05) 0.3 ± 0.1 (0.03)- Valerate [CsH1o02, CH3(CH2)3COOH] - - 7.2 ± 1.7 (0.37) Hi 16.9 ± 2.0 (0.87) 18.9 ± 2.0 (0.97) C02 ND NDe ND Biomass c! ND ND ND Biomass ND Hg ND ND Carbon recovery (%)h 72 98 93 O/R ratio; ND ND ND C1/C2 ratioi ND ND ND 0Values are the mean of triplicate tubes ± SD. For this experiment, DM + Trp was DM with tryptophan as sole amino acid. Determined by taking the difference between and Tmax values (see Table 4). b Parenthetical values are product formed {mM)To per glucose (mM) consumed. c not detected. d -, not determined. e ND,Assu ming 50% of carbon per unit of cell biomass, 1 mg of cell dry weight is equivalent to 41.6 µmol of Ibiomass carbon. Assuming 8% hydrogen per unit of cell biomass, 1 mg of cell dry weight is equivalent to 80 µmol of biomass g hydrogen.h Calculations for carbon recovery: For DM with P04 buffer, (1.27 x 2C) (0.04 x 4C) + (0.02 x 3C) + + (0.32 x lC) + (0.55 x 2C) + (0.01 x 4C) + (0.02 x 5C)/{1 x 6C) x 100 = 72%; for M Trp with P04 buffer, (1.81 + x 2C) + (0.04 x 4C} (0.03 x 3C) + (0.14 x lC) + (0.66 x 2C} (0.05 x 4C) + (0.03D x 4C} + (0.05 x 5C)/(1 xGC} x 100 + + 98%; and, for MM with P04, (2.02 x 2C) + (0.03 x 4C} (0.04 x 3C) (0.05 x lC) + (0.39 x 2C) + (0.07 x 4C) + = + + (0.01 x 4C) + {0.03 x 5C}/(1 x 6C) x 100 = 93%. A ratio of-1 indicates all electrons have been recovered. Glucose (CsHuOs) has 2H for each 0, thus products 1 with >2H per O have been reduced whereas products with <2H per 0 have been oxidized. To calculate O/R value in compound, assign +l per 0 and -1 per 2H in compound. The calculated O/R value is multiplied by the amount (mM) of compound detected. The ratio of total (mM) oxidized compounds to total {mM) reduced compounds is then calculated. A ratio of indicates conversion of a C3 (pyruvate) to C1 C2 compounds and that pyruvate is an + Iintermediate. -1 To calculate ratio, total (mM) of C1 compounds formed is divided by the total (mM) of C2 compounds formed. 81 Information on genes associated within metabolic pathways of C. scindens ATCC 35704. After resolving the nutritional requirement of C. scindens ATCC 35704, the next goal of my project was to analyze its genomic data in order to determine if this information was in agreement with my findings that tryptophan and three vitamins (riboflavin, pantothenic acid and pyridoxal-HCI) were required as growth factors for the growth of C. scindens. The most likely reason of such requirements could be due to missing genes associated with the biosynthetic pathways for these growth factors. For analysis of missing genes, Integrated Microbial Genome (IMG) datasets (https://img.jgi.doe.go v) were used. The genome for C. scindens ATCC 35704 was used to filter the data, and information based on Kyoto Encyclopedia of Genes and Genomes (KEGG) for metabolic pathways was collected. The results from KEGG pathway on tryptophan biosynthesis of C. scindens ATCC 35704 showed four key missing enzymes: enzyme commission (E.C) 4.1.3.27, E.C 2.4.2.18, E.C 5.3.1.24 and E.C 4.1.1.48 which are needed consecutively for tryptophan biosynthesis (Table 6). Similarly, vitamin biosynthesis showed that six enzymes (E.C 3.5.4.25, E.C 3.5.4.26, E.C 1.1.1.193, E.C 4.1.99.12, E.C 2.5.1.78 and E.C 2.5.1.9) in riboflavin biosynthesis; three enzymes (E.C 2.1.2.11, E.C 1.1.1.169 and E.C 4.1.1.11) in pantothenic acid biosynthesis and six enzymes (E.C 4.3.3.6, E.C 3.1.3.74, E.C 1.4.3.5, E.C 2.6.99.2, E.C 1.2.1.72 and E.C 1.1.1.290 ) in pyridoxal biosynthesis; were missing (Table 8). The genomic information based on metabolic pathways provided supporting evidence for requirement of tryptophan, riboflavin, pantothenic acid and pyridoxal-HCI for the growth of C. scindens ATCC 