— w———_——v-— —

 

 

. . — o

mmsmeu OF SéLMQNELLA GALLJINARUM
BY mssams AND H's REVERSAL

 

 

The“: {0? the Degree of M. 5:
MICHIGAN STATE UNIVERSUY
M. Louise Brock

1958

f O I." ..._

a . wing.

IRHIBITION OF SALMOLELLA GALLINARUM

 

BY D'SERINE AND ITS REVERSAL

By

M. Louise grock

AN ABSTRACT

Submitted to the School of Science and Arts of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1958 , ,
i C" *’ " C

. I / .2 A 1/ ’ , 7-h- ,_
Approved by ,( (;,MH¢M9.h/kiv,

Abstract M. Louise Brock

The growth, nutrition and inhibition of Salmonella

 

gallinarum was studied in a defined synthetic medium using a

 

turbidimetric method and viable cell count. This organism is
inhibited by the D isomer of DLrserine which reduces the growth
rate. The inhibition is temporary and is reversed noncompetitively
by nucleic acid derivatives and amino acids, for which inhibition
indices have been determined, and also by RNA, KHCO3 and

Lrserine.

The metabolism of amino acids and nucleic acids is dis-
cussed, and it is suggested that D-serine interferes with the
metabolism of several amino acids, particularly where they are
required for nucleic acid synthesis.

D'Cycloserine and azaserine also inhibit g, gallinarum but

Orcarbamyl-D'serine does not.

ILHIBITION OF SALMOKELLA GALLINARUM

 

BY D'SERINE ARD ITS REVERSAL

By

EL Iouise grock

A THESIS

Submitted to the School of Science and Arts of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of'bficrobiology and Public Health

1958

AC KN 0%? LE D G IVE 1“: TS

The author wishes to express her appreciation
to Dr. D. E. Schoenhard for suggesting this problem
and for his guidance and encouragement in the study.

The advice and encouragement of Dr. T. D. Brock,
in whose laboratory at Western Reserve University

much of the work was performed, is also appreciated.

TABLE

INTRODUCTION . . .

MATERIALS AND I-IETHODS

RESULTS.....

DISCUSSION . . . .

SUP‘MARYo....

BIBLIOGRAPHY . . .

APPENDIX. . . . 0

OF

COIQTEIQTS

PAGE

10
29
1+2
1+3
47

IKTRODUCTIOR

Salmonella gallinarum was first described by Klein in 1889
(Breed, Murray, and Smith, 1957). A fermentation variant of'§,
gallinarum was described by Rettger and Harvey in 1908 and
given the name Bacterium pullorum by Rettger in 1909. This was
later changed to Salmonella pullorum (Breed 3£_213, 1957), and
in the latest edition of Bergey's Manual for Determinative

Bacteriology is included within the species Salmonella

 

gallinarum. For the sake of clarity the species name

 

"pullorum" will be retained here in reference to research
published prior to this recent revision in nomenclature when it
had been distinguished between the species pullorum and
gallinarum.

S, gallinarum is pathogenic for fowl, causing the disease
known as white diarrhea or fatal septicemia; it is in this
regard that it differs mainly from other organisms of the genus

Salm0ne 1.1a.

 

Early attempts to grow S, pullorum.in synthetic media met
with no success. Koser and Rettger (1919) found it would not
grow in an amino acid-salts'glycerol medium and grew only
slightly when complex nitrOgen sources were added. In another
case several strains would not grow on a simple salts medium

supplemented with inorganic nitrOgen sources and salts of

organic acids (Hajna, 1935).

Beard and Snow (1956) included S: pullorum among the
organisms they grew on synthetic media.for a study of antigenic
characteristics, using a protein-free medium of 21 amino acids,
glucose and salts. Of a large number of strains used by
Johnson and Rettger (19A3) for a nutritional study, most did
not require growth factors while some required one or more of
the following: nicotinic acid or its amide, glucose, aspartic
acid, glutamic acid, leucine, and arginine. Several strains
would not grow even when supplemented with 16 amino acids,
vitamins, and other factors. All the strains of’S: gallinarum
they tested could grow without glucose but required thiamine
hydrochloride, while some required 10% 002 in addition to
initiate growth. Lederberg (1997) was able to grow two strains
of §, pullorum in a glucose-salts-asparagine medium if leucine
and.cystine were supplied for one and these plus methionine for
the other. Davis and Solowey (1950), however, found that when
several amino acids and other organic compounds were added
individually to a salts medium, g, pullorum would not grow.

The synthesis of glutamic acid and alanine by several
strains of'S, pullorum.in synthetic media was studied by Jones
and Holtman (1953). and Schoenhard and Stafseth (1953)
described the culture cycle of the organism grown in several

complex media compared with a synthetic medium. This synthetic

medium was then modified by Gilfillan, Holtman and Ross (1955)

to decrease the period of time required for maximum growth, and
among the modifications is the addition of DL-serine. The
latest published work concerning S, pullorum is that of Stokes
and Bayne (1957) who were able to increase the colony size and
growth rate on complex solid media, but they could not decrease
the lag period.

In the process of developing a synthetic medium for the
growth of this organism Schoenhard (personal communication)
found the racemic mixture of DL-serine to be inhibitory. There
have been many reports in the literature of amino acids being
inhibitory to the growth of bacteria. The single amino acids
threonine, lysine, and cysteine, and the combination of serine
and alanine were found to inhibit S, pullorum (Gilfillan,
Holtman and Ross, 1955). and urea was also inhibitory (Ross,
Holtman and Gilfillan, 1956). Castellani (1953) found that 91.-
serine had a possible slight inhibition of the growth of this
organism in a cream pastry filling, but no further work was
done in a synthetic medium.

The D isomer of serine prevents toxin formation by
Clostridium tetani (Mueller and Miller, l9h9) and interferes
with pantothenic acid synthesis in Escherichia.ggli (Maas and
Davis, 1950). DLrSerine inhibits Bacillis anthracis
(Gladstone, 1939). mutants of Bacillis subtilis (Teas, 1950),
lactobacilli (Teeri and Josselyn, 1955), Aerobacter aerogenes

(Dagley, Dawes and Morrison, 1950), Agrobacterium tumefaciens

(Van Lanen, Riker and Baldwin, 1952), Bacterium linens (Friedman,

 

Wood and Nelson, 1955), I-chobacterium tuberculosis var. hominis

 

(Dubos, 1949), and Streptococcus bovis (Prescott EEHEE°9 1957).
In most of these instances the mechanism of action is not known.
This study was undertaken in an attempt to determine the

nature of DLrserine inhibition in.§, gallinarum when the

 

organism is grown in a defined synthetic medium; a large

number of compounds including amino acids, purines. pyrimidines,
nucleic acids, and nucleic acid derivatives were tested for
possible reversal of serine inhibition.

The term inhibition is used in this study to describe the
fact that a test culture has a turbidity less than that of a
control culture at some arbitrary time after inoculation.

The term reversal is used to describe the fact that in the
presence of some added compound inhibition by serine does not
occur, or occurs to a lesser extent, when the turbidity of a
test culture is compared with that of a control culture; it
does not imply by what mechanism the inhibition is prevented or
counteracted.

The inhibition index is used to evaluate the ability of a
compound to relieve serine inhibition. It is obtained by
subtracting that concentration of serine in mM/ml at which
halfbmaximum growth of the control culture occurs from the
concentration of serine giving half-maximum growth in the

presence of a reverser; the ratio of this value to the

concentration of reverser in mH/ml is the inhibition index. The

formula for calculating the indices is shown below.

mM/ml DLrserine for mM/ml DLvserine for
‘% max. growth in - ‘% max. growth
presence ofreverser of control ‘ Inhibition

 

Index
mM/ml of reverser

Culture methods.

isolated from poultry.

I'iATERI AIS AliD IuEThCDS

Strain 6 of Salmonella gallinarum was

It conforms to the physiological

characteristics of g, gallinarum with the exception of

producing a weak alkaline reaction in litmus milk; H S is

produced but citrate is not utilized.

2

The cells are

agglutinated by Salmonella polyvalent 0 serum (Lederle) and are

highly virulent when introduced into day-old chicks.

All the growth and inhibition studies were made using the

following synthetic medium:

Lrleucine

chystine
Lrarginine°HCl
ThiamineOHCl
Calcium pantothenate
MgSO 07H 0

Trace elements‘

87 mg
60 mg.

45 mg

1 mg
2 mg

100 mg

1 ml

distilled water 1000 ml

NHgCl 5 g
NEQNO l g
KQBPQu 5.82g
KEZPQQ 2.18g
Glucose 10 g
K11003 2 g

I’Trace elements solution (Horowitz and Beadle, l9#5):

NaZH+07° 101120

(NHQ)6 M07 021+°4H20

FbC13'6H20

CuSOn anhydrous

MhC12'#320

ZnSOq,’ 7H20

distilled water
dilute 1-100

The amino acids, vitamins,

3-52 mg
2.78 mg
9068 mg
2051 mg
0.72 mg

100 m1

salts and trace elements were

dissolved at 1.5625 times the above concentration in distilled

water and autoclaved.

The glucose was prepared similarly at,

10 times the above concentration and

-6-

autoclaved separately, after which it was added to the sterile
basal medium; 1 to 5 liter amounts of this medium were made at
one time and stored in the refrigerator for future use. The
bicarbonate was also prepared at 10; times the above concentra-
tion, sterilized by filtration through asbestos, and added to
900nfl. or more quantities of the above basal medium containing
glucose; this was then refrigerated and used within two weeks.
Preliminary experiments showed that refrigeration of the
complete concentrated medium containing bicarbonate for this
length of time had no effect on the growth of the organism.

After having been inoculated the concentrated medium was
dispensed in 4nd. quantities into sterile test tubes containing
1nd. of test solution or, for the growth curve experiments, in
#Onfl. quantities into sterile dilution bottles containing 10nd.
of test solution.

The cultures were maintained on brain heart agar (Difco)
slants which were stored in the refrigerator after a thu'
incubation period at 57<L For each experiment cells were
inoculated into 5 ml amounts of synthetic medium and two-drop
amounts transferred at 2# hr intervals. The inoculum was then
taken from the third tube of synthetic medium in which the
organisms had been grown and used to seed the complete medium
at the rate of 1 ml inoculum per liter of 1.25'strength medium.

Compounds to be tested were dissolved in distilled water

and autoclaved in test tubes or bottles at 118C for 20 min.

Estimation 2:.ggowth. Growth was measured either by
visual comparison or by plate count methods. For the visual
method the amount of turbidity in test tubes was compared with
that in a set of standard BaSQu tubes (McFarland's nephelo-
meter) (Stafseth, Stockton, and Newman, 1956), numbered 1
through 10. no attempt was made to estimate an amount of
growth greater than 10. Preliminary experiments showed this
method to be more sensitive than one using a colorimeter to
evaluate low turbidities. The turbidities of cell suspensions
were compared with the TESOh tubes and total cell counts were
made using the Petroff-Hauser counting chamber. The results
of this experiment are shown in figure 1 which indicates that
the Ea804 units are proportional to the logarithm of the total
cell count.

Standard Methods for the Examination g£_Dairy Products

 

(l9#8) was followed regarding the selection and counting of
plates. Dilutions were made in sterile tap water; nutrient

agar (Difco) was used for the plating medium.

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RES ULTS

Growth £252§_i§_the presence g£_ssrine and g_reverser.

