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1970 Tryptophan Catabolism During Sporulation in Bacillus Cereus. Chandan Prasad Louisiana State University and Agricultural & Mechanical College
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PRASAD, Chandan, 1942- TRYPTOPHAN CATABOLISM DURING SPORULATION IN BACILLUS CEREUS.
The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1970 Microbiology
University Microfilms, Inc., Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED Tryptophan Catabolism During Sporulation in Bacillus cereus
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Microbiology
by Chandan Prasad B. Sc. (Hons.) U. P. Agricultural University, India, 1964 M. Sc. U. P. Agricultural University, India, 1966 May, 1970 ACKNOWLEDGME NTS
The author wishes to express his appreciation to the staff and faculty
members of the Department of Microbiology, who in their own ways contri buted to the progress of this work.
It is with great pleasure that acknowledgment is made to Dr. V. R.
Srinivasan for his guidance, patience, and understanding during the develop
ment of this work. He has been a continual source of creative ideas as well
as being a mentor even for things beyond the realm of science. The endless
succession of arguments and discussions between the author and Dr.
Srinivasan continually provided a stimulating atmosphere for research.
The author is indebted to the U. S. Public Health Service and the
American Cancer Society for their financial support of his entire graduate education.
Deep appreciation is extended to M rs. M. W. Bumm and M rs. J. K.
Saito for their excellent technical assistance. The author also thanks Mr.
C. K. Banerjee for his help in putting this dissertation into its final form.
Thanks are extended to Mrs. Lucy Tynes for typing and helping in proof-reading this dissertation. TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... ii
LIST OF T A B L E S ...... vi
LIST OF FIGURES ...... viii
LIST OF ABBREVIATIONS ...... x
ABSTRACT...... xii
INTRODUCTION ...... 1
REVIEW OF LITERATURE...... 3
A. Biochemical Pathways of n --yptophan C atabolism...... 3 1. Aromatic Pathway ...... 4 2. Quinoline Pathway...... 8 3. Quinazoline Pathway ...... 9 4. Tryptophanase Pathway...... '. 11
B. The Functions of Tryptophan Catabolism...... 17 1. Biosynthesis of Niacin and NAD+ ...... 17 2. Utilization of Tryptophan for the Formation of Auxin and Related Compounds...... 21 3. Other Products of Tryptophan M etabolism ...... 24
C. Involvement of Tryptophan Catabolism in Senescense (Aging), Differentiation and Certain Pathological Conditions 27
MATERIALS AND METHODS...... 29
A. Organisms...... 29
B. M e d ia...... 29 1. "G"- M e d iu m ...... 29 2. "An-M edium ...... 30 3. CDGS-Medium...... 30 4. GGMS-Medium...... 31 5. Nutrient Agar...... 31 Page
C. Culture T ech n iq u es...... 32 1. Transfer and Storage of C u ltu r e s...... 32 2. Active Culture Technique...... 32
D. Viable and Spore C ounts...... 32
E. Turbidimetric and pH M ea su rem en ts...... 32
F. Chromatographic Methods...... 33 1. Paper Chromatography...... 33 2. Ion-exchange Chromatography...... 33
G. Ultraviolet and Infrared Spectra...... 34
H. Preparation of Cell-free Extract...... 34
I. Enzyme Assay ...... 34 1. Tryptophan Pyrrolase...... 35 2. Formylase ...... 35 3. Kynureninase ...... 35
J. Enzyme Unit...... 35
K. Incorporation of ^H-Tryptophan into Dipicolinie Acid . . . 36
L. Isolation and Purification of Dipicolinie A c id ...... 36
M. Inhibitors and Reversing A gents...... 37
N. Chemical Analyses...... 38
O. C h em ica ls...... 38
P. Synthesis of N-Formyl-DL-Kynurenine...... 38
RESULTS ...... 41
A. Intermediates of Tryptophan Catabolism in a Sporulating Culture of Bacillus c e r e u s...... 41 1. Isolation and P u rification...... 41 2. Characterization...... 43
iv Page
3. Formation of Kynurenine and Anthranilic Acid by Whole C ells...... 43
B. Enzymes of Tryptophan Catabolism...... 49 1. Enzyme Activities in Cell-free Extracts of B. cereu s ...... 49 2. Effect of Nutritional Conditions on the Development of Tryptophan Catabolic Enzymes During Sporulation...... 51
C. Functional Aspects of Tryptophan C atabolites...... 56 1. Induction of Heat Sensitivity by Nicotinamide and Ethyloxamate...... 61 2. Reversion of Nicotinamide and Ethyloxamate Induced Heat Sensitivity by the Addition of ' Tryptophan Catabolites...... 61 3. Effect of Time of Addition of Different Catabolites on the Development of Heat Resistance...... 63
DISCUSSION ...... 70
LITERATURE C IT E D ...... 77
VITA ...... 92
v LIST OF TABLES
Table Page
1. Micro or gani sm s Po s ses sing Quinaz oline Pathway of Tiyptophan Catabolism...... 12
2. Occurrence of Tryptophanase in a Variety of M icroorganisms...... 16
3. Microorganisms Producing Indoleacetic Acid from Tryptophan...... 23
4. Chromatography and Color Reactions of Some Catabolites of Tryptophan...... 44
5. Tryptophan Pyrrolase, Formylase and Kynureninase During Sporulation...... 52
6 . The Effect of Cultural Conditions on the Pattern of Development of Tryptophan P y r r o la se...... 53
7. The Effect of Cultural Conditions on the Pattern of Development of K ynureninase...... 54
8 . Effect of Media on the Development of Tryptophan Pyrrolase Activity in a Bacillus cereus TR-2 C u ltu re...... 55
9. The Effect of L-Tryptophan on the Development of Tryptophan Pyrrolase Activity in Bacillus cereus 49 Culture Grown in GGMS M edium ...... 57
10. The Effect of Time of Addition of ^H-Tryptophan on its Incorporation into Dipicolinie A c id ...... 59
11. Efficiency of Various Compounds as Precursors of Dipicolinie A c id ...... 60
12. Effect of Nicotinamide and Ethyloxamate on Sporulation of Bacillus cereus T R -2 ...... 62
13. Effect of Some Tryptophan Catabolites on the Inhibition of Development of Heat Resistance by Nicotinamide ...... 64
vi Table Page
14. Effect of Time of Addition of Quinaldic Acid in an Ethyloxamate-treated Culture on the Development of Heat R esistance...... 65
15. Effect of Time of Addition of 3-Hydroxyanthranilic Acid in an Ethyloxamate-treated Culture on the Development of Heat Resistance ...... 6 6
16. Effect of Time of Addition of DL-Kynurenine in a Nicotinamide-treated Culture on the Development of Heat Resistance ...... 6 8
17. Effect of Time of Addition of Xanthurenic Acid in a Nicotinamide-treated Culture on the Development of Heat R esistance...... 69
vii LIST OF FIGURES
Figure Page
1. "Aromatic", "Quinoline" and "Quinazoline" Pathways for Tryptophan D issim ilation...... 5
2. Alternate Pathways of Anthranilate Metabolism in Microorganisms...... 6
3. Quinoline Pathway of Tryptophan Metabolism in Pseudomonas fluorescens and Pseudomonas acidovorans.... 10
4. Some Common Quinazoline Compounds Produced by M icroorganism s...... 13
5. Quinazoline Pathway of Tryptophan Metabolism in Pseudomonas aeruginosa...... 14
6. Biosynthesis of Niacin and NAD+ from Tryptophan ...... 19
7. Microbial Synthesis and Degradation of Indoleacetie Acid and Related Compounds...... 22
8 . Proposed Metabolic Pathways for the Conversion of L-Tryptophan to Indolepropionate by Clostridium sporogenes . . 25
9. An Ultraviolet Spectrum of N-Formyl-DL-Kynurenine A - commercial sample; B - synthesized from DL-Kynurenine . . 40
10. Procedure for the Isolation of Intermediates of Tryptophan Catabolism from a Sporulating Culture of Bacillus cereus.... 42
11. Ultraviolet Spectrum of Authentic Kynurenine and Compound K ...... 45
12. Infrared Spectrum of Authentic Kynurenine and Compound K ...... 46
13. Ultraviolet Spectrum of Authentic Anthranilic Acid and Compound A ...... 47
14. Infrared Spectrum of Compound A ...... 48 Figure Page
15. Biotransformation of Tryptophan and Kynurenine by Bacillus cereus C e lls ...... 50
16. Time Course of Sporulation and Development of Heat Resistance and its Inhibition by Nicotinamide and Ethyloxam ate...... 74
ix LIST OF ABBREVIATIONS
ATP adenosine triphosphate
C centigrade cpm counts per minute
DPA dipicolinie acid (pyridine-2, 6 -dicarboxylic acid)
DPN+ diphosphopyridine nucleotide
DPNH reduced diphosphopyridine nucleotide
EO ethyloxamate g gravity, centrifugal
I. R. infrared
1 liter ml milliliter min minute
M molar mM m illim olar mg milligram p. micro mju millimicro jig microgram
NA nicotinamide
i . NAD nicotinamide adenine dinucleotide
NADH reduced nicotinamide adenine dinucleotide
NADPH nicotinamide adenine dinucleotide phosphate, reduced
x nm nanometer
0. D. optical density
POP 2 , 5-diphenyloxazole
P0P0P 1 ,4-bis-2-(4-methyl-5“phenyloxazolyl) benzene
% percent
PRPP 5-phosphor ibosyl- 1 -pyrophosphate
U.V. ultraviolet w/v weight by volume v/v volume by volume ABSTRACT
Two inter mediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at pre-spore stage. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at pre-spore stage. Kynureninase activity reached a maxi mum during early stages of sporulation and then started declining. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of L-tryptophan or DL-kynurenine. The cultures ex hibited the maximal activity of kynureninase two hours earlier in the presence of DL-kynurenine whereas L-tryptophan delayed the appearance of the maximal activity by two hours. The presence of glucose in G-medium had no effect on the pattern of development of tryptophan pyrrolase during grpwth and sporulation.
On the addition of tryptophan in a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was noticed.
Ethyloxamate and nicotinamide inhibited the development of heat resistance and the biosynthesis of pyridine-2 , 6 -dicarboxylic acid (dipicolinie acid) in
Bacillus cereus spores. Addition of quinaldic acid or hydroxyanthranilic acid to an ethyloxamate-grown culture resulted in an increase in the number of heat resistant spores. Nicotinamide-induced heat sensitivity could be reversed to different degrees by the addition of kynurenine or xanthurenic acid.
xii INTRODUCTION
Differentiation can be defined as thfe process involving the orderly emergence of a series of functions and morphological changes in organisms.
In Bacillus, the differentiation of a vegetative cell into a dormant spore is a complex process during which a cell undergoes marked metabolic and mor phological changes. The regulation of the sequence of structural and bio chemical changes which lead to spore formation is not clearly understood. In an attempt to correlate the biochemical and structural events, several investi gations have been reported on the pattern of development of enzymes of nucleic acid, carbohydrate, acetate, and phosphorus metabolism during growth and sporulation in bacilli {Hanson et al., 1963; Falaschi et al., 1966; Gardner et al.,
1967; Tono et al., 1967; Deutscher et al., 1968; Warren, 1968; Mandelstam,
1969). The development of enzymes follows markedly different patterns during differentiation. Some enzymes appear at specific times during differentiation while others which are previously synthesized may disappear. Particular use has been made of immunological methods to demonstrate progressive changes in the composition of soluble protein during differentiation in chicken liver
(Murison, 1969), amphibia (Cooper, 1950), insects (Teller et al., 1953), echinoderms (Perlman, 1953), plants (Wright, 1963), and bacteria (Leitzmann et al., 1968; Norris, 1969), In each case it has been shown that new species of proteins make their appearance at different stages of development; in some instances proteins which appear in early stages of development disappear later.