35704. However, genomic information indicated that genes were 82 missing relative to the biosynthesis of biotin and thiamine (Table 8), suggesting these two vitamins were not synthesized by C. scindens. This was in direct contrast to the findings of my study. Interestingly, the genetic map for biosynthesis of amino acids on KEGG showed that only histidine was synthesized by C. scindens ATCC 35704 and that one or more genes associated with biosynthesis of the 19 other amino acids synthesis were missing. For . some amino acids like tyrosine and phenylalanine (Table 6), many of the genes necessary for biosynthesis were lacking whereas with other amino acids like glycine and valine (Table 7) key genes needed for the synthesis of critical intermediates were absent and hence those pathways were interrupted. 83 Table 6. Missing genes of C. scindens ATCC 35704 in amino acid anabolism using the Integrated Microbial Genomes datasets Metabolic pathway involved Missing gene for enzyme EC no. (KEGG) Tryptophan biosynthesis Anthranilate synthase 4.1.3.27 Anthranilate phosphoribosyltransferase 2.4.2.18 Phosphoribosylanthranilate isomerase 5.3.1.24 I ndole-3-glycerol-phosphate synthase 4.1.1.48 Tyrosine biosynthesis Tyrosine transaminase 2.6.1.5 Phenylalanine 4-monooxygenase 1.14.16.1 Arogenate dehydrogenase (NADP(+)) 1.3.1.78 Aromatic-amino-acid transaminase 2.6.1.57 Phenylalanine biosynthesis Aromatic-amino-acid transaminase 2.6.1.57 Alanine biosynthesis Alanine transaminase 2.6.1.2 Aspartate 4-decarboxylase 4.1.1.12 Serine biosynthesis Phosphoserine phosphatase 3.1.3.3 Asparagine biosynthesis Aspartate--ammonialigase 6.3.1.1 Cysteine biosynthesis Cystathionine gamma-lyase 4.4.1.1 Methionine biosynthesis 5-methyltetrahydropteroyltriglutamate- homocysteine S-methyltransferase 2.1.1.14 Lysine [Lysine-biosynthesis-protein LysW]-L-2- 6.3.2.43 aminoadipate ligase Glutamate Alanine transaminase 2.6.1.2 84 Table 7. Amino acid biosynthesis of C. scindens ATCC 35704 dependent on other intermediates Metabolic pathway Intermediates Disrupted pathway involved (KEGG) required Glycine biosynthesis Serine or threonine Missing genes for serine and threonine synthesis Threonine biosynthesis Serine Missing genes for serine synthesis Valine, Leucine and Pyruvate Missing genes for pyruvate lsoleucine biosynthesis synthesis on glycolysis Aspartate biosynthesis Oxaloacetate Missing genes for oxaloacetate synthesis on TCA cycle Glutamine, Arginine, Glutamate Missing genes for glutamate Proline biosynthesis synthesis 85 Table 8. Missing genes of C. scindens ATCC 35704 in vitamin anabolism using the Integrated Microbial Genomes (IMG) datasets Metabolic Missing gene for enzyme EC no. pathway involved (KEGG) Riboflavin GTP cyclohydrolase II 3.5.4.25 biosynthesis Diaminohydroxyphosphoribosylaminopyrimidine deaminase 3.5.4.26 5-amino-6-(5-phosphoribosylamino) uracil reductase 1.1.1.193 3,4-dihydroxy-2-butanone-4-phosphate synthase 4.1.99.12 6,7-dimethyl-8-ribityl lumazine synthase 2.5.1.78 Riboflavin synthase 2.5.1.9 Pantothenic 3-methyl-2-oxobutanoatehydroxymethyltransferase 2.1.2.11 acid 2-dehydropantoate 2-reductase 1.1.1.169 biosynthesis Aspartate 1-decarboxylase 4.1.1.11 Pyridoxal Pyridoxal 51-phosphate synthase 4.3.3.6 biosynthesis Pyridoxal phosphatase 3.1.3.74 Pyridoxal 51-phosphate synthase 1.4.3.5 Pyridoxine 51-phosphate synthase 2.6.99.2 Erythrose-4-phosphate dehydrogenase 1.2.1.72 4-phosphoerythronate dehydrogenase 1.1.1.290 Biotin Dethiobiotin synthase 6.3.3.3 biosynthesis Adenosylmeth ion ine--8-ami no-7-oxononanoate transaminase 2.6.1.62 Lysine--8-amino-7-oxononanoate transaminase 2.6.1.105 8-amino-7-oxononanoate synthase 2.3.1.47 Pimeloyl-[acyl-carrier protein] synthase 1.14.15.12 