Growth curves of Salmonella gallinarum in the synthetic
medium with and without a high concentration of serine and with
and without uridine are shown in figure 2. The inoculum was
taken from a culture growing in the logarithmic phase. Five ml
portions of each of the four cultures used were transferred to
test tubes so that turbidity could be estimated in BaSQu units
and compared with viable count; the results are shown in figure
2 and table 1.

Since the dip in the growth curve at 56 hours did not
occur in other experiments and since the turbidities of the
cultures had increased between 2# and 56 hours, the low cell
counts obtained at 56 hours are probably due to errors in
sampling.

The control culture remains in the logarithmic phase which
persists for about 2# hours; there is no initial lag phase in
the serine'inhibited culture, but the growth rate is less than
that of the control culture. In other experiments it was found
that the addition of serine did not affect the final cell count.
The addition of 62.5 ug/ml of uridine completely reversed
serine inhibition after 56 hours by enabling the cells to
attain approximately the same growth rate as the controls,
whereas serinerinhibited cells do not attain the same growth
rate as the controls even at 60 hours.

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TABLE 1

Effect of DL-serine and uridine on growth of'S: gallinarum

 

Hours

16 18 21 2h 56 #8 60

 

Control 2 5 7 10 >10 >10 >10
DL-Serine 400 ug/ml 0 O 0 0 1 1 5
Uridine 62.5 ug/ml 2 5 7 10 >10 >10 >10
DLPSerine #00 ug/ml 0 0 0 0 1 5 :710

+ Uridine 62.5ug/m1

 

Values are turbidity in 1353.301+ units

Experiments performed at several different times showed
that the delayed growth in the presence of DL-serine is not due
to the multiplication of serine-resistant mutants or the
adaptation of the culture to serine. When a culture grown in
the presence of serine was reinoculated into fresh serine'con-
taining medium, the growth of the culture was again inhibited,
indicating that delayed growth following serine inhibition is
not due to selection or adaptation. The results of one of these
experiments are shown in table 2.

Isomer g£_serine effecting_inhibition. The inhibitory
effect of DL-serine was found to be due entirely to the D
isomer. The activities of DL-, L“, D", and D+Lrserine were
compared and the results are shown in table 5. It can be seen
that DLrserine has the same activity as a mixture of equal

amounts of D- and L-serine, and that Lrserine is essentially

TABLE 2

Effect of DLrserine on growth of S, gallinarum

 

Hours

18 25 5O (#2

 

DL-Serine 100 ug/ml
DIrSerine 50 ug/ml
DLrSerine 25 ug/ml

Control

1 5 10
7 >10 710
8 > 10 >10
>10 '710 7-10

«3 e-oa <>

 

At 26 hours the culture in the tube containing
50 ug/ml of DL-serine was diluted and re-
inoculated into the following tubes:

 

Hours

21+ 28 no 48
DIrSerine 800 ug/ml 0 0 0; O
DLrSerine #00 ug/ml 0 0 0 5
DLrSerine 200 ug/ml 0 0 5 >10
DL-Serine 100 ug/ml o 3 >10 >10
DLrSerine 50 ug/ml 5 6 >10 >10
DL-Serine 25 ug/ml 5 8 710 710
DL-Serine 12.5 ug/ml 5 10 >10 >10
Control 5 9 >10 >10

 

not inhibitory. It can also be seen that, comparing the same
concentrations of D- and DL-serine, the D isomer is more than
half as active as the racemic mixture, indicating that D-serine
is partially reversed by L-serine. In the experiments which
follow, DLrserine was used throughout since it is cheaper and
reversal by the L component is slight. Thereene insufficient
data available to determine an inhibition index for L-serine.

TABLE 5

Comparison of the effects of DL-, L-, D-, and D+L-serine on
growth of §, gallinarum

 

 

 

 

18 Hours Total serine concentration in ug/ml
3200 1600 800 #00 200 100 50 #0 50 20 10 5
Isomer ,
DL . o o o o o o 1 3 5 7
L 0 0 0 5 5 7 7 8 8 8 8 8
D 0 0 0 0 0 0 0 0 0 l
D+L o o o o o o 3 5
Control: 8
2# Hours Total serine concentration in ug/ml
3200 1600 800 #00 200 100 50 #0 30 20 10 5
Isomer
DL 0 o o 1 3 5 6 7 9 10
L 0 1 1+ 7 10 >10 >10 >10 >10 ’10 >10 >10
D 0 0 0 0 0 0 0 0 1 #
D+L 0 0 0 0 1 S 7 9

 

Control: >10

Inhibition by the antibiotics azaserine and D-cycloserine.
Two antibiotics which are derivatives of serine were
tested to see if they would affect the growth of S, gallinarum.

Although azaserine and D-cycloserine were not tested extemnxely,

several eXperiments showed that these compounds were inhibitory,
while O'carbamyl-Drserine, an unusual amino acid produced by a
species of Streptomyces, was not.

Azaserine was tested in the synthetic medium reported here

which contained, in addition, L-asparagine in a final

-14..

concentration of 1 mg/ml. Azaserine was highly inhibitory in
concentrations from 5 ug/ml to 100 ug/ml, while lower concentra‘

The results of this experiment, in

5

tions were less active.
which a #8 hour inoculum diluted to approximately 1.5::10

cells/ml was used, are shown in table #.

TABLE #

Inhibition of S, gellinarum by azaserine

 

 

 

Hours

18 24 28 54 48 6o 96

Azaserine 100 .008 .078 .005 .012 .002 .012 0
“g/ml 50 .021 .005 .020 .024 .020 .025 .015
25 .017 .020 .015 .015 .012 .015 .010
12.5 .016 .012 .018 .018 .012 .015 .020

6.25 .028 .017 .050 .050 .025 .025 .025

5.125 .050 .027 .051 .028 .024 .052 .45

1.5 .025 .027 .028 .050 .058 .20 .57

0.75 .050 .025 .058 .22 .55 .22 .48

0.575 .022 .040 .072 .50 .57 .51 .55

0 .058 .070 .14 .58 .54 .51 .49
Turbidity in Optical density, determined

on a Bausch and Lomb Spectronic 20
colorimeter, 525 7\.

Cycloserine and O‘carbamyl-D-serine were tested at a single

concentration of 250 ug/ml each using a 2# hour old inoculum.

O'CarbamylvD'serine was not inhibitory in the presence of either

threonine or asparagine, while cycloserine was inhibitory in
the presence of threonine or asparagine or asparagine plus
cwalanine. The results of this experiment are shown in table 5.

-15-

TABLE 5

Effect of D-cycloserine and O'carbamyl-D-serine on growth of
S: gallinarum

Hours

18 25 28 #1 69 92

 

O'Carbamyl-D-serine 250 ug/ml
+ DL-Threonine 1 mg/ml

o-Carbamyl-D-serine 250 ug/ml
+ L-asparagine 1 mg/ml

D-Cycloserine 250 ug/ml
+ DL-Threonine 1 mg/ml

D-Cycloserine 250 ug/ml
+ L'Asparagine 1 mg/ml

D-Cycloserine 250 ug/ml

l 1 5 >10 >10 >10

1 3 5 >10 >10 >10

4- L-Asparagine 1 mg/ml 0 0 O 0 O 0
+ DLadalanine l.mg/m1

DLrThreonine 1 mg/ml 0 5 6 10 >10 '510
Control 0 0 2 10 >10 710

 

Effect g£_constituents g: the medium.g§_growth and serine

 

inhibition. During the preliminary work on this study of serine
inhibition a synthetic medium was used which was identical to

the one reported here except that it contained L-asparagine in

a final concentration of 1.5 mg/ml. Serine inhibition was
irregular and the results of some of the experiments could not

be duplicated. In attempting to reverse serine inhibition it

was found that when the asparagine content of the medium was
doubled asparagine itself was inhibitory and that this inhibition
was reversed by 50 ug/ml of DL-serine. Consequently an experi‘

ment was set up in which various concentrations of asparagine

-16..

were tested for their effect on serine inhibition. It was dis-
covered that asparagine in low concentrations was able to
reverse serine inhibition, and this explained the irregular
results in asparagine-containing medium. Since asparagine was
not required for growth, it was omitted, and more reproducible
results were obtained. Schoenhard (personal communication) had
previously found that aspartic acid could also reverse serine
inhibition.

In order to determine what effect the other constituents
in the medium had on growth some of these were eliminated
singly and some in combination from the synthetic medium.
Serial transfers of 2 drops from each tube were carried over at
2# hour intervals into the various deficient media. To obtain
continuous growth in the synthetic medium cystine, leucine, and
glucose were found to be essential, while there was some need
for arginine and inorganic nitrogen. Thiamine and pantothenic
acid were nonessential under these conditions of a large
inoculum.

When the constituents were then added back to the various
deficient media in graded concentrations with and without
serine to determine the effect of concentration of these
ingredients on serine inhibition, a small inoculum was again
used. It was found that the concentrations of cystine, leucine,
glucose, arginine, and inorganic nitrogen were not critical

for growth, nor did these compounds have any tendency to

reverse serine inhibition. A four-fold increase in amount of
glucose, amino acids, or inorganic nitrogen over the concentra-
tions normally present in the complete synthetic medium did not
relieve serine inhibition. When the amount of glucose was
reduced, growth was also reduced but serine inhibition was
unaffected. Reduced amounts of amino acids also gave less
growth, and serine was more inhibitory under these conditions.
In these experiments histidine could replace arginine with
nearly identical results on growth and inhibition. Reduced
amounts of inorganic nitrOgen did not affect growth or serine
inhibition, while increased amounts over that normally present
in the complete medium were somewhat inhibitory and potentiated
serine inhibition.

When graded concentrations of K11003 were added to a
bicarbonate‘deficient medium with and without serine it was
found that the growth attained in 21 hours was independent of
the bicarbonate concentration up to 2 mg/ml. At this time
growth was reduced slightly in the presence of # mg/ml of
KH003 and markedly by an 8 mg/ml concentration. At 55 hours
concentrations from 100 ug/ml to # mg/ml gave better growth
than no bicarbonate at all, while the # mg/ml and 8 mg/ml
concentrations potentiated the inhibition caused by 50 ug/ml
and 100 ug/ml of DLrserine. Concentrations of bicarbonate from
100 ug/ml to 2 mg/ml had some ability to reverse serine inhibi-
tion but therezxe insufficient data to determine an inhibition

index.

-18-

In their studies on the nutrition and virulence of §_.
pullorum in a synthetic medium, Gilfillan, Holtman and Ross
(1955) reported that DL-serine was not inhibitory, but their
medium also contained glycine and DL-olalanine. DL‘dalanine
and glycine were also tested singly by the writer and found not
to reverse serine inhibition; the raw data for these experi-
ments are included in tables 25 and 2# of the appendix. The
combination of alanine and glycine was not tested here for its
effect on growth and serine inhibition.

It should be noted that the medium of Gilfillan, Holtman
and Ross (1955) also contained xanthine in a concentration of
5 ug/ml. Since in the work reported here xanthine has been
found to be inactive at 12 ug/ml in reversing a serine concentra"
tion of 50 ug/ml, it is improbable that the presence of xanthine
in their medium would be responsible for the lack of serine
inhibition, but their strain would have to be tested before any
definite statement to this effect could be made.

Reversal 91 inhibition by nucleic acid derivatives.