This problem has been recently reviewed by Kornberg et al. (1968). Several
1 studies have shown the increased synthesis of intermediates and enzymes of tryptophan catabolism during differentiation in mammals, insects and amphibians (Brown et al., 1956; Baglioni, 1959; Amer et al,, 1968; Paik et al., 1968; Haining et al., 1969). However, so far there has been no report on the involvement of tryptophan catabolism during differentiation of a bacterial cell into a spore.
The purpose of the work reported in this dissertation was to study the presence and function of tryptophan catabolism during intracellular differen tiation in Bacillus cereus. Fluorescent catabolites of tryptophan were isolated from a sporulating culture of B. cereus and identified as kynurenine and anthra nilic acid. The enzyme systems responsible for the conversion of tryptophan to anthranilic acid via kynurenine were shown to be present. Addition of nicotinamide or ethyloxamate in a B. cereus culture resulted in the production of heat sensitive spores. Several intermediates of tryptophan catabolism could elevate the heat resistance of nicotinamide or ethyloxamate grown spores. REVIEW OF LITERATURE
A. Biochemical Pathways of Tryptophan Catabolism
Tryptophan constitutes a significant part of microbial, plant and animal proteins as well as intracellular pool of free amino acids. Most of the studies on tryptophan metabolism have been concerned with its biosynthesis. Catabolic studies on tryptophan have been conducted mostly in the mammalian systems.
However, there are a few reports on the investigations of tryptophan catabolism in microorganisms. Tryptophan and its several catabolites have been shown to participate in a number of biosynthetic reactions (Bonner and Yanofsky,
1951; Udenfriend et al., 1956; Joshi et al., 1960; Kowanko et al. , 1962). The initial oxidative dissimilation of tryptophan in Pseudomonas is brought about by the enzyme tryptophan pyrrolase which converts tryptophan to formyl kynur enine. It has been shown recently that formyl kynurenine, which is usually broken down to kynurenine, can also enter into quinazoline pathway (Mann,
1967). From kynurenine, the oxidation of tryptophan proceeds by one of two pathways, the so-called "aromatic" and "quinoline" pathways. In the aromatic pathway, a series of enzymes convert L-kynurenine via anthranilic acid, into succinate and acetate or fumarate and pyruvate (Hayaishi and Stanier, 1951;
Subba Rao et al., 1967; Cain, 1968). In the quinoline pathway, kynurenine is ultimately converted to glutamate, alanine, acetate and CO2 via kynurenic acid (Hayaishi et al., 1961; Behrman, 1962). Any Pseudomonas capable of oxidizing tryptophan utilizes one of the three pathways — aromatic, quinoline and quinazoline (Stanier et al., 1951b; Mann, 1967). These pathways are
3 4
summarized in Figure 1.
1. Aromatic Pathway. In the aromatic pathway of degradation of
tryptophan, kynurenine is metabolized via anthranilic acid (Fig. 2). Anthra
nilic acid is an important intermediary metabolite in both biosynthetic and
catabolic pathways in microorganisms (Tatum etal., 1954; Matchett et al.,
1963). Most bacteria degrade anthranilate through catechol (Higashi etal.,
1960; Hosokawa et al., 1961; Cain, 1966; Mann, 1967). Although 3-hydroxy-
anthranilate is degraded by bacteria grown or induced in its presence there
are no reports that this compound can arise from anthranilate in bacteria
(Ichiyama et al., 1964), Terui et al. (1953, 1961) reported that Aspergillus
niger can metabolize salicylic acid as well as anthranilic acid by a route
involving the formation of 2 ,3-dihydroxybenzoic acid. Recently Subba Rao
et al. (1967) have reported a direct conversion of anthranilate to catechol via
3-hydroxy anthranilate and 2, 3-dihydroxybenzoate in Aspergillus niger. The
Achromobacter isolated by Ladd (1962) oxidized 5-hydroxyanthranilate but
not 3-hydroxyanthranilate after growth on anthranilate but the intermediary position of this hydroxy anthranilate remained obscure since it was not detected
in cultures, or incubation media. Recently, 5-hydroxyanthranilate was isolated
and characterized as an intermediate in the metabolism of anthranilate by
Nocardia opaca (Cain, 1968). Both 5-hydroxyanthranilate and gentisate were
oxidized to pyruvate by extracts of anthranilate-grown cells. Evidence pre
sented by Cain (1968) suggests that gentisate pathway of anthranilate metabo
lism inN. opaca is of secondary importance. Hydroxylation of anthranilate Figure 1. "Aromatic", "Quinoline" and "Quinazoline" Pathways for Tryptophan Dissimilation. Tryptophan i N- Formylkynurenine
1 Kynurenine
Anthranilic Kynurenic N- Formylamino- Acid Acid acetophenone
d o 0 >f H a o »rt N 1 + cd a S3 o • dH < I
Acetate + Succinate Glutamate, 4-Methylquinazoline , or co2 4-Methyl 2-alky 1- Pyruvate + Fumarate Alanine, Acetate quinazoline Figure 2. Alternate Pathways of Anthranilate Metabolism in Microorganisms. Kynurenine
3-Hydroxykynurenine Anthranilic Acid
3-Hydroxyanthranilate 5 - Hy droxyanthr anilate
2,3-Dihydroxy benzoate Gentisate
sV Catechol Maleylpyruvate
cis, cis-Muconate vumaryl pyruvate
V -Carboxymethyl- Pyruvate + Fumarate -butenolide
p -Ketoadipate
si/ Succinate +■ Acetate by "anthranilate hydroxylase" in bacteria (Ichihara etal., 1962; Taniuchi et al., 1964), is a reaction requiring NADH but in which the two atoms of molecular oxygen are incorporated into catechol. The enzymes that catalyze the conversion of catechol to cis, cis-muconic acid (pyrocatechase) have been purified from anthranilic acid-adapted cells of Pseudomonas and from
Brevibacterium fuscum (Nakagawa et al., 1963). These catalyze the cleavage of catechol with the consumption of 1 mole of oxygen per mole of catechol cleaved. The enzyme has been purified (Molecular weight about 80, 000) and found to require ferrous ion. Studies with 0 ^ 8 have shown that the oxygen atoms added to catechol are derived from oxygen (Hayaishi etal., 1957).
The conversion of cis, cis-muconic acid to p -ketoadipic acid involves the participation of two enzymes (Sistrom et al., 1954). These catalyze, respect ively, conversion of cis, cis-rmuconic acid to -carboxymethylbutenolide, and conversion of the latter compound to p -ketoadipic acid which in turn is degraded into succinate and acetate.
The oxidation of anthranilate to pyruvate and fumarate via 5-hydroxy anthranilate, gentisate and maleylpyruvate has been demonstrated in Nocardia opaca (Cain, 1968) and Achromobacter (Ladd, 1962). The conversion of 5- hydroxyanthranilate to gentisate requires 1 mole of oxygen and 2 moles of a reducing agent per mole of hydroxy anthranilate oxidized (Equation 1).
C7 H7 O3 N + O2 + 2 AH2 C7 HQO4 + NH3 + H2 O + 2 A 5-hydroxy gentisate
anthranilate (1 )
The chemical nature of the H-donor required in this reaction is not known.
The gentisate oxygenase in crude dialyzed extracts of N. opaca has a Km of 80 micromolar for gentisate (pH 7. 0 in phosphate buffer) and is completely inhibited by 2 ,2'-dipyridyI (2mM) when preincubated with the inhibitor for
10 min before the addition of the substrate. This inhibition could be reversed by the addition of ferrous sulfate. The enzyme does not oxidize catechol, quinol, 2,3-, 3,4- or 3 ,5-dihydroxybenzoate, but the first four compounds are strong competitive inhibitors of gentisate oxygenase (Cain, 1968).
Maleylpyruvate, a product of gentisate oxygenase reaction, is not significantly attacked by dialyzed extracts unless the reaction mixture is supplemented with reduced glutathione. Cysteine, but not thioglycollate can substitute for glutathione.
2. Quinoline Pathway. The conversion of kynurenine to kynurenic acid takes place by transamination. This reaction involves the loss of an amino group and ring closure between the alpha-carbon atom and the nitrogen atom attached to the benzene ring. It is generally assumed that two steps are in volved, o-amino-benzoylpyruvic acid being formed as an intermediate.
Kynurenine transaminase has been separated from kynureninase, which also requires pyridoxal phosphate. Kynurenine transaminase preparations have been obtained from animal tissues (Mason et al., 1951; Mason, 1957; Ueno
et al., 1963), Neurospora (Jakoby et al., 1956), and Pseudomonas (Miller et al., 1953; Stanier et al., 1951a). The formation of kynurenic acid is not reversible due to the formation of a stable aromatic ring. Miller et al.
(1953) observed that in the absence of o<-ketoglutarate, extracts from
Pseudomonas slowly attacked kynurenine, with the formation of anthranilic
acid, presumably by the kynureninase reaction. This observation corrects 9 the earlier report by Stanier et al. (1951) that the quinolinate and aromatic pathways for the degradation of kynurenine are mutually exclusive.
Hayaishi and his co-workers have investigated the degradation of C1^- kynurenic acid labeled at various carbon atoms by tryptophan-adapted cells of Pseudomonas fluorescens (Hayaishi et al., 1961; Horibata et al., 1961;
Taniuchi et al., 1963). L-glutamic acid, D- and L-alanine, and acetic acid were among the main products of kynurenic acid breakdown. It was found that the carbon chain of glutamic acid came from the benzene moiety of ky nurenic acid. Carbon atoms 6 and 9 of kynurenic acid were the precursors of carbon atoms 4 and 1 of glutamic acid, respectively. Carbon atoms 2 and 3 of kynurenic acid were distributed among carbon atoms 2,3,4 and 5 of glutamic acid. Carbon atom 2 of kynurenic acid was found in carbon atom
2 and 3 of alanine and carbon atoms 1 and 2 of acetic acid. The carboxyl groups of alanine and the alpha-carboxyl groups of glutamic acid contained little radioactivity, and the carboxyl carbon of kynurenic acid converted mainly to COg. An enzyme preparation was obtained from Pseudomonas which catalyzed the conversion of kynurenic acid to a compound that exhibited the properties of 7, 8 -dihydrokynurenic acid -7, 8 -diol in the presence of
DPNH and oxygen. This compound was dehydrogenated in the presence of
DPN+ to 7, 8 -dihydroxykynurenic acid which was isolated and definitely identi fied. Similar findings were reported by Behrman and Tanaka (1959). The pathway for the catabolism of kynurenic acid is presented in Figure 3.
3. Quinazoline Pathway. The first report of this pathway appeared in
1967 in Pseudomonas and certain other microorganisms (Mann, 1967). No Figure 3. Quinoline Pathway of Tryptophan Metabolism in Pseudomonas fluorescence and Pseudomonas acidovorans. Tryptophan
Kynurenine
Kynurenic Acid
7, 8 -Dihydrokynurenic Acid-7, 8 -diol
7, 8 - Dihydroxykynurenic Acid i 'i I I i Glutamate Alanine Acetate attempt has been made to show if this pathway is present in plants and animals. A list of the microorganisms tested for the presence of this path way is presented in Table 1. In quinazoline pathway N-formylkynurenine is converted to quinazoline derivatives (Fig. 4) via formylaminoacetophenone and o-aminoacetophenone. The pathway is presented in Figure 5. The o- aminoacetophenone and 4-methylquinazolines were found to be more constant characteristics of Pseudomonas aeruginosa than their pigments. Strains forming a melanin-like pigment were marked by a clear retardation of this way of metabolism. In Pseudomonas fluorescens, Pseudomonas putida,
Aeromonas and Candida, the quinazoline pathway of tryptophan catabolism is only of minor importance.