Thiamine Glycine oxidase 1.4.3.19 biosynthesis Thiazole tautomerase 5.3.99.10 86 V. DISCUSSION Microorganisms originated and have lived for billions of years in anoxic environments on earth (29). Interestingly, the human gut which is a completely anaerobic environment is a niche for 10 to 100 trillion microorganisms (60). Microorganisms require nutrients like carbohydrates, amino acids, vitamins, co-enzymes, and inorganic ions for growth and development (57). All nutrients have different functions in cellular metabolism, for instance, carbohydrates are a carbon and energy source; amino acids act as building block for protein synthesis; vitamins are essential components for coenzyme synthesis; and inorganic ions act as co-factors and activators for metabolic processes (61). Some microorganisms can synthesize all of their amino acids and vitamins since they have the enzymes required for their synthesis, whereas other microorganisms lack biosynthetic abilities. For example, microbes such as Escherichia coli can grow on minimal medium with glucose as sole source of carbon and ammonium as sole source of nitrogen (62). Thus, there are differences in the nutritional requirements of various species of microorganisms. Clostridium scindens ATCC 35704 is a major bile acid dehydroxylating gut microbe (51), and, therefore, a full understanding of the physiology of this gut microbe is important and necessary. Since little research has been performed in this area, this project is a step towards understanding the overall metabolic potentials of C. scindens ATCC 35704. The current work showed that C. scindens ATCC 35704 required three vitamins (riboflavin, pantothenic acid, and pyridoxal-HCI) for its growth. These same three vitamins are required by C. hylemonae, another bile acid-dehydroxylating bacteria (63). 87 A previous study on C. scindens VPI 12708 also shows similar results as riboflavin and pantothenic acid were required for growth (57). Clostridium bifermentans, another bile acid-dehydroxylating gut microbe, requires biotin, nicotinic acid, pantothenic acid, and pyridoxamine for its growth {64). A non-bile acid-dehydroxylating Clostridium sp., Clostridium perfringens, requires biotin, calcium pantothenate, and pyridoxine for its growth (65). The current work also showed that C. scindens ATCC 35704 required only one amino acid (tryptophan) for its growth. This result matches the amino acid requirement of C. scindens VPI 12708 (57). A previous study on C. hylemonae shows that methionine, valine, and tryptophan required for the growth of this organism (63). Very little is known about the amino acid requirements of other bile acid-dehydroxylating bacteria. Several amino acids have been found to promote the spore germination of certain bile acid hydroxylating bacteria. For instance, C. bifermentans requires L-alanine and sodium ions for spore germination (66), and C. sordellii requires L-phenylalanine and L-alanine for its spore germination {67). The most interesting aspect of this study was that C. scindens ATCC 35704 was unmaintainable after the third passage in minimal medium which indicated that additional amino acids or vitamins were necessary for its continuous growth. Nonetheless, the findings relative to the minimal nutritional requirements of C. scindens ATCC 35704 were supported by information based on KEGG maps of biosynthetic pathways for amino acids and vitamins (Fig. 25-28). 