Cert ain nucleosides and ribonucleic acid had considerable
ability to relieve serine inhibition. Since no clear-cut line
°°uld be drawn between moderately and weakly active reversing
compounds, the inhibition index has been used to indicate the
releutive activities of these compounds, and they are listed in

1able 6.

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Uridine has a more constant index than any of the other
compounds tested, around 15, while adenosine and cytidine are
the next most active compounds followed by the other nucleic
acid derivatives which have indices of l# or less. Figure 5
shows typical inhibition curves plotted from data obtained with
uridine. Although the inhibition indices of uridine indicate
that it may possibly reverse serine competitively, the highest
concentration tested was 100 ug/ml so no definite conclusions
can be drawn. The method used for calculating inhibition
indices is shown in table 7.

Although inhibition indices were not determined for RLA
and DNA because of unknown molecular weights, RNA was less
active than cytidine and more active than cytidylic acid when
these compounds are compared on a weight basis. In contrast to
the activities of the deoxyribose derivatives and RNA, DNA was
actually inhibitory alone but somewhat stimulatory in the
presence of serine, depending on the concentrations used.

Since one of the nucleic acid derivatives, adenine sulfate,
was completely without ability to relieve serine inhibition,
and in fact was somewhat inhibitory while adenosine was very
active, ribose and adenine together were tested against serine.
This combination was only weakly active in relieving inhibition,
while ribose alone was inactive. Deoxyguanylic acid was the
only DhA derivative tested which did not show some serine

reversal within 2# hours.

 

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TABLE 7

method of calculating inhibition indices;
based on curves shown in figure 3

DL-Serine concen-

 

 

 

 

 

Uridine concentration tration at ‘72 Calculations
maximum growth

ug/ml mM/ml ug/ml mM/ml
0 0 5 0.0#8
10 0.041 66 0.63 0'63 £10'48 = 1#
25 0.102 1#0 1.3# 1'3: 103°#8 = 13
50 0.205 250 2.38 2'33 20;.h8 = 11

100 0.#1o >250 72.38 ’2'3gilo'48 = )6

 

The raw data showing the activities of RNA, DhA, and the
nucleic acid derivatives testedane shown in tables 25 through
#7 in the appendix.

Effect QMM, vitamins, and other organic commounds

on serine inhibition. Several amino acids, amino acid amides,

 

and urea were found to relieve serine inhibition to varying
degrees. The most active of these compounds is L-glutamic acid
with an inhibition index of 2, followed by Lraspartic acid, Dir
isoleucine, L-glutamine, L-asparagine, L-histidine, and L-
proline. The inhibition indices of DL-methionine and urea were
also determined but, since these were based on control
turbidities greater than 5, these values cannot be compared
directly with the others. The inhibition indices are given in

tables 8 and 9, and the raw datatne included in tables 48

through 56 in the appendix.

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The ability of urea to reverse serine inhibition in this
strain, although slight, is especially interesting in view of
the findings of Ross, Holtman, and Gilfillan (1956) that urea
is inhibitory to §, pullorum at a concentration of 60 mg/ml,
while a concentration of 15 mg/ml reduced growth somewhat. The
writer found that while urea was very slightly inhibitory alone
at much lower concentrations, from 250 ug/ml to 2 mg/ml, these
amounts of urea were capable of reversing serine inhibition to
varying degrees depending on the concentration used.

In an attempt to reverse serine inhibition by adding
graded amounts of Lrasparagine it was found that high concentra-
tions of this amino acid amide were toxic, and that serine
reversed this inhibitory effect of asparagine. Table 10 shows
that in the presence of 5 mg/ml of L-asparagine no growth occurs

at a concentration of DL-serine less than 2.5 mgyml by 29 hours.

TABLE 10
Inhibition of growth of g, gallinarum.by L'asparagine and its
reversal by DL-serine
12 Hours DL'Serine concentration mg/ml
10 5 2.5 1.25 0.625 0.512 0.156 0.078 0.039 0

 

 

L-Asparagine

 

5 mg/ml ‘7 5 O 0 0 0 O 0 O 0
Control: 7
22 Hours DLrSerine concentration mg/ml

10 5 2.5 1.25 0.625 0.512 0.156 0.078 0.059 0

 

L-Asparagine

5 mg/ml >10 >10 3 O 0 0 0 0 0 0

Control: )10

 

As in the case of serine inhibition, asparagine inhibition is
temporary and the cultures eventually develop a high turbidity,
by approximately 65 hours in this experiment. 0ther compounds
were not tested for this effect.

Interesting results were obtained with Ir'cysteine"I and
glutathione‘. Gilfillan, Holtman, and Ross (1955) reported
that a concentration of 60 ug/ml of cysteine was inhibitory to
their strain of §: pullorum. As is shown in table 11, cysteine

is inhibitory to §, gallinarum in the absence of serine,

 

depending on the concentration used. Cysteine potentiates the
inhibition caused by DL-serine for 50 hours or more, but by 42
hours after inoculation cysteine reverses the serine inhibition.
A similar set of results of potentiation of inhibition followed
by reversal was obtained with glutathione (L-Iglutamyl-L-
cysteyl-glycine). Since glycine alone neither stimulated growth
nor reversed serine inhibition, while L-glutamic acid reversed
serine but did not reduce growth, the activity of glutathione
is probably due to the cysteine component. The complete data
for cysteine and glutathionezne.inc1uded in tables 57 and 58 of
the appendix.

A number of amino acids, vitamins, and other compounds were
found to be unable to reverse within #8 hours the inhibition

caused by a DL-serine concentration of 120 ug/ml. These

*autoclaved only 5 minutes

-26—

TABLE 11

Effect of L-cysteine and DL-serine on growth of §3§§llinarum

18 Hours

L-Cysteine
ug/ml

25 Hours

L'Cysteine
ug/ml

50 Hours

L-Cysteine
ug/ml

#2 Hours

LrCysteine
ug/ml

2000
1000
500

250
125

2000
1000
500
250
125

2000
1000
500
250
125

2000
1000
500
250
125

 

DL“Serine ug/ml

 

 

 

 

 

 

 

800 #00 200 100 50 25 L&f§ 0

0 0 0 0 O 0 0 0

0 0 0 0 0 0 2 4

0 0 0 0 0 0 a 4

O 0 0 0 O O O 5

O O O O 0 2 5 7

0 O 0 O 1 4 6 7
DL-Serine ug/ml

800 11-00 200 100 50 25 12. 5 0

0 0 O 0 O 0 0 l

O 0 O O 2 5 8 8

0 0 0 0 0 1 10 10

0 0 O O l 6 6 >10

0 0 O 0 5 7 10 >10

0 0 0 1 7 8 10 >10

DL-Serine 11 g/ml .

800 #00 200 100 50 25 12.5 0

0 0 0 0 0 0 5 5

O O O 0 9 >10 >10 >10

0 0 O O 5 5 >10 >10

0 O O 0 7 =10 710 >10

0 0 0 0 9 ’10 >10 ’10

O 0 0 3 >10 ’10 >10 ’10
DLPSerine ug/ml

800 #00 200 100 50 25 12.5 0

10 5 5 1 1 0 10 10

>10 >10 >10 9 >10 >10 >10 >10

0 0 l 9 710 >10 >10 >10

0 O 0 7 >10 >10 >10 >10

0 0 0 . 7 >10 >10 >10 >10

0 0 0 10 >10 >10 >10 '710

 

compounds in the highest concentrations tested are listed in
table 12. A combination of the eight vitamins in a total con-
centration of l ug/ml was also ineffective in relieving inhibi-
tion.

TABLE 12

Compounds which did not reverse a DL-serine concentration of
120 ug/ml within 48 hours

Compounds in which the highest concentration tested was l_mg[ml

 

DL- Threonine L-Tyrosine
DLrHomoserine Hydroxyproline
DL- Ornithine Glycine "‘
DLrValine Sodium acetate
lDL-“Alanine Thiourea
DLrflAlanine Allylthiourea’
DLrLysine Succinic acid
DIrTryptophane Sodium formate
L-TryptOphane Methionine sulfoxide

DL-Norleucine

Compounds i2_which the highest concentration tested was l_ug[ml

Riboflavin Thiamine hydrochloride
Folic acid. PAminobenzoic acid
Inositol . Pyridoxine hydrochloride

Kicotinic acid
Compound in which the highest concentration tested was 3; ug( l
Biotin

 

I

inhibitory

-28..

DISCUSSION

The concept g£_inhibition and limitations 33 inhibition

 

studies. An inhibitor is a chemical which decreases the rate
of a reaction. Inhibitors may react preferentially with
particular groupings on enzyme or protein molecules, although
enzymes or proteins are not necessarily involved. Inhibition
may result from the affinity of a compound structurally
similar to the substrate for an enzyme or prosthetic group, or
the inhibitor may have no structural similarity to the sub-
strate. Inhibition may be due also to the removal of a
required substrate, as in the case of a chelating agent re“
moving ions necessary for a particular reaction.

The term inhibition has been applied in other literature
to an increase in the duration of the lag phase, a reduction
in amount of total growth, and also to a decrease in growth
rate. These types of inhibitory actions are clearly distin-
guishable and are discussed more fully by Moore and Boylen
(1952).

Reversal of inhibition may be accomplished in several
ways and can sometimes be used to determine the nature and
specificity of the inhibition. This can be done by removing
the inhibitor or adding a substance which will combine pre'

ferentially with the inhibitor, but since these methods may

present difficulties when the growth of cells is being studied,
the following methods are more useful in demonstrating
specificity: adding increased amounts of the inhibited meta-
bolite or its precursor, supplying the product of the inhibited
reaction or an effective substitute for the product, or
supplying precursors of the blocked enzyme or prosthetic group.

The action of inhibitors can be studied best with the use
of isolated enzymes; nevertheless, actively growing cells can
be used although the results must be interpreted with caution.
In living organisms the penetration of the inhibitor and
reverser must be considered, as well as the ability of the
cells to alter these compounds. If the inhibitor is able to
affect several different processes, a single reverser may not
be effective in relieving each of these inhibitions to the
same degree. Therefore inhibition of growth may be the result
of a number of complex interactions and must be considered
accordingly.

The inhibition index pr0posed by Mollwain (1942) to
evaluate the effectiveness of a reversing agent and used ex-
tensively by Shive (1950) to trace metabolic pathways is the
ratio of the inhibitor concentration to the reverser concentra*
tion, both on a weight basis, at a point of marked inhibition
or half-maximum growth. The inhibition index the writer used
is based on the difference in inhibitor concentration between

the control value and that obtained in the presence of a

reverser, both at a point of half-maximum growth, and is the
ratio of this value to the concentration of reverser; it is
not based on merely a total amount of inhibitor. This
corrects for the amount of metabolite which may be synthesized
within the cell (Woolley, 1952) and also corrects for the
variations obtained in the control tubes from one experiment to
the next. It is also based on a concentration expressed in
moles rather than in grams. In other words, the inhibition
index used here indicates the number of molecules of serine
that are reversed, which is more meaningful for intact cells
than a simple inhibitor/reverser ratio based on weight alone.

An inhibition may be reversed competitively or non-
competitively. If competitive, the inhibition index will
remain constant over a large range of concentrations of
inhibitor and reverser, but if non-competitive, the index is
not constant but will change as the concentrations of inhibitor
and reverser are changed. Perhaps the most widely known
worker in the field of inhibition analysis is Shive (1950) who
has been able to trace several steps in a series of metabolic
transformations by studying competitive inhibitions.