4. Tryptophanase Pathway. Degradation of tryptophan in certain bacteria takes place by the tryptophanase reaction, in which tryptophan is converted to indole, pyruvic acid and ammonia:
Tryptophan------» Indole + Pyruvate + Ammonia
Tryptophanase was first demonstrated by Hopkins and Cole (1903). Wood et al. (1947) prepared the enzyme from E. coli and demonstrated that pyridoxal phosphate was the coenzyme. The reaction appeared to require potassium and ammonium ions and iron (Dawes et al., 1949; Hoch et al., 1966; Griffiths et al., 1970). Major studies on tryptophanase have utilized E. coli as the enzyme source (Happold, 1950; Morino et al., 1967). In recent years it has been reported that a number of bacterial species possess the capability of synthesizing tryptophanase (Arora et al., 1959; Hoch et al., 1965; Bito,
1967; DeM oss et al., 1969). A list of microorganisms possessing Table 1. Microorganism possessing quinazoline pathway of tryptophan catabolism (Mann, 1968).
A. Major pathway
1. Pseudomonas aeruginosa
2. Sarcina lutea Kurs
3. Bacillus cereus Kurs
B. Minor pathway
1 . Bacillus mesentericus Kurs
2 . Candida albicans Kurs
3. Pseudomonas fluorescens
4. Pseudomonas putida
5. Aeromonas hydrophila
6 . Pseudomonas shigelloides
C. Absent
1. Enterobacter aerogenes
2. Escherichia coli O m
3. Staphylococcus albus
4. Staphylococcus aureus Figure 4. Some Common Quinazoline Compounds Produced by Microorganisms. R_ Quinazoline Compound
-H 4-Methylquinazoline
- c h 3 2 ,4-Dimethylquinazoline
2 - c 2 h 5 - Ethyl-4-Methylquinazoline
- c h 2oh 2-Hy dr oxym ethyl- 4- Methy lquinaz oline N- Formylkynurenine
N-Formylaminoacetophenone
NHc
o-Aminoacetophenone
4-Methylquinazoline
o-Acylaminoacetophenone
NH3
-RH
4-Methyl 2-alkylquinazoline 15 tryptophanase activity is presented in Table 2, The formation of indole has been observed in many bacterial species and is utilized as an identification
characteristic. In at least one bacterial species, B. alvei, indole excretion
may arise by either of two mechanisms (Hoch et al., 1965), i. e ., by degra dation of indole-glycerol phosphate, an intermediate in tryptophan biosynthesis, or by degradation of tryptophan, catalyzed by tryptophanase. Although the reaction catalyzed by tryptophanase is essentially irreversible, Newton and
Snell (1962, 1964) have found that E. coli tryptophanase can catalyze the
synthesis of tryptophan from indole and serine. More recent work (Newton
et al., 1964) has led to the crystallization of E. coli tryptophanase. In ad dition to the reactions mentioned above, the crystalline enzyme also catalyzed the desulfhydration of cysteine, the deamination of serine, the conversion of
S-methylcysteine to pyruvate, methylmercaptan, and ammonia, and the
synthesis of tryptophan from indole and cysteine (or S-methylcysteine). The tryptophanase of 13, alvei has also been shown to catalyze the deamination of
serine to pyruvate and ammonia (Griffiths et al., 197 0), but this reaction is catalyzed only 15% as efficiently as the tryptophan degradation reaction. In contrast to the tryptophanase of E. coli which is inducible at high levels of tryptophan (Happold et al., 1935), tryptophanase of B. alvei is a constitutive enzyme, not influenced by the exogenous level of tryptophan or related com pounds (Hoch et al., 1965). A recent investigation by Griffiths and DeMoss
(1970) has shown that tryptophanase from B. alvei is definitely subject to repression by glucose, serine and to an even greater extent by glycerol. The tryptophanase of E. coli is known to be repressible by glucose (Happold, 1950; 16
Table 2. Occurrence of tryptophanase in a variety of bacterial species.
Repressibility Organism Indue ibility by glucose Reference
Aeromonas liquefaciens + + DeMoss et al., 1969
Aeromonas hydrophila 0 0 Mann, 1967
Bacillus alvei - + DeMoss et al., 1969; Griffiths etal., 1970
Bacteroides sp. + + DeMoss et al., 1969
Corynebacterium acnes + DeMoss et al., 1969
Escherichia coli + + Happold et al., 1935; DeMoss et al., 1969
Micrococcus aerogenes + - DeMoss et al., 1969
Paracolobactrum coliforme + + DeMoss et al., 1969
Pasteurella multocida 0 0 Bito, 1967
Photobacterium harveyi + + DeMoss et al., 1969
Pseudomonas shigelloides 0 0 Mann, 1967
Proteus vulgaris + ' b DeMoss et al., 1869
Sphaerophorus varius + + DeMoss et al., 1969
Vibrio cholerae 0 0 Arora et al., 1959
+ present, - absent, 0 not known Freundlich et al., 1962).
It has been suggested that certain bacteria may degrade indole by the following sequence (Sakamoto et al., 1953):
Tryptophan » Indole ) Isatin 1 Formylanthranilic Acid 1 Anthranilic Acid 1 Salicylic Acid J. i co2
B. The Functions of Tryptophan Catabolism
1. Biosynthesis of Niacin and NAD+. Beadle et al. (1947) first showed that a mutant of Neurospora orassa which required niacin for growth could grow maximally if either tryptophan or kynurenine was supplied in the growth medium. It was later found that 3-hydroxyanthranilic acid was as effective as tryptophan or kynurenine in replacing the niacin requirement in the same mutant (Mitchell et al., 1948). Finally, Bonner (1948) isolated 3-hydroxy anthranilic acid from the culture supernatant of this mutant which was grown in the presence of niacin. The role of quinolinic acid in the biosynthesis of niacin in N. crassa was established by Bonner et al. (1949) who could sub stitute quinolinic acid for niacin in a number of niacin requiring mutants of
N. crassa. 3-Hydroxykynurenine was then implicated as an intermediate in the conversion of tryptophan to niacin. Yanofsky and Bonner (1950) isolated a niacin requiring mutant of N. crassa in which either 3-hydroxykynurenine or
3-hydroxyanthranilic acid could be substituted for niacin. As a result of all 18 these observations, Bonner et al. {1951) concluded that tryptophan is con verted to niacin by the kynurenine, quinolinate pathway (Figure 6 ).
Tryptophan-niacin relationship has also been established in a variety
of other microorganisms. Starr (1946) found that Xanthomonas pruni re-
quired an exogenous source of tryptophan when grown on a minimal medium
containing vitamin-free acid hydrolyzed casein. Subsequently, Davis et al.
(1950) showed that the niacin requirement of X. pruni could be satisfied if
either tryptophan, anthranilic acid, kynurenine, 3-hydroxyanthranilic acid,
3-hydroxykynurenine, indole, or quinolinic acid was added to the growth
medium. Wilson et al. (1963) reported the incorporation of C1^-tryptophan
and C^-3-hydroxyanthranilic acid into niacin inJJC. pruni.
In addition to X, pruni, Lingens et al. (1964) have reported the presence
of tryptophan-niacin relationship in Streptomyces antibioticus, Cyanidium
caldarium, Karlingici rosea, and Saccharomyces cerevisiae. Recently,
Desaty et al. (1967) have demonstrated the incorporation of C1^-tryptophan
in nicotinic acid in Fusarium oxysporum. Ahmod et al. (1966) have also
shown using C-^-labeled tryptophan that S. cerevisiae possesses the ability
to convert tryptophan to niacin only when it is grown under aerobic conditions.
In contrast to the microorganisms discussed above, it has been shown that
E. coli, B. subtilis and Pseudomonas species are unable to convert tryptophan
to niacin (Yanofsky, 1954; Lingens et al., 1966).
A tryptophan-niacin relationship has also been established in mammals.
Krehl et al. (1945) found that either tryptophan or niacin added to a diet
completely counteracts the growth retardation caused by the inclusion of 40% Figure 6 . Biosynthesis of Niacin and NAD+ from Tryptophan. L-Tryptophan > N- For my 1- L-Kynurenine
* * 3-Hydroxykynurenine <------Kynurenine
3- Hydroxyanthranilic Acid I Quinolinic Acid
PRPP
Quinolinic Acid Ribonucleotide
Nicotinic Acid co2 PRPP
Nicotinic Acid Ribonucleotide
ATP
Desamido - NAD+ I ATP
Glutamine 1 20 corn grits in a low protein diet. An increase in the excretion of N-methyl- nicotinamide was demonstrated after the administration of tryptophan to rats and humans by Sarett et al. (1947). Heidelberger et al. (1949a, 1949b) found that tryptophan labeled with in the beta carbon was converted into kynurenine by rats. None of the isotopic carbon was incorporated into niacin; although when tryptophan labeled in carbon 3 of the indole ring was used, the label was incorporated into niacin as the carboxyl carbon.
Subsequently, Mehler (1956) found that 3-hydroxyanthranilic acid was converted to quinolinic acid by cell-free extracts of rat liver. The role of
3-hydroxyanthranilic acid in mammalian niacin biosynthesis was further studied by Hankes et al. (1957). Using carboxyl-C1^-3-hydroxyanthranilic acid, they established that the carboxyl carbon of niacin and the 3-carboxyl group of quinolinic acid (in rats) were derived from the carboxyl group of this precursor. Also, Wiss et al. (1950) found that rats were able to convert
3-hydroxykynurenine to 3-hydroxyanthranilate and alanine by a kynureninase type of reaction.
The relationship of kynurenine to 3-hydroxykynurenine was established by DeCastro et al. (1956) when they isolated an enzyme from rat liver mito chondria which would hydroxylate kynurenine to 3-hydroxykynurenine in the presence of NADPH. Nishizuka and Hayaishi (1963) have found that free niacin is not an intermediate in NAD+ biosynthesis. Quinolinic acid reacts directly with PRPP to yield niacin mononucleotide which is then converted to
NAD+ in the presence of ATP and glutamine (Figure 6 ). The pathway for niacin biosynthesis from tryptophan seems to be identical in molds, some 21 plants, and mammals.
2. Utilization of Tryptophan for the Formation of Auxin and Related
Compounds. The isolation of a plant growth hormone from human urine
(Kogl et al., 1934) was followed by its demonstration in Rhizopus suinus
(Gordon et al., 1949), and identification as indole-acetic acid. As indicated
in Figure 7, decarboxylation of tryptophan yields tryptamine, which in turn
can be oxidized to indoleacetaldehyde. The latter product might arise by
decarboxylation of indolepyruvic acid, which can be formed from tryptophan
by oxidative deamination or transamination. Oxidation of indoleacetaldehyde
leads to the formation of indoleacetic acid. Studies on the formation of indole-
acetic acid from tryptophan by microorganisms revealed the presence of
indolepyruvic acid, indolelactic acid, tryptophol, indoleacetamide and several other intermediates in the culture broth (Kaper et al., 1958; Kosuge et al.,
1966; Perley et al., 1966). Indolepyruvic acid undergoes spontaneous decom position leading to the formation of several compounds whose presence on paper chromatograms makes the interpretation difficult. However, these
studies support the hypothesis that indoleacetic acid arises by two different pathways involving indolepyruvic acid and indoleacetamide (Table 3 and
Figure 7).