88 Tryptophan is an amino acid with several metabolic functions (68). It can act as a precursors for biosynthesis of protein and the cofactor NAO (68). The principal pathway for tryptophan biosynthesis is shown in Figure 25. The core precursor for this pathway is chorismate (69}. The chorismate is converted to anthranilate by anthranilate synthase (E.C 4.1.3.27). The anthranilate undergoes condensation followed by decarboxylation process to form indoyl-3-glycerol-phosphate by series of enzymes: anthranilate phosphoribosyltransferase (E.C 2.4.2.18}; phosphoribosylanthranilate isomerase (E.C 5.3.1.24); and indole-3-glycerol-phosphate synthase (E.C 4.1.1.48) (70}. The indoyl-3- glycerol-phosphate is either converted to tryptophan or first converted to indole and then to tryptophan by enzyme tryptophan synthase (E. C 4.2.1.20) (71}. The genes for enzymes involved in the conversion from chorismate to indoyl-3-glycerol-phosphate are missing in C. scindens ATCC 35704 (Fig. 33). This supports my findings where tryptophan was required for growth of C. scindens ATCC 35704. 89 Current Genome Closlndium sc ndens ATCC 3570� D Genes m aostnd um scmdens ATCC 35704 • Enz)mes \'irth KO hits D �lssmg Enz�·mes Pentosephosphate Quinate tll.thway 7P-2-Dehydro- (Photosynthesis)-� 3-deoxy- .----===rn=====-- --. I D-mbmo- MptoMte �I �I��� D- �sefl 4-phOsphate r--2-.5.1-.54..... �� x Phosphoenol- ( Glycol:ysis ) PJmwa� � 0 3-Deh"�""' Tsbikinii t;v lndole �42.12ll Shikimate Shikimate L-Tryptophan O• l4212ll:�!��tn:fit.pra � 3-phosphate 1 �(2-Caiboxvohenylamino)- iibulose �- S-phoO-( l -Ca.ib o ..�)- t=;�� sphoshi=. >.. 5 .. .------. PRPP 0 .3. ���42 Chorismate Fig. 33. KEGG map for biosynthesis of tryptophan in C. scindens ATCC 35704 (71). 90 Riboflavin (vitamin 82) is an essential component in cellular metabolism as it acts as a precursors for flavin coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) {72, 73). The biosynthetic pathway for riboflavin with missing genes associated to C. scindens ATCC 35704 is shown in Figure 26 {71). Two precursors, GTP and ribulose-5-phosphate, are required for riboflavin synthesis (72). These precursors undergo series of reaction before they combine to form riboflavin, FMN, and FAD {72). The enzymes involved in GTP conversion includes: GTP cyclohydrolase II (E.C 3.5.4.25), diaminohydroxyphosphoribosylaminopyrimidine deaminase (E.C 3.5.4.26), �nd 5-amino- 6-(5-phosphoribosylamino )uracil reductase (E.C 1.1.1.193); and the enzyme for ribulose- 5-phosphate conversion involves 3,4-dihydroxy-2-butanone-4-phosphate synthase (E.C 4.1.99.12) (71). The products from GTP and ribulose-5-phosphate undergo condensation, and the reaction is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (E.C 2.5.1.78) (71). Eventually, riboflavin is formed by action of riboflavin synthase (E.C 2.5.1.9} (71). All the enzymes necessary for riboflavin biosynthesis appear to be absent in C. scindens ATCC 35704 (Fig. 34). Therefore, this provides supporting evidence for my findings. 91 Current Genome C� !ndJm• scn OlP OFMNH2 0FADH2 Fig. 34. KEGG map for biosynthesis of riboflavin in C. scindens ATCC 35704 (71). Pantothenate (vitamin Bs) is a core precursor for cofactors like coenzyme A (CoA) and acyl carrier protein (74). These cofactors are important in a large number of metabolic reactions (74). The novel pathway for biosynthesis of pantothenate is shown in Figure 27. Pantothenate is synthesized from aspartate and 3-methyl-2-oxobutanote, an intermediate in valine synthesis (74, 75). First, pantoate is derived from 3-methyl-2- oxobutanote in a two-step reaction by action of 3-methyl-2-oxobutanoate hydroxymethyltransferase (E.C 2.1.2.11), and 2-dehydropantoate 2-reductase (E.C 1.1.1.169) (71). Second, �-alanine is formed by decarboxylation of aspartate by aspartate 1-decarboxylase (E.C 4.1.1.11) (71, 74). Finally, the pantoate and �-alanine undergo condensation by action of pantothenate kinase (EC 2.7.1.33) for pantothenate synthesis 92 (74). Apart from pantothenate kinase (EC 2.7.1.33), all the enzymes involved in pantothenate synthesis appear to be absent in C. scindens ATCC 35704 (Fig. 35) which is a supportive indicator for my findings. Current Genome Clostrrdrum scmdens ATCC35704 Genes in Clostnd•um scindens ATCC 35704 D Enz}mes w1:h hits • KO 0 sstng Enz�mes r.,• 2-Dehydropento&'9 ___. 