The conclusion that a substance which overcomes an inhibi-
tion may be directly or indirectly involved in a blocked
reaction is not necessarily correct in each case, as has been
pointed out by Davis (1956). He showed that while valine and

isoleucine could relieve the inhibition of growth of E. coli

-31-

caused by homoserine, these amino acids acted not by enabling
an inhibited reaction sequence to proceed but by preventing
the penetration of homoserine.

It has also been pointed out by Cohen and honod (1957)
that in the competitive reversal of norleucine inhibition by
methionine in the growth of E: 22213 norleucine does not
inhibit the utilization of methionine, nor does norleucine
inhibit a possible function of methionine in the biosynthesis
of valine, leucine or isoleucine as had been postulated.
Instead, norleucine has been found to substitute for methionine,
resulting in the synthesis of "false proteins," but norleucine
does not impair the synthesis of methionine.

Inhibitory action.gf.D-serine. The D isomer of DL-serine

 

inhibits g, gallinarum by reducing the growth rate, and indirect
evidence indicates that the degree of reduction is dependent on
the concentration of DL-serine used. Serine does not reduce

the amount of total growth.

Although only broad generalizations can be made from the
limited growth curve data, it appears that the serine“
inhibited culture, shown in figure 2, page 11, grows at a
fairly constant rate. The apparent lag following an initial
growth of about 2 generations may reflect the actual growth
pattern of the serine-inhibited cultures. 0n the other hand,
errors in sampling may have resulted in high cell counts

obtained at 12 hours, in which case the serine‘inhibited

cultures would continue to grow logarithmically from the time
of inoculation. The latter interpretation is the simpler
one. If further experiments showed that a lag period does
exist after approximately 12 hours, it may result from the
cells having used up some product of a serine-inhibited
reaction, after which inhibition by the D isomer would be
manifested by a period of negligible growth.

The delay of reversal activity in the presence of uridine,
also shown in figure 2, suggests that uridine may be con-
verted to some other compound before complete reversal occurs.
Since growth curves were not determined for other compounds,
no conclusions can be drawn concerning the direct effects on
growth of compounds other than uridine.

Because none of the constituents of the synthetic medium
in the concentrations normally present have any ability to
relieve serine inhibition, serine does not appear to inhibit
growth by preventing the incorporation of an essential nutrient.
Although it is not known if D‘serine is actually taken up by
the cells, indirect evidence indicates that any interference
it has with metabolism occurs within the boundaries of the cell.
It has been shown that the inhibitory effect of DL-serine is
due to the D isomer. But because the organism does not re-
quire an exogenous supply of Lrserine, D-serine would not
inhibit by interfering with the uptake of L’serine but could

possibly interfere with the endogenous utilization of its

isomer or some other compound synthesized within the cell.
D'serine does not interfere with the utilization of L-serine
in Pasteurella pestis (Levine, 1954). D’serine could be
substituting for its L isomer in a reaction, for example where
serine acts as a one-carbon donor through folic acid.

Composition 23 the pynthetic medium. In the study of the

 

 

organic compounds, inorganic nitrogen, and bicarbonate require-

ments of §, gallinarum and their effects on serine inhibition

it was found that concentrations of the individual compounds
in the medium could be varied over a wide range, and that some
of the concentrations of compounds in the original medium were
not Optimum. Since it was intended merely to show that the

medium itself was not masking serine inhibition, the medium

was not modified for further work. In order to reduce the tins
required for maximum growth, and possibly to obtain a greater
amount of growth, it is suggested that in further studies the
amount of cystine be increased two- to four’fold and the
amounts of inorganic nitrogen compounds be halved. The medium
could be simplified by the omission of thiamine and calcium
pantothenate if a large inoculum is used.

Relationship of serine inhibition to amino acid metabolism.

 

Since D-serine is an amino acid it might be interfering with
the metabolism of other amino acids. Serine has been found to
be present in the cell walls of §: pullorum along with 15 other

amino acids including glutamic acid, aspartic acid, proline,

-34..

arginine, and methionine; asparagine and glutamine were not
present (Salton, 1952). Several of these compounds are inter-
convertible by reactions involving transamination and anddation
In the urea cycle citrulline and aspartic acid combine to
form arginosuccinic acid from which arginine is derived; the
hydrolysis of arginine yields ornithine and urea. Aspartic
acid is also a precursor of carbamylaspartio acid from which
pyrimidines are synthesized, and it is involved in purine
synthesis along with the bicarbonate ion. Since arginine is

required for the growth ofJS. gallinarum but was not demonstra-

 

ted to reverse serine inhibition, nor was ornithine, the
function of urea in relieving serine inhibition does not seem
to involve this cycle if it is operative in this organism.

Ferguson and Hook (1943) tested 75 strains of Salmonella

 

including 1 strain of Salmonella gallinarum and found none of

 

them to have urease activity, although they suggest that it
would be not at all surprising to find a variant in view of

the fact that some Salmonella organisms digress from the

 

typical metabolic reactions. It is possible that strain 6 of
§fggallinarum used here possesses a small amount of urease
activity, undetectable by the usual method, which can hydrolyze
urea to ammonia and C02, both of these compounds then being
available for other reactions. The ammonia may be incorporated
into glutamic acid or aspartic acid or, a more interesting

speculation, it might recombine with C0 in the presence of

2

ATP to form carbamyl phosphate required in pyrimidine synthesis.
Carbamyl phosphate may also be formed from free amino groups
derived from glutamine, asparagine, and amino acids. Glutamic
acid also functions as a constituent of the folic acid co-
enzymes which are required in purine synthesis.

The methyl carbon of methionine is probably derived from
the,phydroxymethyl group of serine in E, ggli_(Gibson, 1952).
The role of methionine in nucleic acid metabolism is not
clear, but it is known to function as a methyl donor to folic
acid enzymes and is able to relieve inhibitions of folic acid
and RNA synthesis in.§, ggli and other microorganisms. Some

interesting interrelations of methionine, serine, and CO are

2

found in.§b coli and Streptococcus bovis. Gibson (1952)

 

reported that methionine synthesis in.§b coli was inhibited by

a 20% concentration of CO and was reversed by DL-serine,

2
while Prescott, Ragland, and Stutts (1957) found that DL-serine
inhibition in §, bggig_was reversed by a 0.0002% concentration
of 002 supplied as NaHCQS.

In some experiments it was found that low concentrations
of DLPserine, from 5 to 25 ug/ml, occasionally stimulated
growth. This type of a stimulatory effect has been reported
to occur with low concentrations of many inhibitors. Some
bacteria are able to utilize Dramino acids, many of which

occur naturally in bacterial cells, and it has been postulated

by Work (1957) that these D-amino acids may function in the

osmotic barrier of the cell. é: gallinarum may possess a

 

limited ability to racemize or oxidatively deaminate D-serine
to its L isomer, although no reports to this effect have been
found.

Inhibition §y_antibiotics containing serine. In conneC”
tion with serine inhibition it is interesting to note that two

serine‘containing antibiotics produced by Streptomyces sp.

 

inhibit the growth of §: gallinarum. Azaserine, an L-serine

 

containing analog of L-glutamine, was highly active, while
D-cycloserine was also inhibitory. O’Carbamyl-L-serine is
also an analog of L-glutamine but was not available for
testing; however, its D isomer, O‘carbamyl-D'serine which is

also produced by Streptpmyces was found not to be inhibitory

 

at a concentration of 250 ug/ml. Azaserine and 0-carbamyl-L-
serine have been shown in other bacteria to interfere with a
step in purine synthesis where glutamine acts as an amino donor.

Reversal of inhibition by nucleic acid derivatives. The

 

fact that the nucleic acid derivatives were more active in
relieving serine inhibition than any other compounds tested
would seem to indicate that serine interferes, directly or
indirectly, with nucleic acid metabolism.‘ In each case the
nucleoside was more active than the cornmmonding nucleotide or
free base in relieving inhibition, and the adenine, guanine,
and cytosine ribosides were more active than the corresponding

deoxyribosides.

It is not known whether or not these compounds must enter
the cell to be active in relieving serine inhibition, but
Cohen and Earner (1956) found that free pyrimidines and
pyrimidine ribosides could be incorporated by g: £2li_mutants
requiring thymine. The fact that adenine sulfate is not only

inhibitory to S, gallinarum but potentiates serine inhibition

 

indicates that this compound is either able to penetrate into
the cells or it interferes with an extracellular reaction,
possibly the incorporation of some other substance into the
cells. Since Harkins and Freiser (1958) have shown that the
acidic LH group of adenine (position 7 on the imidazole ring)
forms metal complexes with the divalent ions copper, nickel,
and cobalt, it may be that adenine inhibits by forming chelates
with essential metals. They reported that adenosine does not
behave in quite the same manner as adenine, but it and ribose
can also react with copper. no attempts were made to reverse
the inhibitory effect of adenine by increasing the concentra‘
tion of metals in the medium. Inhibition did not occur with
the other two free bases tested, uracil and xanthine.

Since it is not known whether the nucleic acid derivatives
which reverse serine actually penetrate the cell as intact
molecules or are converted to some other active products
inside or outside the cell, no conclusions can be drawn con‘
cerning the mechanism by which these compounds reverse the

inhibition.

-38-

Because both precursors and products of a blocked reaction
may relieve an inhibition non-competitively, and the compounds
considered here were non-competitive, these compounds may be
either precursors or products of a possible blocked reaction
or may be involved in more than one reaction. It is also
possible that they are not related to a blocked reaction.

The inhibition indices are higher for most of the nucleic
acid derivatives than for the amino acids or amino acid amides
tested, many of which are known to function in nucleic acid
synthesis. From this one could conclude that the nucleic acid
derivatives are either more closely associated with the
blocked reaction(s) than the amino acids or their amides, or
that they penetrate the cells more easily. Figure # illustrates
the origins of carbon and nitrogen in the structures of the
purine and pyrimidine rings and points out the importance of
the amino acids discussed here in relation to nucleic acid
synthesis.

Because the inhibition indices were obtained using growing
cells and the compounds did not relieve serine inhibition
competitively, further conclusions regarding at what step(s)
serine inhibits are not warranted, and one can only speculate
beyond this point.

This discussion indicates that there are a number of
complex interactions which may involve serine and other com-
pounds. However, in a study of this sort where the growth of

cells is the sole criterion to evaluate the effect of a number

Purine

 

 

I
\ .~ -
\ HCO3 x
\ C I
:7/ \4 glycine /,/
\ .
aspartic acid N \ :\C II //
- "H” -" _' "’ II ”‘71 I /+ folic acid,
C of serine C ” - C /\ C possibly
/\ y/X /\ C of serine
’ \.
/ N i N \
/l . | . ‘\
glutamine . glutamine
Eyrimidine
,'/"\
free RH} N i C
......— _ .__ 4'] I aspartic acid
free CO

2 “M

I

Figure #. Origin of carbon and nitrOgen in purine and
pyrimidine rings.

-1“)-

of different kinds of compounds, the results are entirely de‘
pendent on the ability of the organisms toutilize in some
manner the compounds supplied. The inhibition index as a tool
in evaluating the activities of amino acids and nucleic acid
derivatives is limited, and the values are only relative and
subject to change depending on the conditions used. They show
only the growth response to a particular compound in the
presence of serine, and since this response is dependent on
permeability of the compounds before or after any possible
conversion that may occur, the growth response may reflect only
the degree of permeability rather than the "true" activity of a
particular compound. For example, assuming equal penetrability,
uridylic acid might be incorporated into RNA much more easily
than uridine, but since in this study these compounds were
supplied exogenously, the organisms seem to be able to take up
uridine more readily than the phosphorylated compound, and
hence the riboside appears to have the greater activity.