Indoleacetamide might be formed from tryptophan by an oxygenase-type reaction similar to that observed in the other amino acids. Hydrolysis of indoleacetonitrile or indoleacetamide would yield indoleacetic acid. The formation of indolepyruvic acid from tryptophan is catalyzed by tryptophan transaminase. The presence of tryptophan transaminase in bacteria has been Figure 7. Microbial Synthesis and Degradation of Indoleacetic Acid and Related Compounds. N- ac ety 1 try ptami ne N- ac etyl tryptophan 1 Tryptamine Tryptophan Indoleacetamide
Indolepyruvic Acid Indoleacetic * Acid
Indoleacetonitrile
Tryptophol 4* Indoleacetaldehyde
Acetyltryptophol Indoleacetic Acid
Indol e- 3- ac ety l- E - L-ly sine
Indolecarboxaldehyde
Anthranilic Acid 23
Table 3. Microorganisms producting indoleacetic acid from tryptophan.
Organism Pathway Reference
1 . Achromobaeter sp. unknown Libbert et al., 1969
2 . Agrobacterium tumefaciens indolepyruvate Kaper et al., 1958
3. Alcaligenes sp. unknown Libbert et al., 1969
4. Bacillus cereus (KVT) indolepyruvate Perley et al., 1966
5. Flavobacterium sp. unknown Libbert et al., 1969
6 . Pseudomonas savastanoi indoleacetamide Kosuge et al., 1966
7. Rhizopus suinus unknown Gordon et al., 1947 24
reported for Escherichia coli (Rudman et al., 1953), Rhizobium leguminosarum
(Hartmann et al., 1967), Agrobacterium tumefaciens (Sukanya et a l., 1964),
and Clostridium sporogenes (O'Neil et al., 1968). The physiological role of this enzyme is not certain. The function of this enzyme can be easily explained
in the organism producing indoleacetic acid from tryptophan. In Clostridium
sporogenes which converts tryptophan to indolepropionate (Figure 8 ), it is possible that this pathway represents a physiologically important means for the recycling of NAD+ during growth.
3. Other Products of Tryptophan Metabolism. An important pathway of tryptophan metabolism in animals is conversion to 5-hydroxytryptamine.
Administration of 5 r hydroxy tryptophan led to increased tissue 5-hydroxytryp tamine in animals (Udenfriend et al., 1957). 5-Hydroxytryptamine is formed by the following reaction:
Tryptophan * 5-Hydroxytryptophan » Serotonin
The evidence clearly indicates that oxidation of the aromatic ring precedes decarboxylation, because tryptamine is not formed under the same conditions nor does it serve as a precursor of 5-hydroxytryptamine. Conversion of L- tryptophan to 5-hydroxy- L-tryptophan has been demonstrated in studies with cell suspension of Chromobacterium violaceum (Mitoma et al., 1955); this organism does not possess a "kynurenine" pathway and can utilize 5-hydroxy- tryptophan for production of its characteristic pigment, violacein (Beer et al.,
1954).
Melatonin, a hormone found in bovine pineal glands which produces lightening of frog skin, has been shown to be N-acetyl-5-methoxytryptamine Figure 8 . Proposed Metabolic Pathway for the Conversion of L-tryptophan to Indolepropionate by Clostridium sporogenes (O'Neil and DeMoss, 1968). Tryptophan
✓ °<-ketoglutarate NADH PLP glutamate NAD+
Indolepyruvate
-NADH
NAD+
Indolelactate
-H20
Indoleacrylate
+2H
Indolepropionate 26
(Lerner et al., 1959). Further study showed that the presence of an enzyme
in pineal glands that catalyzes the o-methylation of N-acety 1-5-hydroxytryp tamine to yield melatonin. The enzyme also catalyze the o-methylation of
several other hydroxy indoles but at a lower rate. The enzyme utilizes S- adeno sy lmethionine.
5-Methoxytryptophol decreases ovarian weight and the incidence of estrus in rats (Mclsaac et al., 1964) and has been isolated from beef pineal
(Mclsaac et al., 1965). The occurrence of 5-methoxytryptophol in pineal tissue is of interest in the light of its known biological action. However, the biosyn thetic pathway has not been completely elucidated. In a recent paper, Otani et al. (1969) have proposed the following sequence of reactions leading to the formation of 5-hydroxytryptophol.
Tryptophan
1 5-Hydroxytryptamine
monoamine oxidase 4* 5 - Hydroxy indole ac etaldehyde
Hydroxy indole -o-methyltransf erase
5- Methoxy tryptophol
Tryptophan is also utilized for the biosynthesis of alkaloids of the
Harman group, and others including yohimbine, cinchonine, strychnine, serpentine, and reserpine (Robinson, 1955; Leete, 1969). 27
C. Involvement of Tryptophan Catabolism in Senescence (Aging), D iffer
entiation and Certain Pathological Conditions
The deteriorative processes which naturally terminate the functional organizations in biological systems are collectively called senescence. In many respects, the development of senescence may be considered a continuum from juvenility to maturity. In contrast to senescence, aging may be attributed to the changes which occur in time without reference to the progress of the built-in decay process in the cell.
The indirect evidences presented by Macnicol (1968) suggest a correlation between tryptophan catabolism and senescence of tobacco (Nicotiana tabacum L.) leaves. He showed that the 6 -hydroxykynurenic acid content was found to increase throughout development and senescence of the leaf finally reaching six times of that present in the young leaf.
The role of intracellular turnover of proteins in the process of differ entiation, senescence, and aging has been the subject of numerous investi gations from both the quantitative and qualitative viewpoints (Oeriu, 1964;
Medvedev, 1967; Kornberg et al., 1969). However, most of the reports con cerning protein synthesis and/or turnover during differentiation dealt with an attempt to study the changes in the over-all pattern of proteins. Very few studies attempting to separate and contrast the anabolic and catabolic com ponents of the dynamic equilibrium of individual proteins in relation to differ entiation have appeared in the literature.
The mammalian enzyme which has been most thoroughly investigated and which has yielded the most useful results is tryptophan pyrrolase. Very 28 little, if any, tryptophan pyrrolase is found in fetal liver, but activity appears soon after birth. Tryptophan pyrrolase is induced in adult rat liver by tryp tophan and adrenocortical hormones by two different mechanisms. Tryptophan injection and administration of adrenocortical hormones do not produce an increase in the activity of fetal liver (Nemeth, 1959). It has been suggested that tryptophan increases tryptophan pyrrolase activity by favoring the forma tion of the enzyme from apoenzyme and hematin (Feigelson et al., 1961;
Greengard et al., 1961; Feigelson et al., 1962) but there is also substantial evidence that the enhanced enzymatic activity is due to de novo synthesis of enzyme. For example, the normal increase during development in tryptophan pyrrolase as well as the adaptive increase in the adult is inhibited by puro- mycin, an inhibitor of protein synthesis (Nemeth et al., 1962).
Abnormal tryptophan metabolism has been shown in various clinical conditions, for instance, avitaminosis B £ and Bq (Axelrod et al., 1945;
Glazer et al., 1951; Charconnet-Harding et al., 1952; Yess et al., 1964), diabetes (Rosen et a l., 1955; McDaniell et al., 1956), haemoblastosis
(Musajo et al., 1956), anaemia (Altman et al., 1953; Hankes et al., 1968), upset of estrogen balance (Sprince et al., 1951; Brown et al., 1961; Rose,
1966a; Rose, 1966b; P rice et al., 1967), and cancer of the bladder (Allen et al., 1957; Wallace, 1959; Price et al., 1962; Bryan et al., 1964; Bryan et al., 1965; Wolf et a l., 1965), and the prostate (Wolf et al., 1968). In most cancerous tissues there was an increased excretion of tryptophan catabolites. MATERIALS AND METHODS
A. Organism
Most of the experiments were carried out with Bacillus cereus TR-2, a phage resistant strain derived from B. cereus (Ristroph, 1967). In some experiments B. cereus 49 {obtained from J. Wolfe, University of Leeds) was used. These organisms were maintained on nutrient agar (Difco) slants.
B. Media
1. ”G" - Medium. A semi-synthetic medium described by Stewart and Halvorson (1953) and modified by Greenberg (1954) was used for the growth and sporulation of B, cereus TR-2. This medium will hereafter be referred to as "Gn-medium.
The following is the final composition of "G"-medium;
Compound Percent (w/v)
Glucose 1 . 0 x 1 0 " 1
Yeast Extract 2 .0 x lO- 1
k 2 h p o 4 5 .0 x 10~ 2
CaCl2 *2H20 8 . 0 x 1 0 " 3
(NH4 )2 S0 4 2 . 0 x 1 0 - 1
MgS04 2 . 0 x 1 0 “ 2
MnS04* H20 5 .0 x 10" 3
ZnS04 ’7H20 5. 0 x 10~ 4
CuS04 * 5H20 5. 0 x 10- 4
FeS04* 7H20 5 .0 x 10- 5
29 30
This medium was prepared by making stock solutions of 10% yeast extract, 10% glucose, 5% K2 HPO4 , and 0. 8% CaCl2 * 2 H2 O. These solutions were then autoclaved (at 121 C temperature and 15 lbs pressure per sq inch for 15 min) and the glucose and yeast extract were stored in the cold. A stock solution of minerals was made by mixing a solution of 0 . 0 1 % FeSO^
7H 2 0 , 0.1% CuS04* 5H 2 0 , 1% MnS04 *H 2 0, 4% MgS04, and 0 .1% ZnS04-
7H20 with an equal volume of 40% (NE^^SO^., After the preparation of stock solutions, the medium to be used for growth was prepared by adding an amount of the mineral solution equivalent to 1 % in distilled water of the final volume of the medium and by the addition of 2 % yeast extract and 1% each of K2 HPO4 , Ca.Cl2' 2 H2 O, and glucose. The final pH of the medium was
7. 0-7. 4.
2. '^"-Medium. This is a synthetic medium developed by Srinivasan and Halvorson (1963) which allows morphogenesis of B. cereus without growth.
"A"-medium consists of all the minerals in G-medium (Stewart and Halvorson,
1953) and the following (%,w/v):
Sodium citrate 0.05
Sodium acetate 0. 05
Sodium glutamate 0.05
Sodium bicarbonate 0.05
These organic acids and sodium bicarbonate were sterilized separately and added aseptically to sterilized G-minerals.
3. CDGS Medium. This medium was developed by Nakata (1964) for the growth and sporulation of B. cereus T in the absence of yeast extract. It 31 consists of all the minerals in the same proportions as in G-medium (Stewart and Halvorson, 1953) and the following:
m g/m l
L-glutamic acid 1.84
L-leucine 0.80
L-valine 0.30
DL-threonine 0.336
DL-methionine 0.140
L- histidine 0.050
Dextrose 2 . 0
Amino acid mixtures were sterilized by passing through a bacterial filter and dextrose was sterilized by autoclaving a concentrated solution.
4. GGMS Medium. This medium was used for the growth and sporulation of B. cereus 49. It consisted of all the minerals of G-medium and the following
(%, w/v):
Dextrose 0.5
Sodium glutamate 0.5
Dextrose and sodium glutamate were sterilized separately and then added aseptically to cold sterile G-minerals.