1----0 O (R)-3,3-DimethyimaA1e --,..---1oo•63•2•.1�'--1...0( R)·Pa ntothenate 3.S. 1 .22 Fig. 35. KEGG map for biosynthesis of pantothenate in C. scindens ATCC 35704 (71). 93 Vitamin 86 refers to a group of compounds: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5-phosphorylated forms pyridoxine 5-phosphate (PNP), pyridoxamine 5- phosphate (PMP), and pyridoxal 5-phosphate {PLP) (76). PLP is an active form of vitamin 86 and an vital cofactor in amino acid and carbohydrate metabolism (59, 77). The biosynthesis of pyridoxal is shown in Figure 28. The pyridoxal biosynthesis occurs using various substrates by different mechanisms (59). The first pathway involves erythrose 4-phosphate which is converted to 3-amino-2-oxopropyl phosphate by sequential enzymatic reactions. The latter undergoes conversion by pyridoxine 5- phosphate synthase (E.C 2.6.99.2) to form pyridoxine phosphate which then undergoes conversion by pyridoxal 5-phosphate synthase (E.C 1.4.3.S) to form pyridoxal-5- phosphate (71). The second pathway involves glyceraldehyde 3-phosphate and ribulose 5-phosphate which are converted to pyridoxal 5-phosphate by pyridoxal 5-phosphate synthase (E.C 4.3.3.6) (71). Finally, the pyridoxal 5-phosphate formed from both pathways is converted to pyridoxal by pyridoxal phosphatase (E.C 3.1.3.74) (71). In C. scindens ATCC 35704, all the enzymes mentioned above (Fig. 36) appeared to be missing which provided evidence for the requirement of pyridoxal for growth by this organism. Surprisingly, the genomic information on amino acid biosynthesis showed that C. scindens ATCC 35704 could synthesize only one amino acid (histidine) and also showed missing genes for additional two vitamins, biotin and thiamine. The reasons behind these contradicting results are still unknown but may be related to genome annotation. In addition, it should be noted that further studies to clarifythe vitamin requirements may want to consider performing tests in a culture medium with tryptophan as the sole amino 94 acids instead of DM where all 20 amino acids are present. This might help shed some light on the roles, if any, that amino acids play relative to the synthesis of vitamins. CllTenl Genome CIOstr.d�m scndens3570� ATCC 0 Genes Ctostn Ol�rilieh .)de � 1 3- 5-� I = ' 8 �wav ( Penklse � ) Fig. 36. KEGG map for biosynthesis of pyridoxal in C. scindens ATCC 35704 (71). 95 Early studies on the 7a-dehydroxylation activity on C. scindens were performed in nutritious media like BHI, tryptic soy broth and cooked meat medium (27, 29, 78). An important part of this study was the discovery of 7a-dehydroxylation activity by C. scindens ATCC 35704 under defined as well as minimal culture conditions (Fig. 19). In microbiology, defined medium typically contains growth supportive nutrients in known chemical compositions and in known chemical forms whereas a minimal medium is a defined medium with only specific nutrients essential to the growth of the organism that is present. In a study conducted by Paul, the 7a-dehydroxylation activity by C. scindens ATCC 12708 was observed when the organism was grown in OM (57). However, this activity has not been tested in a minimal medium. In this study, the conversion of cholate to deoxycholate in minimal medium indicated that C. scindens ATCC 35704 expressed genes responsible for 7a-dehydroxylation