A number of possible areas for the interference of serine
with metabolic processes have been suggested, but cell-free
extracts will probably have to be used in order to confirm or
deny these hypotheses. It is hOped that these experiments

will stimulate further research on this problem.

-41..

SUMMARY

DIrSerine inhibits the growth of Salmonella gallinarum.in
a synthetic medium by reducing the growth rate. The inhibition,
due to the D isomer, is temporary and is reversed by nucleic
acid derivatives, particularly uridine, adenosine and cytidine,
and amino acids, particularly glutamic acid and aspartic acid.
DIrSerine inhibition is also reversed by L-serine, KHCC5 and
RNA, while adenine sulfate is inhibitory; DNA is inhibitory
alone but is capable of reversing low concentrations of serine.
It is suggested that D’serine interferes with the metabolism
of several amino acids, particularly where they are required
for nucleic acid synthesis.

This organism is also inhibited by azaserine and D‘cyclo-
serine but not by O'carbamyl'D*serine.

Some miscellaneous observations on the nutrition of’§:

gallinarum and the effect of varying the concentrations of the

 

constituents of the synthetic medium have been noted.

-42..

BIBLIOGRAPHY

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Breed, R. 5., Murray, E. G. D., and Smith, N. R. 1957
Bergey's manual gf_determinative bacteriology, 7th ed.
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Castellani, A. G. 1953 Inhibiting effects of amino acids and
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195'199-

Cohen, G. N. and Monod, J. 1957 Bacterial permeases.
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Cohen, S. S. and Earner, H. D. 1956 Studies on the induction
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Dagley, S., Dawes, E. A., and Morrison, G. H.]£¥fihibition of
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Davis, F. and Solowey, M. 1950 The utilization of some organic
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Bacteriol, 22, 361-566.

 

Dubos, R. J. 1949 Toxic effects of DL—serine on virulent
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Ferguson, W. N. and Book, A. E. 19%} urease activity of
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Friedman, M. 5., Wood, W. A., and helson, W. O. 1953 The
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BaCteriOIQ §_6_' 568—5710

 

Gibson, F. 1952 Methionine synthesis by Escherichia coli.
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Gilfillan, Re Fe, BOltman, Dc Fe, and R085, Re To 1955 A
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Gladstone, G. P. 1959 Inter-relationships between amino acids
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Hajna, A. A. 1935 Decomposition of salts of organic acids by
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Harkins, T. R. and Freiser, H. 1958 Adenine-metal complexes.
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Johnson, E. A. and Rettger, L. F. 1943 A comparative study of
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Bacteriol.,Eé, 127-135.

 

 

 

Jones, A. W. and Holtman, D. F. 1953 Synthesis of glutamic
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Lederberg, J. 1947 The nutrition of Salmonella. Arch.
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- 4g -

Levine, H. B., Heimberg, R., Dowling, J. H., Bvenson, Ln,
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Maas, W. K. and Davis, B. D. 1950 Pantothenate studies. I.
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thlwain, H. 1942 Bacterial inhibition by metabolite
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Effects of carbon dioxide on the growth of Streptococcus
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-hs-

Shive, W. 1950 The ufllization of antimetabolites in the study
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Nature, 179, 841-897.

- 46 -

APPENDIX

TABLE 13

Effect of uridine and serine on growth of Salmonella gallinarum

 

 

Time in log Turbidity in
hours cells/m1 BaSQu units
Control 0 5.88 O
16 2
18 7.54 5
21 7.94 7
2# 8.2# 10
#8 9.28 >10
60 >10
DIrSerine #00 ug/ml 12 6.56 0
16 0
18 6.98 0
21 6.56 o
29 6.62 0
36 6.83 1
#8 7.0 l
60 7.67 5
Uridine 62.5 ug/ml 12 7.20 O
16 2
18 7.73 5
21 7.90 7
24 8.18 10
36 8.32 >10
#8 9.29 >10
60 >10
DL-Serine 400 ug/ml 12 6.58 0
+ Uridine 62.5 ug/ml 16 0
18 6.63 0
21 6.59 O
29 6.69 O
36 6.54 1
A8 7.75 5
60 9.11 >10

-14}?—

Effect of serine isomers

15 Hours

Isomer
DL
L
D
D+L

18 Hours
Isomer
DL
L
D
D+L

24 Hours
Isomer

28 Hburs
Isomer
DL-

L
D

D+L

41 Hours
Isomer
DL
L.
D
D+L

64 Hours
Isomer
DL.
L.
D
D+L

TABLE 14

on growth of Salmonella gallinarum

 

 

 

 

 

 

 

 

 

 

 

 

 

Total serine ug/ml
290 1600 800 #00 200 100 50 1+0 30 20 10 5
O 0 0 0 0 0 0 l 3 5
O O l l 3 5 5 5 6 6 6 6
0 0 0 0 0 0 0 0 0 O
O O O 0 0 0 l 3
Control: 6
Total serine ug/ml
3200 1600 800 400 200 100 50 40 30 20 10 5
0 0 0 0 0 0 1 3 5 7
0 0 O 3 5 7 7 8 8 8 8 8
O 0 0 0 O O 0 0 O 1
O O 0 O 0 0 3 5
Control: 8
Total serine ug/ml
3200 1600 800 400 200 100 .59 40 30 20 10 <5
0 0 O l 3 5 6 7 9 10
0 1. 4 7 10 >10 )10 )10 >10 710 >10 >10
0 0 0 0 0 0 0 0 l 4
0 0 O O l 5 47 49
Control: >10
Total serine ug/ml
3200 1600 800 400 200 100 50 40 30 20 10 5
O 0 0 1 7 9 10 10 >10 >10
0 5 9 10 >10 >10 >10 >10 >10 710 >10 >10
0 0 0 0 O 0 0 0 3 9
0 0 0 0 1 _9_ 10 >10
Control: >10
Total serine ug/ml
3290 1600 800 400<_§90 100 50 40 ‘30 20 10 5
0 0 0 3 >10 >10 710 710 710 >10
0 >10 710 >10 >10 >10 >10 >10 >10 710 >10 710
0 0 0 O 0 0 0 0 10 >10
0 0 O 0 10 >10 710 710
Control: >10
Total.serine ug/ml
3200 1600 800 400 200 100 #50 40 30 20 10 _5
>10 >10 >10 >10 >10 >10 >10 210 >10 >10
‘>10 .>10 >10 >10 >10 >10 >10 >10 710 >10 >10 >10
10 10 >10 >10 9 9 9 >10 >10 >10
>10 >10 >10 >10 >10 )10 >10 >10
Control: >10

- 48 -

TABLE 15

Effect of omitting organic and inorganic constituents of
synthetic medium on the growth of Salmonella gallinarum

 

Compound omitted Number of times transferred

1 2 3 A 5 6 7

 

IrLeucine 210 >10 >10 .>10 >10 10 0

L-Cystine 7 0 0

L-Arginine >10 710 >10 >10 >10 >10 9
Thiamine >10 7 10 $10 >10 710 7- 10 7 10
Pantothenic acid 710 >10 >10 >10 >10 >10 .710
Arginine + Leucine >10 >10 10 10 10 7 10
Pantothenic acid
4- Thiamine >10 >10 > 10 > 10 > 10 > 10 >10
Inorganic nitrogen 210 >10 10 10 10 5
Glucose 10 1 0
Control -
nothing omitted 710 .>10 >10 >10 >10 >10 '710

 

Turbidity in BaSQh units at 24 hour intervals, except for tube 7

TrgfiséérgongZ drops to each successive tube except from tube 6
to tube 7 when 2 drops of a 1:1000 dilution was transferred

-49...

TABLE 16

Effect of variation of arginine concentration on growth and
serine inhibition of galmonella gallinarum

 

 

 

 

gm DL-Serine ug/ml
100 50 0
L-Arginine ug/ml 160 5 5
8O 0 5 5
#0 O 5 5
20 0 6 5
10 O 5 5
0 5 5
2 O 9 9
0 O 3 1+
#8 Hours DIrSerine ug/ml
100 50 O
L-Arginine ug/ml 160 >10 >10
80 9 >10 >10
1,0 9 >10 >10
20 9 >10 >10
10 9 ‘10 >10
9 >10 >10
2 7 >10 >10
6 10 >10

 

67 Kauai

All tube. >10

-50—

TABLE 17

Effect of variation of histidine concentration on growth and
serine inhibition of Salmonella gallinarum

 

 

 

 

#2 Hours DL—Serine ug/ml
100 50 0
L'Histidine ug/ml 160 O 3 5
8O 0 3 6
40 O 9 6
2O 0 3 7
10 O 3 6
4 0 3 5
2 0 3 5
0 O O 4
48 Hours DL-Serine ug/ml
100 50 0
L-Histidine ug/ml 160 7 10 > 10
80 7 10 >10
40 9 10 710
20 7 10 >10
10 7 10 >10
# 7 10 710
2 7 10 >10
0 6 10 710

 

62 Hours

All tubes >10

-51-

TABLE 18

Effect of variation of cystine concentration on growth and
serine inhibition of Salmonella gallinarum

 

 

 

 

 

 

 

#2 Hours DIr-Serine ug/ml 67 Hours DIr-Serine ug/ml
100 50 o 100 50 o

L-Cystine ug/ml 2100 O 0 >10 Cystine ug/ml 240 3 10 >10
120 o o 7 120 5 >10 >10

60 o o 5 60 5 ,10 710

30 0 0 4 5o 5 >10 710

15 o o 5 15 5 ,10 710

6 o o 3 6 o 10 >10

3 o o 3 3 o 5 710

o o o o o o o o

48 Hours DLPSerine ug/ml 96 Hours DIrSerine ug/ml
100 50 o _ 100 50 o

IrCystine ug/ml 260 0 0 >10 cystine ug/ml 240 >10 >10 >10
120 0 0 >10 120 >10 > 10 7 10

60 o o 710 60 >10 >10 »10

30 0 0 10 50 >10 >10 710

15 o o 9 15 >10 ~r10 710

6 0 0 9 6 710 ->10 >10

3 O 0 9 3 710 'rlO 710

o o o o o o o o

 

 

TABLE 19

Effect of variation of leucine concentration on growth and
serine inhibition of galmonella_gallinarum

 

 

 

 

 

 

 

#2 Hours DIrSerine ug/ml 67 Hours DL-Serine ug/ml
100 50 O 100 50 O

IPLeucine 560 o o 5 leucine 560 5 >10 >10
“E/ml 180 o o 6 “g/ml 180 4 >10 >10
90 O 0 6 9O 5 >10 >10

#5 O O 6 #5 3 >10 >10

22.5 0 O 6 22.5 0 5 >10

9 O 0 ' 5 9 0 3 710

#.5 O O 5 #.5 0 0 710

o o o o o o o 710

#8 Hours DIrSerine ug/ml 96 Hours DLrSerine ug/ml
100 50 O 100 50 O

IrLeucine 360 0 1 >10 Leucine 560 >10 >10 >10
ug/ml 180 0 3 710 ”Lg/ml 180 >10 >10 >10