5. Nutrient Agar.
grams/liter
Beef Extract 3. 0
Peptone 5.0
Agar 15.0 32
C. Culture Techniques
1. Transfer and Storage of Cultures. The culture was maintained on
nutrient agar slants and transferred periodically. All slants were stored at
4 C after incubation at 30 C until at least the majority of cells on the slants
had sporulated.
2. Active Culture Technique. Unless otherwise stated the active culture
method described by Nakata and Halvorson(1960) was routinely used. Es
sentially the method is used to synchronize the culture for growth and
sporulation by two successive transfers. The initial culture was started
with an inoculum of about 1 x 106 heat shocked <80 C for 30 min) spores from
a nutrient agar slant. This culture was left overnight at room temperature
(26-27 C) without shaking. After 12-15 hours of incubation, the culture was
shaken at 30 C on a rotary shaker until a near maximum population of about
108 cells/m l was obtained. This took about 20-40 min. A second flask con
taining G-medium was inoculated from the initial culture with a volume equal
to 10% of the volume of the new medium, and was incubated at 30 C for two hours. This culture was used as the final inoculum for the test flasks.
D. Viable and Spore Counts
Both viable and spore counts were made by plating proper dilutions of
samples on nutrient agar. The spore count represented cells which were able to produce colonies after heating the sample at 80 C for 30 min.
E. Turbidimetric and pH Measurements
Growth was estimated by measuring the optical density of the c’llture 33 at 630 nm using Spectronic-20 (Bausch & Lomb). The pH of the culture was determined on a Corning pH meter model 7, The time at which pH of the culture reaches a minimum is referred to as to (tG = 4 hr). Under our ex perimental conditions t0 was also the time at which exponential growth ceased.
F. Chromatographic Methods
1. Paper Chromatography. The following solvent systems were used for ascending paper chromatography of tryptophan catabolites on Whatman no. 1 paper:
(i) n-butanol-acetie acid-water (4:1:1, by volume)
(ii) 20% KC1 (w/v, in water)
(iii) 80% 1-propanol (v/v, in water).
The different intermediates on paper were detected by their characteristic fluorescence under UV light and Rf value.
Dipicolinic acid (DPA) was separated by ascending paper chromatography on Whatman no. 1 paper in two different solvent systems:
(i) Sec. butanol-formic acid-water (15:3:2, v/v)
(ii) Trimethylamine (25% in CHgOH)-ethanol-water (1:4:4, v/v)
2. Ion-exchange Chromatography. The catabolites of tryptophan were fractionated on a column (1.3 cm diameter x 21 cm length) of Amberlite IR-
120 (H+ form, 200-500 mesh) by the method of Benassi et al. (1967) with the following modifications: The column was run at room temperature (25-26 C) and elution was accomplished with a gradient between 0.1 M pyridine-formate pH 2 . 6 and 0 .4 M pyridine-formate pH 6 .5. Formic acid (95% v/v) was used to adjust the pH of different pyridine solutions. Two hundred fractions (each 34
5 ml volume) were collected at a flow rate of 20 ml per hour.
G. Ultraviolet and Infrared Spectra ___
Ultraviolet spectra were taken on Beckman DB spectrophotometer
(Beckman & Co., California) and infrared spectra were measured on a
Perkin-Elmer model 137 spectrophotometer (Perkin-Elmer Corp., Norwalk,
Conn.) using KBr pellets.
H. Preparation of Cell-free Extract
Washed cells from 1 1 culture were resuspended in 10 ml of 0. 04 M
KH2 PO 4 adjusted to pH 7.0 with 1 M KOH and disintegrated by sonication with a Branson ultrasonic drill. Pulses of 30 seconds were used with 1 min in tervals between the pulses. The temperature of the samples was not allowed to rise above 10 C during sonic treatment. Cells were sonicated until 60-80% cell breakage was obtained. The samples which were used for the assay of tryptophan pyrrolase were broken in the presence of 0.3 mM L-tryptophan.
The sonicate was centrifuged at 35, 000 g. for 30 min in a RC-2 Servall re frigerated centrifuge and the supernatant liquid ^hs dialyzed overnight against
0. 04 M KHgPO^ buffer adjusted to pH 7.0 with 1 M KOH; sufficient L-tryptophan was added to the buffer to bring its concentration to 0. 03 mM. The non- diffusible material was used for the enzyme assay.
I. Enzyme Assay
All the enzyme assays were carried out at 30 C. In all cases, the assay volumes were 2.5 mL 35
1. Tryptophan Pyrrolase. Tryptophan pyrrolase (EC 1.13.1.12.,
L-tryptophan:oxygen oxidoreductase) was assayed at 30 C in a Gilford recording spectrophotometer model 2400 by measuring the rate of increase in optical density at 321 nm. The reaction mixture (2.5 ml) contained L-tryptophan
(0.02 M, pH 7. 0) 0.45 ml, dithiothreitol (0. 04 M) 0.1 ml, K2 HPO4 (0. 5 M, pH 7. 0) 0.5 ml, cell-free extract equivalent to 10 mg protein and water to make 2 .5 ml.
2. Form ylase. Form ylase (EC 3.5.1.9., Aryl-formylaraine amido- hydrolase) was assayed by the rate of kynurenine formation at 30 C in a Gilford recording spectrophotometer model 2400 according to the method of Knox (1955) by the rate of decrease in O.D. at 321 nm. The reaction mixture (2.5 ml) contained N-formyl-DL-kynurenine (0. 01 M, pH 7. 0) 0.2 ml, K2 HPO4 (0.5 M, pH 7.0) 0.5 ml, cell-free extract equivalent to 10 mg protein and water to make 2 .5 ml.
3. Kynureninase. Kynureninase (EC 3.7.1. 3., L-kynurenine hydrolase) was assayed by the disappearance of kynurenine at 30 C in a Gilford recording spectrophotometer model 2400 according to the method of Dalgliesh, Knox and
Neuberger (1951) by the rate of decrease of O.D. at 360 nm. The reaction mixture (2.5 ml) contained DL-kynurenine 0.75 /imole, pyridoxal phosphate
20 ug, Tris-H C l (pH 8.5) 250^imole and water to make 2 .5 ml.
J. Enzyme Unit
One unit of enzyme is defined as the amount which catalyzes the appearance or disappearance of 1 mjimole of product or substrate per hour at
30 C under conditions of assay described in the text. 36 q K. Incorporation of H-Tryptophan Into Dipicolinic Acid
In order to estimate the incorporation of 3 H-tryptophan into DPA, the following procedure was employed. Bacteria in the various phases of growth and sporulation were harvested by centrifugation (2 C) at 17,000 x g for 10 min in a RC-2 Servall centrifuge, then washed by resuspension in 20 ml of ice-cold A-medium and centrifugation as above. Finally, the bacterial pellet was suspended in enough A-medium to give original cell concentration. Ten ml of this cell suspension was transferred to a sterile Erlenrayer flask in duplicate and enough %I-tryptophan was added to give 1 . 0 x 1 0 7 cpm /m l of culture. The culture was incubated on a rotary shaker maintained at 30 C and allowed to sporulate. Spores were harvested by centrifugation {2 C) at
17, 000 x g for 10 min in a RC-2 Servall centrifuge, then washed once with
20 ml of 0. 01 M K2 HPO4 buffer (pH 7. 0) containing 0. 04 M L-tryptophan.
DPA was isolated and purified from the spores as described in the text.
For isotope competition experiments, cells were harvested from a 4- hour old culture and processed as described above. The ability of a compound to compete with the incorporation of 3 H-tryptophan in DPA was tested by adding
100 }ig per ml of the competitor along with the isotope. The culture was al lowed to sporulate. DPA was isolated and purified. The effect of different competitors was compared by measuring the specific activity of DPA in spores.
L. Isolation and Purification of Dipicolinic Acid
Washed spores were resuspended in water autoclaved at 121 C for 15 min and cooled to room temperature (26-27 C). Then 0.1 ml of 1. 0 N acetic acid was added and the suspension was allowed to stand for 1 hour at the same temperature. The supernatant was collected after centrifugation at 17, 000 x g for 10 min in a RC-2 refrigerated Servall centrifuge. The supernatant was lyophilized and the residue was resuspended in a very small amount of water
(usually 0.2 - 0.4 ml). The suspension was applied to a Whatman no. 3 paper and ascending chromatogram in trimethylamine (20% in CH3 OH) - ethanol- water (1:4:4, v/v) was run. The U. V. absorbing band corresponding to standard DPA was cut and stitched on a fresh Whatman no. 3 paper. A second ascending chromatogram using this paper was run in Sec. butanol-formic acid-water (15:3:2). The U. V. absorbing band corresponding to DPA was cut and eluted with hot 0.01 N HC1. The amount of DPA was measured by e s timating the O. D. at 270 nm. The radioactivity was counted on a Beckman scintillation spectrometer by transferring 0 . 1 ml of test solution to a glass counting vial containing 10 ml of scintillation fluid (6 gram POP and 250 mg
POPOP per lite r of dioxane).
M. Inhibitors and Reversing Agents
The effects of inhibitors on growth, sporulation and heat resistance of spores were studied by adding the compound to an active culture immediately after the final transfer was made (referred to as "O" time). The ability of a compound to reverse the effect of an inhibitor was usually tested by the ad dition of inhibitor and the test compound to an active culture at 0 time. To study the effect of time of addition, the inhibitor was added at 0 time and the test compound at the indicated time. 38
N. Chemical Analyses
Kynurenine and anthranilic acid were determined by the method of
Mason and Berg (1952) after deproteinization of the reaction mixture with trichloroacetic acid.
The amount of dipicolinic acid in samples was determined by one of the following procedures: (a) by the colorimetric procedure of Janssen, Lund and Anderson (1958), or (b) by the measurement of absorption at 270 nm after purification of the sample by paper chromatography.
Protein was estimated by the method of Lowry, Rosebrough, Farr and
Randall (1951) using crystalline bovine serum albumin as the standard.
O. Chemicals
Dipicolinic acid, DL-kynurenine, ethyloxamate and kynurenic acid were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. L- tryptophan and 3-OH-anthranilic acid were obtained from Nutritional Bio chemical Corporation, Cleveland, Ohio. Anthranilic acid was bought from
W. H. Curtin and Company, New Orleans, Louisiana. Quinaldic acid and nicotinamide were the products of J. T. Baker Chemical Company,
Phillipsburg, New Jersey. N-formyl-DL-kynurenine was synthesized accord ing to the method of Auerbach and Knox (1957).
P. Synthesis of N-Formyl-DL-Kynurenine
N-formyl-DL-kynurenine was synthesized by the method of Auerbach and Knox (1957) as described below:
To a solution of 520 mg (0. 0025 moles) of DL-kynurenine in 1. 25 ml of 98% formic acid was added 0.75 ml of formic acid-acetic anhydride mixture
(0.0005 mole). Formic acid-acetic anhydride mixture was prepared by mixing
0.5 ml of freshly distilled acetic anhydride and 1. 0 ml of 98-100% formic acid
in an ice bath and allowed to stand at room temperature {26-27 C) for 30 min before use. The reaction mixture containing DL-kynurenine and formic acid- acetic anhydride was thoroughly mixed and allowed to stand at room tempera ture for 2 hours. Fifty volumes of anhydrous ether was added to the above mixture. The mixture was stirred and allowed to stand at 0 C for 2 hours.
