Table Of ContentTHE MECHANISM OF TRYPTOPHAN SYNTHESIS BY
LACTOBACILLUS ARABINOSUS; THE ROLE OF
ANTHRANILIC ACID
LIONEL EDWARD pULAND
Bachelor of Science, 1947
University of New Hampshire
Master of A rts, 1949
Indiana University
Submitted to the Faculty of the G-raduate School
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
in the Department of Bacteriology
Indiana University
June, 1951
ProQuest Number: 10296453
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AGKNOWLSUGMMTS
The author takes th is opportunity to express his
gratitude to Professor R. 0# Bard whose in terest and
c ritic a l criticism s contributed greatly to the execution
of this study*
The author also wishes to thank Professor L* S.
MeClung for his counsel and aid extended throughout th is
study*
SPECIAL ACK2JOVlHjEDGMSIITS
The author also wishes to express his appreciation
to Professors J. H* Billman, C* E, Kaslow and E* E*
Campaigne of the Department of Chemistry, Indiana
U niversity, and Professor R* L* Shriner, Department of
Chemistry, University of Iowa for generously supplying
some of the compounds used in this study*
VITA
Lionel Edward Bhuland was bom September 26, 1920 in
Mahone Bay, Nova Scotia* He attended the public schools of
Gorham, New Hampshire, graduating from Gorham High School in
1938. He was a student at the University of New Hampshire
u n til 1942, at which time he le ft for m ilitary service in
the United States Army, (serving in the Infantry and Combat
Engineers). He was relieved from duty in January, 1946 and
completed the Bachelor of Science degree at the University of
New Hampshire in 1947. He enrolled at Indiana University as
a graduate student in Bacteriology in September 1947, receiv
ing the degree Master of Arts in Bacteriology in 1949. He
held a teaching assistantship or teaching fellowship from
September, 1947 to June, 1951* He is a member of the Society
of American B acteriologists and Sigma Xi*
To
Gloria
TABLE OF CONTENTS
Page
INTRODUCTION.............................................................................................. 1
METHODS............................................................................................................ 5
Bacteriological .................................................................. 5
Analytical ........................... 8
Chemical .................................................................. 8
Hie rob i o l o g i c a l .................................................... 11
P rep aratio n ........................................................................... 12
EXPERIMENTAL RESULTS........................................................................... 13
Growth E x p erim en ts........................... 14
A nthranillc Acid M etabolism ...................................... 16
Assay of cell hydrolysates and products of
in vitro metabolism ....................... 25
Cell h y d ro ly s a te s ............................ 25
Spectrophotometric analysis • « . . • • 29
Indole Metabolism • ............................................... 30
Selection of L* arabinosus, strain 17-5AA • • 33
DISCUSSION................................ 37
SUMMARY ....................................................................................................... 42
REFERENCES.................................................................................................. 43
INTRODUCTION
Tryptophan has long been known for its role as an
essential amino aeid in the nutritio n of microorganisms and
higher forms of life . It has been demonstrated, however,
that in the case of some microorganisms, the tryptophan re
quirement can be satisfied by synthesis of that compound from
inorganic nitrogen (Dunlop, 1949) , or from nitrogenous com
pounds intermediate in structual complexity between inorganic
nitrogen and tryptophan.
Snell, Strong and Peterson (1937) indicated, during
studies with a number of lac tic acid bacteria, that tryptophan
is required for the growth of Lactobacillus arabinosus. It
has been further shown (Snell, 1943) that anthranilic acid
supports the growth of of L. arabinosus in the absence of
tryptophan. The growth response, however, is not as great as
that obtained with tryptophan, indicating that anthranilic
acid, as such, does not substitute for tryptophan. An under
standing of the n utrition al role of anthranilic acid substitu
tion for tryptophan in the ease of bacteria may be gained
from consideration of data resulting from experiments with the
mold, Neurospora.
Tatum, Bonner and Beadle (1943) observed that a trypto
phan-requiring mutant (strain 10575) of Neurospora grows well
with indole but not with anthranilic acid in the absence of
z
tryptophan, whereas a second genectically different mutant
u tilize s either indole or anthranilic acid for growth under
sim ilar conditions# These workers concluded that a genetic
block occurs between anthranilic acid and indole in strain
10575, and that tryptophan is synthesized through a metabolic
pathway including both anthranilic acid and indole. This
hypothesis was given further support by the demonstration
(Tatum ejfc a l# , 1943) that strain 10575, whieh does not grow
with anthranilic acid, accumulates a relativ ely large amount
of anthranilic acid in the medium. Although it was not clearly
stated in the la tte r paper, reference to an earlier publication
(Beadle and Tatum, 1941) indicates that an inorganic nitrogen
source was supplied in addition to tryptophan or indole, which,
in this case, was required for the growth of strain 10575,
Further studies concerning the synthesis of tryptophan
from indole (Tatum and Bonner, 1944) by Neurospora indicated
that within a range of 10-50 mg of DL-serine per 50 ml of re
action mixture, the rate of indole disappearance is a function
of the concentration of serjne. Tryptophan was isolated from
the above reaction m ixture, constituting evidence for synthesis
of tryptophan from indole and serine. TJmbreit, Wood and
Ounsalus (1946) used a cell-free extract of Neurospora sitophila
to demonstrate the conversion of indole to tryptophan in the
presence of serine; pyridoxal phosphate was found to be the
coenzyme of this enzymatic condensation. These data with
Neurospora indicate that the biosynthesis of tryptophan from
anthranilic acid proceeds via indole , the la tte r in condensa
tion with serine yielding tryptophan.
3
The in itia l observation of Snell (1943) that anthranilic
acid supports growth of L* arabinosus in the absence of trypto
phan suggests the existence in bacteria of a metabolic pathway
sim ilar to that described for Neurospora* Schweigert, Sauber-
lic h , Bauman and Elvehjem (1946) demonstrated that indole also
supports growth of L* arabinosus and that the growth response
to indole and anthranilic acid on an equimolar basis is only 30
to 90 per cent of the response obtained with tryptophan*
Stimulation of indole-grown cells by serine indicates a sim ilar
ity to the indole-serine system that has previously been demon
strated in Neurospora* It has also been shown (Schweigert,
1947) that acetate and serine accelerate the growth of L.
arabinosus In the presence of anthranilic acid or indole*
Additional evidence supporting the hypothesis that the
pathway of tryptophan synthesis from anthranilic in bacteria is
sim ilar to the pathway in Neurospora, was advanced by Rydon
(1948) using Bacterium typhosum [Salmonella typhosal * Studying
a strain found to grow in the absenee of tryptophan, Rydon re
ported inhibition of growth with 4- and 5- methyl anthranilic
acids* However, the inhibition was relieved by the addition
of either indole or anthranilic acid, whereas j>-aminobenzoic
acid was ineffective* From these data, it was concluded by
Rydon (1948) that anthranilic acid is the precursor of indole
in the biosynthesis of tryptophan by bacteria*
These data clearly indicate that the tryptophan require
ment of L* arabinosus can be satisfied by either indole or
anthranilic acid. The observation that serine stim ulates the
growth of L. arabinosus (Schweigert, 1947) in an amino acid