90 O 3 >10 90 >10 >10 >10

#5 O O 710 #5 >10 >10 >10

22.5 0 0 >10 22.5 >10 ’10 >10

9 o o 710 9 >10 >10 >10

4.5 o 0 >10 4.5 0 >10 >10

0 O O 3 O O O 710

 

 

TABLE 20

Effect of variation of glucose concentration on growth and
serine inhibition of Salmonella gallinarum

 

 

 

 

 

 

 

#2 Hours DLrSerine ug/ml 62 Hours DIrSerine ug/ml
100 50 .0 100 50 0

D-Glucose #0 0 0 3 Glucose #0 0 0 >10
“3/“ 20 o o 6 “3/ ml 20 o 0 >10
10 0 0 5 10 0 0 >10

5 0 O 5 5 O 0 710

2.5 o o 5 2.5 o o 510

l 0 0 5 0 0 >10

0.5 0 0 5 0.5 0 0 >10

0 0 0 1 0 0 0 5

#8 Hours DLrSerine ug/ml 96 Hours DL-Serine ug/ml
100 50 0 100 50 0

D-Glucose #0 0 0 10 Glucose #0 >10 >10 >10
"lg/ml 20 o 0 >10 mg/ ml 20 >10 >10 >10
10 0 0 >10 10 O 1 >10

5 0 0 >10 5 0 0 ‘>10

2.5 0 0 >10 2.5 0 0 >10

1 0 0 10 1 0 0 710

0.5 0 0 10 0.5 0 0 >10

0 0 0 5 0 0 0 3

 

 

-51‘"-

TABLE 21

Effect of variation of inorganic nitrogen concentration on growth
and serine inhibition of Salmonella gallinarum

 

 

 

 

 

§l_§22££, DL-Serine ug/ml
NH#01 NHuNO'3 100 50 0
20 mg/ml # mg/ml 0 0 O
10 mg/ml 2 mg/ml o o 5
5 Ins/ml l Ins/ml 0 3 5
2.5 mg/ml 0.5 mg/ml 0 3 7
1.25mg/ml 0.25mg/ml 0 3 7
625 ug/ml 125 ug/ml o 5 7
312.5 ug/ml 62.5 ug/ml 0 3 7
0 0 0 0 5
Q2 Hours . DLrSerine ug/ml
NHhCl NHnN03 100 50 0
20 2113/1111 4 mg/ml 5 7 >10
10 mg/ml 2 mg/ml 6 >10 >10
5 mg/ml 1 mg/ml >10 >10 ‘>10
2.5 nag/ml 0.5 rag/ml >10 >10 >10
1. 25mg/ml O. 25mg/ml >10 >10 >10
625 ug/ml 125 ug/ml ‘710 710 >10
312.5 ug/ml 62.5 ug/ml >10 >10 >10
0 0 >10 >10 >10
96 Heurs

A11 tubes >10

TABLE 22

Effect of variation of bicarbonate concentration on growth and
serine inhibition of §§lmonellg_gallinarum

 

 

 

 

21 Hours DL-Serine ug/ml
100 50 O

K11003 rug/ml 8 o o 5
# 0 1 5

2 O 1 7

1 o 1 7

0.5 O 1 7

0.25 O 1 7

O. l O l 7

O 0 l 7

55 Hours DIrSerine ug/ml
100 50 O

KHco3 mg/n1 8 o o 5
# o 1 >10

2 5 >10 >10

1 >10 >10 >10

0.5 >10 >10 >10

0.25 >10 >10 >10

0.1 >10 >10 >10

0 5 7 10

 

-56-

TABLE 23

Effect of glycine and serine on growth of galmonella gallinarum

 

 

 

 

 

 

2# Hours DL-Serine ug/ml
#00 200 100 50 0
Glycine mg/ml # 0 0 0 O 0
2 0 0 0 0 5
1 O o O 3 5
0 0 0 0 5 . 5

58 Hours DIr-Serine ug/ml
#00 200 100 50 0
Glycine mg/ml # 0 0 0 0 6
2 0 0 0 8 10
1 0 0 0 10 >10
0 0 0 10 >10 >10

#8 Hours 1 » DL'Serine ug/ml
_ ' #00 200 100 50 0
Glycine mg/ml # 0 0 0 0 10
' 2 o o o 510 >10
1 0 0 ‘ 0 >10 >10
0 0 0 >10 >10 )10

 

TABLE 2#

Effect of glycine, DL-dalanine and low concentrations of DL-serine
on growth of §, gallinarum

 

DL-Serine ug/ml

 

22M 20 10 7.5 5 2.5 1.25 0.625 0
Glycine 1000 1 2 5 4 5 5 5 5
“g/ml 100 2 3 3 3 3 3 3 3
10 2 3 3 3 3 3 3 3

2 3 3 3 3 3 3 3

o 2 3 3 3 3 3 3 3

 

DLrSerine ug/ml

 

 

20 10 7.5 5 2.5 1n25 0.625 0
DLnxalanine 1000 1 1 1 1 1 1 1 1
ug/ml 100' 2 2 2 2 2 1 1 1
10 2 3 3 3 3 3 3 3
l 2 3 3 5 5 5 5 5
2 3 3 5 5 5 5 5

.fll Hours All tubes >10

-58..

TABLE 25

Effect of uridine and serine on growth of Salmonella gallinarum

 

 

 

 

 

 

 

12 Hours Dir-Serine ug/ml
800 #00 200 100 50 25 0
Uridine ug/ml 100 # # # 5 6 7 6
50 0 l # 5 7 7 6
25 0 0 2 # 6 7 6
10 0 0 0 0 3 7 5
l 0 0 0 0 0 5 5
0 0 0 0 0 0 1 5

26 Hours DL‘ Serine ug/ ml
800 #00 200 100 50 25 O
Uri dine ug/ml 100 > 10 >10 >10 >10 >10 >10 >10
50 0 0 >10 >10 710 >10 71.0
25 0 0 >10 >10 7 10 >10 >10
10 0 6 6 7 710 710 710
1 0 0 0 ~ 0 7 >10 > 10
0 0 0 0 0 0 6 >10

#0 Hours DI:- Beri ne ug/ ml
800 #00 200 100 50 25 0
Uridine ug/ ml 100 210 >10 >10 >10 >10 >10 >10
50 0 0 710 7 10 7 10 > 10 >10
25 0 0 ‘710 >10 710 >10 >10
10 O 710 >10 >10 710 >10 710
l 0 0 0 ‘710 > 10 7 10 710
0 0 0 0 2 710 ‘7 10 >10

m Growth in all tubes

TABLE 26

Effect of adenosine and serine on growth of Salmonella gallinarum

 

 

 

18 Hours DL-Serine ug/ml
800 #00 200 100 50 25 0
Adenosine ug/ml 100 0 2 2 # 7 10 9
50 0 0 l 3 7 9 7
25 0 0 l 3 '7 7 7
10 o 0 o 3 7 7 7
1 0 0 0 0 1+ 1+ 7
0 0 0 0 0 1 # 5
2# Hours A DIrSerine ug/ml

800 #00 200 100 50 25 0

 

 

 

Adenosine u g/ ml 100 O 1 7 >10 3 10 >10 > 10
50 0 l 8 >10 ‘710 .>10 >10
25 0 0 # 10 >10 >10 710
10 0 - 0 3 9 >10 >10 >10
1 0 0 0 # 9 >10 >10
0 0 0 0 0 7 10 >10
#8 Hours DL' Serine ug/ m1
800 #00 200 100 50 25 0
Adenosine ug/ ml 100 710 210 >10 710 210 > 10 >10

50 >10 1>10 710 >10 >10 >10 >10
25 710 210 710 710 710 710 710
10 >10 >10 >10 >10 710 ~"1.0 710
l. 5 # 710 710 >10 >10 >10
0 0 0 5 >10 >10 710 >10

 

-60..

TABLE 27

Effect of cytidine and serine on growth of Salmonella gallinarum

 

 

 

 

 

 

 

17 Hours DLrSerine ug/ml
800 #00 200 100 50 25 0
Cytidine ug/ml 100 0 0 l # 7 7 6
50 0 0 0 3 5 7 5
25 0 0 0 1 3 7 5
10 0 0 0 0 5 5 5
l O 0 0 0 0 5 5
0 0 0 0 O 0 l 5
26 Hogs DL‘Serine ug/ml
800 #00 200 100 50 25 0
Cytidine ug/ml 100 0 O 2 710 710 >10 >10
50 0 0 10 710 >10 >10 >10
25 0 0 . 0 10 710 710 710
10 0 0 0 7 710 >10 >10
1 0 0 0 2 7 >10 710
0 0 0 0 0 2 6 710
W DL-Serine ug/ml
800 #00 200 100 50 25 O
Cytidine ug/ml 100 0 0 >10 310 >10 >10 >10
50 0 ‘310 '210 >10 310 >10 >10
25 0 2 0 >10 710 >10 710
10 0 0 210 >10 >10 710 >10
1 0 0 0 310 >10 >10 710 2
0 0 0 0 8 710 710 710
22 Hours Growth in all tubes except tube in

column 3, row 3

-61...

TABLE 28

Effect of cytidylic acid and serine on growth of
Salmonella gallinarum

17 Hours

Cytidvlic acid
ug/ml

26 Hours

Cytidylic aci d
ug/ml

#0 Hours

Cytidylic aci d

22 Hours

 

DL‘Seri ne ug/ml

 

 

 

 

 

 

800 #00 200 100 50 25 0

100 0 0 0 5 5 6 3

50 0 0 0 3 5 5 5

25 0 0 0 3 5 5 5

10 0 0 0 0 5 5 5

l 0 0 0 0 0 5 5

0 0 0 0 0 0 l 5

DL-Serine ug/ml

800 #00 200 100 50 25 O

100 710 0 5 >10 >10 7 10 710

50 _ 0 o 0 .10 >10 >10 210

25 0 0 O > 10 >10 >10 ’ 10

10 0 0 0 5 7 10 ’10 ’ 10

1 0 0 0 0 6 10 7 10

0 0 0 0 0 0 6 7 10

DL- Serine ug/ml

800 #00 200 100 50 25 0

100 710 0 > 10 710 )10 >10 > 10

50 710 0 5 >10 >10 >10 ’ 10

25 0 5 5 >10 710 >10 >10

10 0 0 0 >10 >10 71.0 > 10

1 O 0 0 10 710 >10 > 10

0 0 0 O 2 710 710 7 10
Growth in all tubes

-62..

TABLE 29

Effect of uridylic acid and serine on growth of
Salmonella gallinarum

 

 

 

12 Hours DL-Serine ug/ml
800 #00 200 100 50 25 O
Uridylic acid 100 0 O O O 5 6 5
“E/ml 50 o o o o 5 5 5
25 o o o o 5 5 5
10 o o o 0 3 5 5
1 o o o o 0 5 5
0 0 O O O O 1 5
26 Hours DL’Serine ug/ml

800 400 200 100 50 25 O

 

 

 

 

Uridylic acid 100 o o 1 10 >10 >10 >10
“8/ “1 5o 0 o o 10 >10 >10 >10
25 o o 6 6 >10 >10 >10
10 o o o 0 >10 >10 >10
1 o o o o 6 10 >10
0 o o o o o 6 :a0
#0 Hours DIrSerine ug/ml
800 #00 200 100 50 25 o
uridylic acid 100 O O 5 >10 >10 >10 >10 A
“E/M1 50 o o 2 >10 >10 >10 >10
25 0 0 >10 >10 >10 >10 >10
10 o o o 10 >10 >10 >10
1 o o o 10 >10 710 710
o o o o 2 >10 >10 >10
22 Hours Growth in all tubes

-63...