even in a nutritionally challenged environment. Surprisingly, besides deoxycholate, other bile acid-derived metabolites were observed in defined and minimal culture conditions (Fig. 19). Similar metabolites were also observed when C. scindens ATCC 35704 was grown in BHI medium (Fig. 20). Currently, it is not known if these metabolites were formed by conversion of cholate or deoxycholate. However, previous studies have shown that several bile acid intermediates are formed during the multi-step 7a-dehydroxylation process and include: 3-oxo-64- cholic acid, 7-oxo-cholic acid, 3-oxo-cholic acid, allo-deoxycholic acid, 3-oxo-L'l4- deoxycholic acid, 3-oxo-deoxycholic acid, and allo-3-oxo-deoxycholic acid (27, 78). In this study, defined medium was used for end product detection to avoid components that might give erroneous results (79). The anaerobic fermentation of 96 carbohydrates generates a variety of end products (e.g., alcohols, organic acids, and gases such as hydrogen and carbon dioxide) (80). A typical glucose fermentation pathway for Clostridium spp. is shown in Figure 6. The outcomes from glucose fermentation in defined and minimal conditions represent that C. scindens ATCC 35704 is a saccharolytic clostridia with ethanol and acetate as a major end-products. A previous study on C. scindens shows that peptone-yeast extra� glucose fermentation produced ethanol, acetic acid, and H2 as an end products (53). Another study shows that C. scindens ATCC 35704 fermented glucose to produce acid (81). Since ethanol and acetate are commonly derived from pyruvate during sugar fermentation , the present findings suggest that C. scindens ATCC 35704 ferments glucose via the Embden-Meyerhof-Parnas pathway (82). However, the genomic information on KEGG shows different conclusions as there are several missing genes associated with this pathway (Fig. 37). Thus, it appears there is often conflicting information between results from lab work and KEGG outcomes. A detailed understanding on the whole genome of C. scindens ATCC 35704 is essential in order to fill in the gap between invitro tests and present genomic data. Theoretically, the end products of 1 mol of glucose fermentation are 1 mol of acetate, 1 mol of ethanol, 1 mol of C02, 1 mol of H1, and 1 mol of formate (Fig. 30). The comparison of substrate end product profilein different culture mediua was calculated from the ratio of product formed (mM) per glucose (mM) consumed. In OM with P04 buffer, 1 glucose fermentation produced 0.55 acetate, 1.27 ethanol, 0.87 H2, and 0.32 formate. In OM with tryptophan as sole amino acid with P04 buffer, 1 glucose fermentation produced 0.66 acetate, 1.81 ethanol, 0.97 H2, and 0.14 formate. In MM with P04 buffer, 1 glucose 97 fermentation produced 0.39 acetate, 2.02 ethanol, and 0.37 Hi. The major end products (acetate, Hi, and formate) formed in defined as well as minimal medium were lower than the excepted theoretical values whereas ethanol formation was higher in all cases. The higher amount of ethanol as an end product suggested that the metabolic reactions in C. scindens ATCC 35704 were more towards ethanol production when the nutrients in the culture medium were limited. Also, the amount of formate production by C. scindens ATCC 35704 decreased from OM with P04 buffer to MM with P04 buffer. This suggested that the organism could switch from acid to alcohol production when grown in nutritionally challenged environment. A non-bile acid-dehydroxylating Clostridium sp., Clostridium pasteurianum, which is a classic acid producer showed similar switching mechanism from acid to solvent production (butanol) when grown in minimal medium (83}. In contrast, C. bifermentans, a