The amorphous product was collected on a sintered glass filter, washed with ether and dried immediately to avoid hydrolysis. Yield: 580 mg (97%), mp
162 C (d). Ultraviolet spectra at pH 7. 0 has maxima at 260 nm and 321 nm with molecular extinction coefficient of 10980 and 3750 respectively (Fig. 9).
The compound gave a single spot on paper chromatogram run in acid, basic and neutral solvent system s. Figure 9. An Ultraviolet Spectrum of N-Formyl-DL-Kynurenine. A - commercial sample. B - synthesized from DL-Kynurenine. OPTICAL DENSITY 0 360 400 AE EGH N m/x IN LENGTH WAVE 320 280 240 RESULTS
A. Intermediates of Tryptophan Catabolism in a Sporulating Culture of
Bacillus cereus
1. Isolation and Purification. Figure 10 presents the essential steps in the extraction and purification of tryptophan catabolites. G-medium in a
40 1 carboy was inoculated with 2 1 of actively growing culture of B. cereus and was maintained at 30 C under forced aeration. At 4 hours 20 grams of
L-tryptophan was added and the culture was allowed to reach the prespore stage. Then 200 ml of glacial acetic acid were added to the culture and the mixture kept overnight at 4 C. The contents of the carboy were filtered through
Whatman no. 1 paper and the precipitate was discarded. One pound of activated charcoal (Matheson, Coleman and Bell, East Rutherford, New Jersey) was added to the filtrate and stirred overnight at room temperature (26-27 C). The above mixture was once again filtered through a Whatman no. 1 paper and the precipitate was collected on the filter paper. The residue was shaken for 2 hours with 2 1 of pyridine-methanol-water (1 :1 :2 , by volume) at room tempera ture (26-27 C) and filtered. The precipitate was discarded and the filtrate was concentrated to dryness at 40-50 C under reduced pressure. The residue was resuspended in 200 ml of distilled water and lyophilized. The crude material obtained after lyophilization was dissolved in the minimum amount of 0.1 M pyridine formate buffer pH 2. 6 and loaded on an Amberlite IR-120 column and eluted with pyridine-formate buffer as described under materials and methods.
The eluate from the column contained five major components. Two of them
41 Figure 10. Procedure for the Isolation of Intermediates of Tryptophan Catabolism from a Sporulating Culture of Bacillus cereus TR-2. CULTURE
Acidified with glacial acetic acid and filtered
Precipitate Fil ;rate
Adsorbed on charcoal and filtered
Precipitater Filtrate (Discard) Eluted with pyridine- methanol- water (1 :1 :2 , v/v)
I Residue Eluate
Removed the solvent. The residue dissolved in water and lyophilized. 4' Crude Material
Chromatographed on Amberlite IR-120 and Whatman no. 1 paper
Kynurenine, Anthranilic Acid were selected for further purification and characterization and were termed compounds A and K. These were purified by ascending chromatography on
Whatman no. 1 paper repeatedly in two different solvent systems.
2. Characterization. Rf values of the two compounds in different sol vent systems are presented in Table 4; for comparison the values of kynurenine and anthranilic acid are also included in the table. A red purple color with ninhydrin reagent indicated the presence of primary amino group in compound
K. A yellow reaction to bromocresol green suggested a carboxyl group in both the compounds. Color reactions with Ehrlich's reagent (2% p-dimethyl- aminobenzaldehyde, w/v, in 5% HC1) and Pauly's reagent (0.5% sulphanilic acid, w/v, in 2 % HC1 and 0.5% aqueous NaNC>2 in equal volumes followed by
10% Na2 CC>3 , w/v) showed that A and K were possibly anthranilic acid and kynurenine respectively. The U. V. and I.E. spectra of A and K were super- imposable on those of anthranilic acid and kynurenine respectively (Figures
11-14). The I. R. spectrum of compound A was comparable with that of anthranilic acid from Sadtler Handbook,
3. Formation of Kynurenine and Anthranilic Acid by Whole Cells. The ability of cells, harvested at different times during the progress of sporulation, to oxidize tryptophan and kynurenine was determined in the following manner.
One liter aliquots of an active culture were removed at intervals and chilled to 4 C. The cells were harvested by centrifugation at 12, 000 g for 10 min in a RC-2 Servall refrigerated centrifuge. Cells were washed once with G-medium and once with A-medium (Srinivasan and Halvorson, 1963) containing chloram phenicol (250 jig/m l). Washed cells were resuspended in A-medium supplemented 44
Table 4. Chromatography and colour reactions of some catabolites of tryptophan.
Anthranilic Properties DL-kynurenine K Acid A
Rf values in
solvent A 0.44 0.44 0.90 0.90
solvent B 0.32 0.33 - -
solvent C 0.73 0.73 0.58 0.58
Fluorescence under u. v. light strong strong blue blue pale pale violet violet blue blue
Colour reactions with:
ninhydrin red purple red purple - -
Ehrlich's Reagent orange orange yellow yellow
Pauly's Reagent brown brown - -
Ascending chromatography on Whatman #1 paper was used. The solvent systems employed were: A. 1 - butanol-acetic acid-water (4:1:1, by volume); B. 80% 1-propanol (in water, by volume); C. 20% KC1 (in water, w/v). Chromatographs were run for 16-20 hours in solvents A and B and for 3-4 hours in solvent C. Figure 11. Ultraviolet Spectrum of Authentic Kynurenine and Compound K. OPTICAL DENSITY 400
DL-KYNURENINE COMPOUND K COMPOUND 320
AEEGH IN WAVELENGTH
240
160 Figure 12. Infrared Spectrum of Authentic Kynurenine and Compound K. TRANSMITTANCE (%) 00 00 00 50 Mi 00 0 80 700 800 900 1000 CM-i 1500 2000 3000 4000 VLNT (MICRONS) AVELENGTH W Figure 13. Ultraviolet Spectrum of Authentic Anthranilic Acid and Compound A. OPTICAL DENSITY 0 240 400 320 AEEGH N m/i IN WAVELENGTH 200 CMON A COMPOUND — ANTHRANILIC ACID Figure 14. Infrared Spectrum of Compound A. TRANSMITTANCE (%} 00 3000 4000 Uuj-L'J. -i 1 i I i I I i 1 1 i i- - .i J ' L - j u .U ' i n I . 00 0 2 1500 VLNT (MICRONS) AVELENGTH W M i10 900 1000 CM-i ,.-J i IL- tl I,I,l.U-IJ, t-l l, .1I i t IjLl-l iEEE 800 — l I ,1, l I I i.1 ssS» ESSS 700 __ l, I with the same amount of chloramphenicol. The following experiment was
carried out in 50 ml Erlenmyer flasks which were incubated for one hour on a
rotary shaker maintained at 30 C. The reaction mixture consisted of 6 ml of
cell suspension (20 mg protein) and 1 ml of substrate (10 mM). After one
hour 1 ml of 1 0 0 % trichloroacetic acid was added to the flask and the mixture
shaken for another 5 min. The contents of the flask were transferred to a
centrifuge tube. The flask was washed with 2 ml of water and the washing was
also transferred to the same centrifuge tube. The mixture was centrifuged at
12,000 g for 10 min in a RC-2 Servall centrifuge maintained at 4 C. The super
natant was used for the assay of kynurenine and anthranilic acid.
The production of kynurenine from tryptophan and anthranilic acid from tryptnphan or kynurenine by chloramphenicol-inhibited cells of B. cereus, harvested during various stages of sporulation is presented in Figure 15. The rate of formation of kynurenine and anthranilic acid started rising at 2t ( 2 0
mpmole/hour/mg protein) and reached a maximum (50-60 mjumole/hour/mg protein) at ts- However, if kynurenine was used as the substrate, the rate of formation of anthranilic acid increased about eight fold from that at 2t and reached its maximum between tg and tio* Nevertheless, the pattern of develop ment of the oxidative ability in the cells to form anthranilic acid from either tryptophan or kynurenine remained unchanged.
B. Enzymes of Tryptophan Catabolism
1. Enzyme Activities in Cell-free Extracts of B. cereus. The specific activities of three enzymes involved in tryptophan catabolism were determined Figure 15. Biotransformation of Tryptophan and Kynurenine by Bacillus cereus TR-2 (See the text for the details of this experiment). OPTICAL DENSITY (6 0 0 m p ) G O CLUE Hours) ( CULTURE OF AGE - YUEIE RM TRYPTOPHAN FROM KYNURENINE 2- KYNURENINE FROM ACID ANTHRANIUC 1- - H OTCL DENSITY -OPTICAL 5 pH 4- TRYPTOPHAN FROM ACID ANTHRANIUC 3- 0 15 10 140 -
mp MOLE PRODUCT FORMED/hour/mg PROTEIN 51
in extracts of cells harvested at different stages of sporulation. These data
are shown in Table 5. Two markedly different patterns of development of the
enzymes were observed. The activities of tryptophan pyrrolase and formylase
started increasing at and reached a maximum attg. On the other hand,
kynureninase attained its maximal activity at14 and then gradually declined.
2. Effect of Nutritional Conditions on the Development of Tryptophan
Catabolic Enzymes During Sporulation. Tables 6 and 7 present the specific
activities of tryptophan pyrrolase and kynureninase in the extracts of B. cereus
TR-2 cells grown in G-medium in the presence or absence of L-tryptophan or
DL-kynurenine. There was a net increase in the activity of tryptophan pyr
rolase when cells were grown in the presence of L-tryptophan or DL-kynurenine
without any change in the pattern of the development of the enzyme. On the
addition of DL-kynurenine, the immediate substrate of kynureninase, the cul
ture exhibited the maximal enzymatic activity of kynureninase about 2 hours
earlier than the control. On the other hand, in the presence of tryptophan the
maximal activity of kynureninase was delayed by two hours.
The effect of glucose and tryptophan on the development of tryptophan pyrrolase activity in a B. cereus TR-2 culture is presented in Table 8 . The presence of 0.1% glucose in G-medium had no effect on the pattern of develop ment of tryptophan pyrrolase during growth and sporulation. The effect of tryptophan was studied in a chemically defined medium (CDGS) to determine whether it results in a change in the activities of tryptophan pyrrolase in the vegetative and sporulating cultures. On the addition of tryptophan in CDGS medium no significant changes in the pattern of development of tryptophan 52
Table 5. Tryptophan pyrrolase, formylase and kynureninase during sporulation*.
Specific Activity (units/mg. protein) Time (hours) Tryptophan pyrrolase Formylase Kynureninase
*2 41 7 43
t4 41 41 167
76 6 6 115 *6
t8 156 124 38
* 1 0 117 83 52
* Assay methods and units of activity are described in the text. 53
Table 6 . The effect of the cultural conditions on the pattern of development of tryptophan pyrrolase.
Specific Activity (units/mg. protein)
Set A _ Set B G-medium + G-medium + Age G-medium L-tryptophan G-medium DL-kynurenine
* 2 37 19 32 60
t4 37 34 60 108
6 8 89 1 2 2 t 6 156
t 8 137 196 73 119
* 1 0 105 198 62 113
A 10 1 G-medium culture of B, cereus TR-2 was grown in a 15 1 carboy as described in materials and methods. At time the culture was divided into two 5 1 portions in separate carboys. In one carboy L-tryptophan (100 jig/ml) or DL-kynurenine (100 yug/ml) was added and the other was maintained as the control. At various intervals samples were taken for the assay of protein and enzymic activity in extracts as described in the text. 54
Table 7. The effect of the cultural conditions on the pattern of development of kynureninase*.