TABLE 50

Effect of adenylic acid and serine on growth of
Salm0ne 1.1a gallinarum

18 Hours

Ade nylic aci d
ug/ ml

24 Hours

Adenylic aci d
u g/ml

1+8 Hours

Adenylic acid

100
50
25
10

100
50
25
10

100
50

25
10

DL‘Serine ug/ml

 

 

 

 

 

800 400 200 100 50 25 O
O O 2 1+ 7 10 ' 8
O O l 5 7 9 8
o o o 2 5 9 7
O O O l 5 9 7
O O O O 5 ’+ 5
O O O 0 l l} 5
DL~Serine u g/ml
800 400 ‘ 200 100 50 25 o
O l 1+ >10 7 10 710 710
o 1 9 >10 710 710 710
0 O 9 10 510 710 710
0 0 3 7 >10 >10 >10
0 O O 3 9 > 10 7 10
O 0 O O 7 10 >10
DL-Seri no ug/ ml
800 #00 200 100 50 25 0
710 > 10 710 >10 >10 >10 710
710 710 710 710 > 10 >10 7 10
>10 710 710 710 210 210 710
7 7 >10 710 >10 21.0 710
O O 5 ?10 7 10 210 7 10
O O 2 710 710 710 7 10

 

-64.-

TABLE 51

Effect of deoxycytidine and serine on growth of fialmonella

 

 

 

 

gallinarum
22 Hours DL‘Serine ug/ml
800 400 200 100 50 25 0
Deoxycytidine ug/ml 100 O O O 5 10 10 9
5O 0 O 0 5 9 9 7
25 O O O O 9 9 6
10 o o o o 7 7 5
1 o o o o 5 6 5
O O O O O 0 5 5
28 Hours DL‘ Serine ug/ml

800 #00 200 100 50 25 O

 

 

Deoxycytidins ug/ml 100 O O 0 7710 >lO‘>10 510
50 O O O 8 >10 >10 ’10
25 O 0 O 5 )10 >10 >10
10 O O O 0 >10 >10 10
l O O O O 10 >10 10
O O O O O 6 10 10
1+2 Hours DL‘Serine ug/ml

800 #00 200 100 50 25 O

 

 

Deoxycytidine ug/ml 100 0 O 0 >10 .>10 '>10 >10
50 O O O 710 >10 '710 ‘le
25 0 O 0 >10 >10 ‘710 710
10 O O O 0 >10 >10 710
l O 0 O 0 >10 '710 710
o o o o o 710 >10 >10
66 Hours Growth in all tubes

-65..

TABLE 52

Effect of thymidine and serine on growth of Salmonella_gallinarum

 

 

 

22 Hours D L~Serine ug/ml
800 l+00 200 100 50 25 O
Thymidine ug/ml 100 O O 0 4 9 9 6
5O 0 O O 5 8 8 6
25 O 0 O 3 7 8 6
10 0 O O O 1+ 7 5
l O O O O 3 5 5
O 0 O O O 0 5 5

28 Hours DIrSerine ug/ml

800 400 200 100 50 25 o

 

 

Thymidine ug/ml 100 o o o 9 >10 >10 >10
50 O O O 8 >10 >10 71.0
25 0 O O 8 >10 >10 >11.0
10 O O O O 10 >10 10
l 0 O O 0 8 >10 10
O O O O O 6 10 10
42 Hours DLrSerine ug/ml

800 l+00 200 100 50 25 O

 

 

Thymidine ug/ml 100 o o 0 >10 ;10 >10 710
50 0 O 0 >10 >10 >10 71.0
25 0 O 0 >10 >10 >10 71.0
10 O O O O ’10 ’10 710
l O O O 0 >10 7'10 >10
0 0 O O O > 10 3‘ 10 >10
66 Hours Growth in all tubes

..66—

TABLE 55

Effect of thymidylic acid and serine on growth of
Salmonella gallinarum

24 Hours

Thymidylic acid
ug/ml

38 Hours

Thymidylic acid
ug/ml

#8 Hours

Thymidylic acid
ug/ml

 

100
50
25

100
50
25

100
50
25

DLcSerine ug/ml

 

"M—n-‘O 46.2“.“ A..-
I
in

 

 

 

 

400 200 100 50 0

0 5 5 5

0 0 5 5 5

0 0 5 5 5

O O O 5 5
DL-Serine ug/ml

A00 200 100 50 o

8 710 710 8

0 8 710 710 710

O O ’10 710 710

o o 10 710 710
DL-Serine ug/ml

#00 200 100 50 O

5 10 > 10 > 10 510

0 >10 ’10 210 710

0 3 710 710 710

0 O >10 710 710

 

-67..

TABLE 3t+

Effect of guanosine and serine on growth of Salmonella gallinarum

 

 

 

18 Hours DL-Serine ug/ml
800 #00 200 100 50 25 0
Guanosine ug/ml 100 0 O 0 l 5 7 8
5O 0 O O l 4 7 7
25 O O O l 4 5 5
10 o o o o 1+ 5 5
1. o o o o 2 h 5
0 O O O O l ‘0» 5
2# Hours DL‘Serine ug/ml

800 l+00 200 100 50 25 O

 

 

Guanosine u g/ ml 100 O O O 1+ 7 10 >10 710
50 O O O 4 >10 710 210
25 O 0 O l-l' 710 710 7 10
10 O O O 4 10 710 710
1 o o o o 8 10 710
0 0 0 O 0 7 10 710
1+8 Hours DL-‘Serine u g/ ml

800 [+00 200 100 50 25 O

 

Guanosine u g/ ml 100 5 5 10 710 7 lO 7 10 710
50' 5 5 5 710 > 10 710 7 10

25 5 5 5 7 10 >10 7 10 >10

10 2 5 710 710 7 10 7 10 710

1 O O 1 >10 7 10 >10 >10

0 0 O 0 5 7 10 >10 7 10

 

-68-

w‘...‘ —---I v.1. '. 'r l A 0 II»
I.

TABLE 55

Effect of guanylic acid and serine on growth of
Salmonella gallinarum

 

 

 

 

 

18 Hours DL-Serine ug/ml
800 l+00 200 100 50 25 O
Guanylic aci d 100 0 O O l 5 1«I» 5
u 8/ ml 50 o o o 1 5 u 5
25 0 0 0 O 5 1+ 5
10 o o o o 3 h 5
l O O O O l 1+ 5
0 O O O O l 1+ 5
24 Hours Dir-Serine ug/ml
800 [+00 200 100 50 25 0
Guanylic acid 100 O 0 O 5 8 10 >10
'1 8/ "11 5o 0 o o 5 8 10 710
25 O O O O 7 10 710
10 O O 0 O 7 10 >10
1 O O O O 7 10 >10
0 0 0 O 0 '7 10 710
1+8 Hours DL~Serine ug/ml
800 [+00 200 100 50 25 O
Guanylic acid 100 5 5 5 710 7 10 710 710
“8/ “1 5o 5 5 10 2 10 >10 > 10 >10
25 > 10 5 10 > 10 910 710 >10
10 5 5 8 >10 '2 10 7 10 710
1 0 O 1 7 10 7 10 710 710
O 0 O O 5 7 10 “210 7 10

 

-69...

r“ -..A._~ _-' .‘_.—r. -C

TABLE 56

Effect of deoxyadenosine and serine on growth of
Salmonella‘gallinarum

 

 

 

 

 

 

 

22 Hours DLrSerine ug/ml
800 #00 200 100 50 25 o
Deoxyadenosine 100 O 0 O l 8 9 #
“3/“ 50 o o o 8 8 4
25 o o o o 7 8 1+
10 O O O 0 4 7 h
1 o o o o 3 7 5
O 0 O 0 0 O 5 5

28 Hours DL-Serine ug/ml
800 400 200 100 50 25 O
Deoxyadenosine 100 O O O 6 '>10 >10 9
“3/ ml 50 o o o 5 >10 >10 9
25 o o o 5 >10 >10 9
10 o o o o 10 >10 9
1 O O O O 9 >10 9
O 0 O 0 O 5 10 10

#2 Hours DLrSerins ug/ml
800 #00 200 100 50 25 O
Deoxyadenosine 100 O O 5 >10 >10 ;>10 >10
“3/ ml 50 o o 6 >10 >10 >10 >10
25 O O 5 >10 >10 >10 710
10 0 O 1 10 >10 >10 >10
1 O O O 1 >10 >10 710
0 O O O 0 >10 >10 >10

66 Hours Growth in all tubes

TABLE.57

Effect of deoxycytidylic acid and serine on growth of
Salmonella gallinarum

 

22 Hours

Deoxycytidylic acid 100
ug/ml 50

25

lO

1

O

28 Hours

Deo c 'd lic acid 100
xyugygly 50

25

10

1

O

42 Hours

Deoxycytidylic acid 100
ug/ml 50

25

10

l

0

66 Hours

DIrSerine ug/ml

 

 

 

 

 

800 #00 200 100 50 25 o
o o o 3 6 5

o o 4 6 5

O 0 O O k 6 5

O O 0 0 5 5 5

O O O O 5 5 5

O O O O 0 5 5

DL-Serine ug/ml

800 #00 200 100 50 25 0
O O O O 8 >10 10

O O 0 O 10 >10 10

O 0 O O 10 >10 10

O O O O 8 >10 10

0 O 0 O 8 10 10

O O O O 5 10 10

DL-Serine ug/ml

800 #00 200 100 50 25 O
o o o 0 >10 510 710

0 O O 10 >10 ‘710 >10
0 O O 0 >10 )10 710

O 0 O 0 >10 >10 )10

0 O O 0 30 >10 >10

0 O O O 710 710 710

 

Growth in all tubes

-71-

Effect of deoxyadenylic acid and serine on growth of

22 Hours

Deoxyadenylic acid
ug/ml

28 Hours

De oxyadenyli c aci 6.
ug/ m1

#2 Hours

De oxyadenylic acid
ug/ml

66 Hours

TABLE 58

Salm0ne lla gallinarum

 

100
50
25
10

100
50
25
10

l
0

100
50
25
10

 

 

 

 

 

DL-Serine ug/ml
800 400 200 100 50 25 O
O O O 5 7 8
O O O h 7 4
O O 0 O 5 7 8'
o o o o 3 7 4
O 0 O O 0 5 5
O O O 0 0 5 5
Dir-Serine ug/ml
800 #00 200 100 50 25 O
O O O O 10 >10 8
O O O O 10 >10 8
0 O O O 9 >10 8
0 O O O 9 >10 10
O O O O 5 10 10
O 0 O O 5 10 10
DL'Serine ug/ml
800 400 200 100 50 25 0
O O 5 10 710 >10 >10
0 O 10 >10 710 710
o o o 10 >10 >10 ,10
O O O 5 )10 >10 >10
0 O O 0 >10 >10 >10
0 O O 0 >10 710 710

 

Growth in all tubes

'. no...