bile acid-dehydroxylating bacteria, showed increased amount of ethanol and acetic acid production and decreased amount of propanol, and propionic, iso-butyric and iso-valeric acids with increased glucose concentration (84). Reasons for the slight shift towards ethanol synthesis are presently unclear because ethanol formation consumes reducing equivalents, which one would assume are needed for the may anabolic pathways that are active during growth under minimal nutritional conditions, and results in no ATP formation whereas acetate formation consumes no reducing equivalents and results in ATP generation (Fig. 30}. Overall, it appeared that the end products formed were lower than expected and this could probably be because all the pyruvate generated in this process was not converted to end products (i.e., used for anabolic purposes) or was converted to other end products which were not detected by 98 HPLC analysis. Clearly, more work is needed on understanding sugar fermentation and its regulation in this important gut anaerobe. Ci.rentGenome C!Qslnd SCfldWS AT CC 35704 .im a Genes Clostncumscmdens ATCC 35704 m I Enz;mes \'1llhKO hrls ( ��c.$1EIOM)4 a f.'ss 1ng Enz1mes I --- - ....- llf------ov ,,.- - - .... 0-01-.1P (�) ThPP l-lactate Fig. 37. KEGG map for glucose fermentation with typical end products by C. scindens ATCC 35704 (71). 99 In human, the roles of bile acids are not limited to food digestion as bile acids has been found to function as nutrient-signaling hormones which regulates glucose, lipids, and other metabolic as well as immune functions (10). The bile acid-dehydroxylation process carried out by a group of gut microbes generates secondary bile acids, specifically deoxycholate, which has several important functions such as acting as a signal molecule in the activation of the epidermal growth factor receptor (EGFR), contributing to human diseases (e.g., gallstones, colon cancer), and inhibiting the growth of the intestinal pathogen Clostridium difficile (10, 85). Since C. scindens shows relatively high activity levels for bile acid dehydroxylation, it is considered a model organism for understanding this metabolic process. Further research on this model organism is necessary to understand the association of bile acid dehydroxylation with other metabolic processes. The bile acid dehydroxylation process is a series of oxidation and reduction reactions which requires coenzymes and cofactors. Since tryptophan is a precursor for cofactor NAO, pantothenate is a core precursor for cofactor coenzyme A (CoA), and pyridoxal phosphate is a vital cofactor in amino acid and carbohydrate metabolism, it is likely that there is a intimate linkage between the nutritional requirements and bile acid dehydroxylation process in C. scindens ATCC 35704. Furthermore, the conversion of chelate to deoxycholate observed in C. scindens cultivated in minimal medium suggests that bile acid conversion is a crucial mechanism of this organism even when it is nutritionally challenged. 100 Glucose Cholic Acid 7-alpha Fennentation Dehydroxylation Pathway I 1 motGlucose I • EMP Pathway AOPXNAO" - ATP NA0H J• - · . ? f ___.. - .. - .. _ _ �----� --- Fig. 38. Hypothetical pathway for coupling glucose fermentation to bile acid dehydroxylation by C. scindens. VI. CONCLUSION The current study provides evidence that C. scindens ATCC 35704 has the ability to grow in a minimal medium suggesting that many biosynthetic pathways were indeed functional in this organism. The findings on bile acid dehydroxylation process by C. scindens ATCC 35704 verified that these reactions occur even cells are in very anabolic stressed conditions. 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