Specific Activity {units/mg protein ______Set A______Set B______G-medium + G-medium + Age______G-medium L-tryptophan G-medium DL-kynurenine
t l 2 0 2 0 2 2 28
*2 45 44 41 191
t4 71 44 160 133
1 1 0 *6 48 65 130
t8 51 58 47 132
* 1 0 50 60 37 132
* Details of the experiment were the same as described in Table 6 . 55
Table 8 . Effect of media on the development of tryptophan pyrrolase activity in a Bacillus cereus TR-2 culture.
Age of Tryptophan Exp. culture in pyrrolase No. Medium hour pH Morphology activity*
1 G-medium 1 . 0 7 .0 i vegetative cells 5
2 . 0 6 .9 4, vegetative cells 7 5. 0 5.1 f sporulating cells 52
G-medium 1 . 0 7 .2 [ vegetative cells 2 minus 2 . 0 7.1 vegetative cells 1 2 dextrose 5 .0 7.1 sporulating cells 8 6
2 CDGS 2 . 0 6 .4 vegetative cells 8 4 ,0 ™ ' 6 .4 vegetative cells 14 9. 0 6.4 sporulating cells 115
CDGS + 2 . 0 6 .4 vegetative cells 1 0 L-tryptophan 4. 0 6 .4 vegetative cells 13 ( 1 0 0 ml) 9.0 6 .4 sporulating cells. 118
* Specific activity (enzyme units/mg protein), i- decreasing, T increasing.
Ten liter cultures of B. cereus TR-2 were grown in 15 1 carboys as described in materials and methods. The cultures were inoculated with 1 1 of actively growing vegetative cells. Washed vegetative cells grown in G- mediuTi were used to inoculate CDGS cultures. At various intervals samples were taken for the assay of protein and enzymic activity in extracts as described in the text. 56 pyrrolase was noticed.
The results from a typical experiment performed with B. cereus 49,
an organism which has properties intermediary between B. cereus and B.
megaterium, is presented in Table 9. This organism was chosen because of
its ability to grow and sporulate well in a chemically defined medium (GGMS)
composed of glucose, glutamate and G-minerals. The presence or absence
of tryptophan in the medium did not affect the level of activities of tryptophan pyrrolase in the vegetative and pre-sporulating cells.
C. Functional Aspects of Tryptophan Catabolites
Bacterial spores are generally far more resistant than their vegetative counterparts to certain processes such as sterilization or disinfection. Studies on the development of heat resistance in spores were undertaken by Murrell
et al. (1964); they made an extensive study on spore composition and heat resistance in a series of different bacilli. Dipicolinic acid has been definitely shown to be one of the several factors involved in the heat resistance of bac terial spores (Halvorson et a l,, 1960; Murrell et al., 1964).
The involvement of tryptophan in the biosynthesis of pyridine ring (nico tinic acid) has been well established in animal, plant and microbes except in the case of E. coli and B acillus (Brown, 1968; Brownstein, 1968). In the members of the family Bacillaceae» tryptophan is synthesized from aspartate and glycerol (Xsquith et a l., 1966)( Recently Desaty et al. (1967) reported the simultaneous occurrence of two different pathways for the pyridine ring synthesis in Fusarium oxysporum. In the light of the findings of Desaty and coworkers, it is interesting to speculate that bacilli may have two different 57
Table 9. The effect of L-tryptophan on the development of tryptophan pyrrolase activity in Bacillus cereus 49 culture grown in GGMS medium.
Age Cell Specific activity (enzyme units/mg protein) (hours) morphology GGMS GGMS + L-tryptophan
4 vegetative cells 18 18
8 vegetative cells 27 34
2 2 sporulating cells 80 8 6
Two 10 1 cultures of B. cereus 49 were grown in 15 1 carboys as described in materials and methods. One of the cultures was grown in GGMS medium and the other in GGMS medium containing L-tryptophan (100 jug/ml). At various intervals samples were taken for the assay of protein and enzymic activity in extracts as described in the text. 58 i pathways for the biosynthesis of nicotinic acid {aspartate pathway) and dipico-
linic acid (tryptophan pathway).
To approach this problem the technique of isotopic competition was
employed (Roberts et al., 1955). In this technique 3 H-tryptophan was added
to B. cereus culture along with the unlabeled metabolites and the resulting
decrease in the radioactivity of DPA was taken as a measure of the partici
pation of the respective metabolites in the biosynthesis of DPA. Before testing
the participation of different metabolites in the biosynthesis of DPA, it was
necessary to find out: (a) the time at which 3 H-tryptophan should be added
to the culture to get its maximum incorporation into DPA and (b) whether
these unlabeled metabolites when added to the culture would allow normal
growth and sporulation. Unlabeled metabolites added at 4 hour at the rate of
1 mg/10 ml of culture, allowed normal growth and sporulation. Table 10
presents the effect of time of addition of 3 H-tryptophan on its incorporation
into DPA. The incorporation of 3 H-tryptophan into DPA was maximum if it was added around 4 hours. However, the total radioactivity incorporated into
DPA was very low which may possibly be due to the excessive intracellular turnover of protein.
Isotope competition experiments were performed in "A" as well as nG,f media. The results are presented in Table 11. It seems quite likely that kynurenine, 3-hydroxyanthraniIic acid and quinaldic acid are involved in some process of DPA synthesis. However, these results cannot be considered con clusive due to a very low incorporation of 3 H-tryptophan into DPA.
Since the incorporation of 3 H-tryptophan into DPA was very low, this 59
Table 10. The effect of time of addition of ^H-tryptophan on its incorporation into DPA.
Time of addition of ^H-tryptophan cpm/mumole DPA
t l 1 0 0
*2 2 1 0
*3 2 0 0
*4.5 260
*5.5 255
Ho 1 2 0
Cells were grown in G-medium. Enough ^H~tryptophan was added at various times to give 106 cpm/ml and cells were allowed to sporulate. DPA was isolated and purified as described in materials and methods. The radio activity was counted in a Beckman Liquid Scintillation Counter. I
60
Table 11. Efficiency of various compounds as precursors of DPA.
cpm per mumole DPA
Unlabeled G--medium A-medium Competitor Exp. 1 Exp. 2 Exp. 1 Exp. 2
None 198 2 1 0 228 204
DL-Kynurenine 144 126 30 42
N-Formyl-DL-
Kynurenine - - 6 6 -
Kynurenic Acid 8 8 8 600 282 -
Quinaldic Acid 156 60 0 84
8 -Hydroxyquinaldic
Acid - - - 6 6
3-Hydroxyanthranilic
Acid 108 126 - 48
Cells were grown in G-medium for 4 hours. Cells were spun down and resuspended in A medium when desired. Enough ^H-tryptophan was added to get 106 cpm/ml of culture. Unlabeled metabolites (100 ug/ml) were added along with the labeled tryptophan and cells were allowed to sporulate. DPA was isolated and purified as described in materials and methods. The radio activity was counted in a Beckman Liquid Scintillation Counter. 61 problem was approached by the use of chemicals which induce the formation of heat sensitive spores. Several methods are now available for the production of heat sensitive spores. These include sporulation in low calcium medium
(Slepecky et al., 1959; Black et al., I960), in the presence of phenylalanine
(Church et al,, 1959), ethyl malonate (Gollakota et al., 1960), ethyloxamate..
(Gollakota et al., 1963), picolinamide (Upreti et al., 1969), or nicotinamide
(Kumar and Gollakota, personal communication).
The present studies were designed to show the extent to which several intermediates of tryptophan catabolism contribute to the increase of heat resistance of spores of Bacillus cereus grown in the presence of ethyloxamate or nicotinamide.
1. Induction of Heat Sensitivity by Nicotinamide and Ethyloxamate.
Nicotinamide (NA) or ethyloxamate (EO) was added at zero hour to an active culture of B. cereus growing at 30 C on a rotary shaker. Cell counts and DPA estimations were made after 24 hours of incubation. Table 12 presents the results of a typical experiment showing the effect of NA and EO on sporulation and heat resistance. Neither of the two inhibitors affected the growth and sporulation. However, the proportion of heat resistant spores was very low in the cultures treated with NA (1 to 4% of control) or EO (1 to 10% of control) with a simultaneous decrease in DPA content.
2. Reversion of Nicotinamide and Ethyloxamate Induced Heat Sensitivity by the Addition of Tryptophan Catabolites. Before testing the reversion of NA or EO induced heat sensitivity by tryptophan catabolites, it had to be established that these compounds would allow normal growth and sporulation of B. cereus 62
Table 12. Effect of nicotinamide and ethyloxamate on sporulation of Bacillus cereus TR-2.
After 24 hours of incubation at 30 C.
Additions at viable heat stable % heat stable zero hour cells/m l c e lls/m l c e lls
None 5 .1 x 108 5. 0 x 108 98
Nicotinamide ( 2 mg/ml) 5.0 x 108 1 . 2 x 1 0 7 2
Ethyloxamate (1 . 2 mg/ml) 4. 0 x 108 1. 7 x 10 6 1
Inhibitors were added to an active culture at zero hour and the cell counts were made after incubating the culture for 24 hours at 30 C on a rotary shaker. 63
under the conditions of our experimentation. This was tested and found to be
true when the supplements were added at zero hour or 4 hours, except quin-
aldic acid and xanthurenic acid which were added only at 4 hours. Each of
the following substances was added at the rate of 1 mg per 1 0 ml of the cul
ture: L-tryptophan, DL-kynurenine, anthranilic acid, 3-hydroxyanthranilic
acid, kynurenic acid, quinaldic acid and xanthurenic acid.
NA or EO were added at zero hour to actively growing cultures of B.
cereus. At 4 hours, a number of common tryptophan catabolites (100 pg/ml,
neutralized to pH 7.0 with 1 M NaOH) were added separately to inhibited cul tures and incubated at 30 C on a rotary shaker. After 24 hours heat stable
spore counts were taken. The results of a typical experiment is presented in
Table 13. There was a 300-500 percent increase in the number of heat resistant
spores in an EO-grown culture in the presence of quinaldic acid. In a NA-
inhibited culture, the increase in the heat resistant spores by addition of an thranilic acid, kynurenine or xanthurenic acid was 150, 200 and 200-500 per cent, respectively.
3. Effect of Time of Addition of Different Catabolites on the Development of Heat Resistance. The EO-treated culture was the most sensitive to reversion by quinaldic acid immediately after the growth stopped (4.5 hours) (Table 14).
As the sporulation progressed the ability of quinaldic acid to reverse EO effect decreased and it completely disappeared at 10 hours (t6 ). The addition of quinaldic acid during growth phase resulted in cell lysis and inhibition of sporulation. On the contrary, hydroxy anthranilic could reverse the effect of
EO only if it was added during growth phase (Table 15), 64
Table 13. Effect of some tryptophan catabolites on the inhibition of develop ment of heat resistance by ethyloxamate and nicotinamide.
Ethyloxamate Nicotinamide (1.2 mg/ml) (2 mg/ml) compound added heat stable % heat stable % (100 jig/ml) c e lls/m l of control cells/ml of control
None (control) 7 .6 x 107 100 2 . 0 x 106 100
L-tryptophan 3. 5 x 107 46 - -
DL-kynurenine 2.6 x 107 35 4.1 x 106 205
Anthranilic Acid 2 .3 x 107 30 2. 9 x 106 146
3-hydroxyanthranilic
Acid - - 1.5 x 106 75
Kynurenic Acid 1. 4 x 107 19 2 . 0 x 107 100
Xanthurenic Acid 5. 0 x 107 65 1. 0 x 107 500
Quinaldic Acid 3 .3 x 108 435 2 . 0 x 106 100
All the cultures were grown in G-medium at 30 C on a rotary shaker. Inhibitors and reversing compounds were added at zero and 4 hours respectively to an active culture. Cell counts were made after 24 hours. 65
Table 14. Effect of time of addition of quinaldic acid in an ethyloxamate- treated culture on the development of heat resistance.
Time of addition After 24 hours of incubation at 30 C. in hours (100 jig/ ml) viable cells/m l heat stable % of control c e lls / ml
None (control) 4. Ox 108 6.7 x 106* 100*
4.5 (t0. 5) 1. 2 x 108 3.5 x 107 522
5.0 (tx) 3.4 x 108 2.1 x 107 313
5-5 ^1.5) 3.2 x 108 1.5 x 107 224
6.1 (t2> 3. 3 x 108 1. 2 x 107 179
10.0 (t6) 3.5 x 108 6. 4 x 106 96
Ethyloxamate (1.2 mg/ml) was added at zero hour to an active culture of B. cereus grown at 30 C on a rotary shaker. 66
Table 15. Effect of time of addition of 3-hydroxyanthranilic acid in an ethyloxamate-treated culture on the development of heat resistance.
Time of addition After 24 hours of incubation at 30 C. in hours viable heat stable % (100 /ig/m l) cells/m l cells/m l of control
None (control) 2 .1 x 108 9.5 x 106* 100*
2.5 2.2 x 108 1.2 x 108 1200
4.5 2.2 x 108 1. 0 x 107 105
5.5 2.6 x 108 4. 0 x 106 42
11.5 2.3 x 108 3.5 x 10® 37
Ethyloxamate (1.2 mg/ml) was added at zero hour to an active culture of B, cereus grown at 30 C on a rotary shaker. Kynurenine or xanthurenic acid, when added to a NA-treated culture at 4 hours, resulted in an increase in the number of heat resistant spores.
The present experiment was designed to find out if time of addition of these reversing agents had any effect by itself. Both of these supplements were effective only if they were added to the culture before pH started rising
(to or 4 hours). The results of a typical experiment are presented in Tables
16 and 17. 68
Table 16. Effect of time of addition of DL-kynurenine in a nicotinamide- treated culture on the development of heat resistance.
After 24 hours of incubation at 30 C. Time of addition in hours viable heat stable % (100 /ig/m l) cells/m l cells/m l of control
None (control) 5. 0 x 10® 2 .6 x 106* 100*
2 (t-2 .0) 4 .8 x 10® 6. Ox 105 23
M t0) 5.1 x 10® 5.2 x 106 200
6 (t2> 4.6 x 108 8. 0 x 105 31
10 (t6) 4.9 x 10® 9. 0 x 105 35
Nicotinamide (2 mg/ml) was added at zero hour to an active culture of B. cereus grown at 30 C on a rotary shaker. 69
Table 17. Effect of time of addition of xanthurenic acid in a nicotinamide- treated culture on the development of heat resistance.
Time of addition After 24 hours of incubation at 30 C. in hours viable heat stable % {100 jig/m l) cells/m l c e lls/m l of control
None (control) 5.2 x 108 2 .6 x 106 100
2 4 (t0) 4.9 x 108 5.6 x 106 215 6 (t2) 4. 8 x 108 1.4 x 106 54 10 Nicotinamide (2 mg/ml) was added at zero hour to an active culture of B. cereus grown at 30 C on a rotary shaker. DISCUSSION The chemical and physical evidence presented in this investigation indicated clearly the formation and the presence of kynurenine and anthranilic acid in a sporulating culture of B. cereus. The formation of these intermedi ates from tryptophan has been previously linked with differentiation in mammals, insects and amphibians (Dalgliesh, 1952; Brown et a l.„ 1956; Baglioni, 1959; Amer et a l., 1968; Paik et al., 1968). th e studies on the biotransformation of tryptophan and kynurenine by cells in the presence of chloramphenicol showed that the onset of tryptophan catabo lism coincided with the spore formation (Figure 15). The maximal rate of formation of anthranilic acid from kynurenine was four times higher than that for the formation of anthranilic acid from tryptophan. This difference may be attributed to the reaction(s) responsible for the formation of kynurenine from tryptophan. The two enzymes responsible for the formation of kynurenine from tryptophan are: tryptophan pyrrolase and formylase. A change in the absolute amount of these enzyme(s) or their kinetics may bring about this phenomenon. This phenomenon can also be explained by assuming a permeability difference in the cell for the two substrates. It is a well-known phenomenon that inducible enzymes are repressed by glucose. Since glucose was present in G-medium and it was utilized during vegetative growth, it was of interest to determine whether the onset of tryptophan catabolism concomitant with sporulation was an artifact due to glucose repres sion. Attempts to grow B. cereus TR-2 in chemically defined medium in the 70 71 absence of glucose were futile. To circumvent the difficulty of experimentation on glucose effect, the organisms were grown in a complex medium without glucose. The levels of activities of tryptophan pyrrolase in cellular extracts were compared with those of the extracts from cells grown in the presence of glucose. The results indicated that the low level of enzyme activity in the vegetative cells cannot be due to its repression by glucose during vegetative growth. Furthermore, the addition of tryptophan to a glucose-containing defined-medium did not significantly alter the enzyme levels. Similar results were obtained with a different organism B. cereus 49. Our experimental re sults are highly indicative that the induction of tryptophan catabolism occurs concomitantly with the differentiation of the organism after completion of the vegetative growth. An analysis of the enzyme activities in cell-free extracts showed that tryptophan pyrrolase and formylase attained the maximal activity at late sporu lation phase whereas kynureninase reached its maximal activity at a very early stage of sporulation. However, in experiments with whole cells, we noticed a maximal activity of all three enzymes at later stages of sporulation. As an explanation of these apparently anomalous results, it is interesting to speculate that kynureninase may be synthesized during early stages of sporulation but remains inactive until later. The inactivation in vivo may be accomplished by the formation of an enzyme inhibitor complex which dissociates from the enzyme on sonication. The other possibility may be the existance of an apoenzyme, in vivo, which forms the holoenzyme in the presence of substrate or co-factor(s). Our understanding of the mechanisms involved in the control of tryptophan pyrrolase is still in statu nascenti. The complexity of this problem has been discussed in detail in two recent reviews {Paul, 1968; Recheigl, 1968), "Under conditions of starvation, when no net synthesis is possible, turnover (of protein and RNA) is probably the only mechanism available for achieving adaption. " This remark (Mandelstam, 1960) very well applies in the case of sporulation. A continued increase of tryptophan pyrrolase activity until the pre-spore stage, in the absence of an appreciable net protein synthesis, shows that either there is a stable m-RNA for this enzyme or there is a continuous synthesis and breakdown of short-lived m-RNA. The participation of stable m-RNA in the synthesis of mammalian tryptophan pyrrolase is well documented (Garren et a l., 1964). The involvement of stable m-RNA for the synthesis of certain enzymes during sporulation has previously been suggested (Rosas del Valle et a l., 1962; Aronson et a l., 1964; Mandelstam, 1969a). The synthesis of pyridine ring has been shown to occur by two different pathways aspartate pathway (Isquith et a l., 1966) and tryptophan pathway (Bonner, 1948). Recently it was reported that an organism may possess both pathways of pyridine ring synthesis at the same time (Ahmod et a l., 1966; Desaty et a l., 1966). On the basis of the above experiments it was interesting to investigate whether DPA could be synthesized by tryptophan pathway in B. cereus. Experiments were designed to measure the incorporation of ®H-trypto phan into DPA. Since DPA constitutes 10-15% dry weight of spores, one should assume a tremendous incorporation of label in DPA. A low incorporation of label into DPA may be due to dilution by cold tryptophan produced from 73 intracellular protein turnover. Isotope competition experiments indicated that kynurenine, 3-hydroxyanthranilic acid and quinaldic acid may be involved in the biosynthesis of DPA. Another approach to get an insight into this problem was to produce heat sensitive spores and see if tryptophan catabolites can increase the heat resistance. Our studies on the inhibition of development of heat resistance and the biosynthesis of DPA by EO and NA are in good agreement with the previous observations (Gollakota and Halvorson, 1963; Kumar and Gollakota, personal communication). Figure 16 illustrates the time course of bacterial sporulation, development of heat resistance and its inhibition by NA and EO. In the actively growing culture NA must be added before tg to obtain a fair degree of inhibition. Incidently, tg is also the time when active synthesis of DPA starts. Kynurenine and xanthurenic acid were able to reverse the NA- induced heat sensitivity to some extent if added at the time when the culture switched from growth phase to sporulation phase. It may be that kynurenine and xanthurenic acid or their metabolic product(s) are affecting some step(s) involved in commitment to heat resistance. "In the differentiating systems, it is common that inductors are effective only during competent periods of cellular differentiation (Needham, 1942).11 It is quite likely that similar phenomena are operative during sporulation of bacilli. This is reflected upon by our observation that EO was effective only if added during growth phase, while NA could inhibit the heat resistance if added up to tg. Quinaldic acid added between tq and tg, to an EO-inhibited culture, increased the number of heat resistance spores.' However, the Figure 16. Time Course of Sporulation and Development of Heat Resistance and its Inhibition by Nicotinamide and Ethyloxamate. REVERSION BY QUINALDATE REVERSION BY 3-HYDROXY-ANTHRANILATE REVERSION BY KYNURENINE OR XANTHURENATE SENSITIVITY TO ETHYLOXAMATE SENSITIVITY TO NICOTINAMIDE DIPICOLINATE SYNTHESIS GROWTH* ■SPORULATION 75 efficiency of reversion decreased as the sporulation progressed. On the other hand, hydroxyanthranilic acid was effective only if it was added during growth phase. It is interesting to speculate that quinaldic acid may be affecting some late reaction(s) involved in the development of heat resistance and a decrease in the reversing effect may simply be due to decreased permeability to this compound. Two major lines of reasoning have been put forward to explain the resistance of bacterial spores to heat: (a) involvement of a dehydrated or hydrophobic spore cortex (Warth et a l., 1962; Warth et a l., 1963a; Warth et a l., 1963b; Murrell et a l., 1964) and (b) stabilization of sppre biopolymers by cross-linking between macromolecules (Black et a l., 1962; Murrell et a l., 1964) or by chelation involving DPA, divalent cations and spore amino acids, and peptides (Fleming, 1963; Riemann, 1963; Tang et a l., 1968). It may be that a complex involving DPA, divalent cations, amino acids, peptides and some other low molecular weight compound(s) may act as a protective barrier of low polarity, low hygroscopicity and low chemical activity. This protective barrier may mask the vital spore polymers from denaturation or inactivation. Investigations on the presence of low molecular weight compounds in bacterial spores have shown the existence of DPA (Powell, 1953), glutamic acid, 3-phosphoglyeerie acid and sulfolactic acid (Nelson et a l., 1969). The functions of the last three components in a bacterial spore are still unknown. DPA is related to the development of heat resistance but it is not an essential step in this process (Murrell et a l., 1964). It has been shown that the two strains of Clostridium perfringens with almost the same level of DPA differed by a factor of 48 in heat resistance (Weiss et a l., 1966). 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He is a candidate for the Doctor of Philosophy degree in May, 1970. 92 EXAMINATION AND THESIS REPORT Candidate: Chandan Prasad Major Field: Microbiology Title of Thesis: Tryptophan Catabolism During Sporulation in Bacillus cereus Approved: M ajor Professor and Chairm an . D ean of the G raduate School EXAMINING COMMITTEE: Q r ~fs Date of Examination: May 11, 1970