.u’d‘

. "I '
yank—-r-"r

...—- A-._~.‘ o
'1
o
o

Effect of xanthine and serine on growth of Salmonella gallinarum

24 Hours

Xanthine ug/ ml

58 Hours

Xanthine ug/ ml

148 Hours

Xant hine ug/ ml

TABLE 39

 

DL-Serine ug/ml

 

 

 

 

 

400 200 100 50 O

50 O O O 1 l

25 O O l 5 5

12. 5 0 O l 5 5

O O O 0 5 5
DL-Serine ug/ ml

#00 200 100 50 0

5O 0 6 8 >10 >10

25 O 1 >10 >10 >10

12.5 0 8 >10 ‘710 >10

0 O O 10 .>10 >10
DL-Serine ug/ ml

400 200 100 50 0

5O 0 >10 7 10 >10 >10

25 0 >10 >10 710 ~’10

12.5 0 >10 >10 >10 '710

0 O 0 >10 ; 10 > 10

 

'03-... an t t
h

§~‘ ‘ ‘hn~ 1
;.
”:__ ..

TABLE 40

Effect of deoxyguanosine and serine on growth of
Salmonella gallinarum

21+ Hours

De oxy guanosine ug/ml

58 Hours

De oxyguanosine ug/ ml

1+8 Hours

De oxyguanosine ug/ ml

 

100
50
25

100
50
25

100
50
25

DL~Seri ne ug/ ml

 

 

 

 

 

400 200 100 50 O

0 O 0 5 5

o o 1 5 5

0 O l 5 5

0 O 0 5 5
DI:- Serine ug/ ml

400 200 100 50 O

O 0 >10 >10 710

O 0 >10 >10 >10

0 5 >10 >10 >10

0 O 10 >10 >10
DL‘Serine ug/ml

#00 200 100 .50 O

0 5 >10 )10 >10

0 >10 >10 >10 > 10

O O ‘710 710 7>10

 

-74..

Effect of uracil and serine on growth of Salmonella gallinarum

2h Hours

Uracil ug/ml

58 Hburs

Uracil ug/ml

#8 Hours

Uracil ug/ml

100
50
25

100
50
25

100
50
25

TABIE [+1

 

DL‘Serine ug/ml

 

 

 

 

 

400 200 100 50 O

O 0 l 5 5

O 0 1 5 5

o o o S 5

o o o 5 5
DL-Serine ug/ml

400 200 100 50 O

0 1 10 >10 >10

0 5 10 >10 ’10

o 5 10. >10 >10

0 0 10 710 >10
DL’Serine ug/ml

#00 200 100 50 0

0 >10 >10 >10 >10

0 7 10 710 > 10 710

0 >10 >10 710 >10

0 O > 10 > 10 7 10

 

Effect of ribose, adenine and serine on growth of

24 Hours

D-Ribose’
100 ug/ml
50 ug/ml
25 ug/ml

O

58 Hours

D-Ribose

100 ug/ml
50 ug/ml
25 ug/ml
O

48 Hours

D-Ribose
100 ug/ml
50 us/ml
25 ug/ml
0

TABLE 1+2

Salmonella gallinarum

 

Adenine sulfats'

100 ug/ml
50 ug/ml
25 ug/ml

O

Adenine sulfate
100 ug/ml
50 ug/ml
25 ug/ml
O

Adenine sulfate
100 ug/ml
50 ug/ml
25 ug/nl
O

 

 

 

 

 

DIrSerine ug/ml

400 200 100 50 O

0 _ 0 5 7 5

O O 5 O 5

0 O 1 5 5

O 0 O 5 5
DL‘Serine ug/ ml

400 200 100 50 0

O 10 210 >10 >10

0 10 710 5 >10

0 10 10 >10 >10

0 O 10 >10 710
DIrSerine ug/ml

400 200 100 50 0

0 >10 >10 >10 >10

10 >10 >10 >10 710

0 '>10 >10 >10 >10

0 0 >10 710 >10

 

I"Autoclaved together 118°C 20 minutes

-76-

 

' .
, __g.. . __

‘5‘

Effect of deoxyguanylic acid and serine on growth of

21+ Hours

Deoxyguanylic acid
ug/ml

58 Hours

Deoxyguanylic acid
ug/ml

1+8 Hours

De oxyguanylic acid
ug/ ml

TABLE

1+3

S almone lla gallinarum

 

100
50
25

100
50
25

100
50
25

DIrSerine ug/ml

 

 

 

 

 

#00 200 100 50 O

O O 0 5 5

O O O 5 5

O O O 5 5

O O O 5 5
DID-Serine ug/ml

#00 200 100 50 O

O 0 >10 710 710

o o ’10 >10 510

o o 10 >10 >10

0 O 10 710 >10
DL'Serine ug/ml

#00 200 100 50 O

O 5 >10 >10 >10

0 O >10 >10 >10

0 0 >10 710 510

O 0 710 >10 710

 

-77-

.1"

Effect of adenine and serine on growth of Salmonella gallinarum

2# Hours

Adenine sulfate
ug/ml

58 Hours

Adenine sulfate

ug/ml

48 Hours

Adenine sulfate
ug/ml

100
50
25

100
50
25

100
50
25

TABLE

1+4

DIr Serine ug/ ml

 

 

 

 

 

400 200 100 50 O

O O O 5 5

O O O 5 5

0 0 O 5 a

O O O 5 5
DLrSerine ug/ml

#00 200 100 50 O

0 O O 8 6

o o 8 >10 10

O o 8 710 10

0 O 10 ‘710 >10
DIrSerine ug/ml

#00 200 100 50 0

O 0 0 >10 510

o 0 >10 >10 710

o 0 >10 ,10 >10

0 O .710 710 710

 

-78..

Effect of ribose and serine on growth of Salmonella gallinarum

2# Hours

D-Ribose ug/ml

:8 Hours

D-Ribose ug/ml

l+8 Hours

D- Ribose ug/ m1

100
50
25

100
50
25

100
50
25

TABLE #5

 

Dir-Serine ug/ml

 

 

 

 

 

 

#00 200 100 50 O

O O O 5 5

O 0 O 5 5

O O O 5 5

O 0 O 5 5
DL‘Serine 113/1211

#00 200 100 50 O

O 0 8 >10 >10

0 0' 9 >10 >10

0 O 10 >10 )10

O 0 10 >10 >10
DL‘Serine ug/ml

400 200 100 50 O

O 0 >10 >10 710

O 8 >10 )10 >10

0 8 ,)10 >10 >10

0 0 >10 >10 >10

 

-79-

“ET
5

'l"

TABIE 46

Effect of ribonucleic acid and serine on growth of
Salmonella_gallinarum

22 Hours

Ribonucleic acid
ug/ml

28 Hours

Ribonucleic acid

ug/ml

#2 Hours

Ribonucleic acid
ug/ml

66 Hours

100
50

25
lO

100
50

25
10

100
50

25
10

DLrSerine ug/ml

 

 

 

 

 

 

800 #00 200 100 50 25 O

O O 3 8 10 10 5

O O l 7 lo 10 5

O O O 5 10 10 5

o o o 1 7 9 5

O O O O 5 5 5

o o o o 0 5 5

DL-Serine ug/ml

800 #00 200 100 50 25 O

0 1+ 7 ) 10 >10 > 10 9

O O 3 710 >10 >10 9

0 O 0 >10 >10 >10 9

0 O O # >10 >10 10

O O O O 8 )10 10

O O O O 6 10 10

DL- Se rine ug/ m1

800 400 200 100 50 25 0

710 >10 >10 )10 )10 >10 >10

210 710 710 >10 >10 > 10 "710

10 10 10 710 '>lO >10 ‘710

0 0 6 710 710 710 7 10

0 O O O .)10 '>10 >10

0 O O 0 >10 > 10 710
Growth in all tubes

-80.—

"'37!
3‘

"—W: "—

. ......“ "v:

\wu

TABLE #7

Effect of deoxyribonucleic acid and serine on growth of
ia‘lmone lla galli narum

 

 

 

 

 

21 Hours DL'Serine ug/ml
120 60 12 O
Deoxyribonucleic acid 100 0 O 0 0
“W1 50 o 7 6 3
10 O 7 7 5
0 0 5 5 5

36 Hours DIrSerine ug/ml
120 60 12 0
Deoxyribonucleic acid 100 O l 3 1
“3/“ so 10 >10 >10 5
10 ’10 >10 >10 8
0 7 > 10 >10 10

61+ Hours DI:— Serine ug/ml
120 60 12 0

 

Deoxyribonucleic acid 100 0 >10 .>10 >10
“5/ ml 50 >10 >10 >10 >10

10 >10 >19 >10 >10

0 >10 >10 >10 >10

 

..81—

. m,
.HI 1"... "

 

TABLE 1+8

Effect of glutamic acid and serine on growth of
Salmonella gallinarum

1:2 Hours DL—Serine ug/ml E:
120 90 60 3o 10 o

 

 

 

 

LrGlutamic acid ug/ml 120 O O O 2 5 7
10 O O O O 5 7
0 0 O O O O 5

26 Hours DL—Serine 113/131

120 90 6O 30 10 O

 

 

L-Glutamic acid ug/ml 120 3 5 7 9 10 710
10 3 3 5 7 10 ‘>10
0 o o o o 5 >10
1+0 Hours DID-Serine ug/ml

120 90 60 30 10 0

 

L-Glutamic acid ug/ ml 120 >10 >10 210 710 >10 710
10 :710 710 '710 710 >10 >10
0 O 0 5 10 >10 ‘710

 

2 Hours Growth in all tubes

-82-

Effect of asPartic acid and serine on growth

 

12 Hours

L‘Aspartic acid ug/ml 120
10
O

26 Hours

L-«Aspartic acid ug/ml 120
10
0

40 Hours

L-Aspartic acid ug/ml 120
10

 

 

 

 

 

TABLE 49
of
Salmonella gallinarum
DLrSerine ug/ml
120 90 6O 30 10 O
0 O 0 0 5 7
O O O O 4 6
O O O O O
DIrSerine ug/ml
120 90 60 5O 10 O
l l 5 7 10 >10
0 O l 6 8 710
0 0 O O 710
DLrSerine ug/ml
120 90 6O 30 10 0
10 710 710 '710 710 710
l 10 >10 7 10 >10 >10
0 O 5 10 7' 10 7 10

0

2 Hours

 

Growth in all tubes

-83...

-_ 9mg; lo: 7‘ J

Effect of isoleucine and serine on growth of Salmonella gallinarum

2# Hours

DL‘Isole ucine mg/ ml

38 Hours

DIrIsoleucine mg/ ml

1+8 Hours

DL-Isoleucine rag/ml

2.0
1.0

0.5

2.0
1.0

0.5

2.0
1.0

0.5

TABLE 50

DL~Serine ug/ ml

 

 

 

 

 

400 200 100 50 o

1* 5 7 7 7

3 5 7 7 7

1 5 7 7 7

0 0 0 5 5
DL-Serine ug/ ml

400 200 100 50 0

>10 >10 710 >10 >10

710 >10 .710 >10 >10

5 710 710 >10 >10

0 0 10 >10 7 10
DL~Serine ug/ ml

1+00 200 100 50 O

> 10 2 10 > 10 >10 710

> 10 7 10 710 > 10 7 10

>10 710 >10 7 10 >10

0 O 7 10 710 710

 

-04-

 

TABLE 51

Effect of glutamine and serine on growth of Salmonella gallinarum

 

 

 

 

 

12 Hours DL~Serine ug/ml
120 90 60 50 10 0
L—Glutamine ug/ml 120 0 0 0 2 5 7
90 0 0 0 2 5 7
60 0 0 0 1 5 6
50 0 0 0 0 5 6
15 0 0 0 0 2 5
10 0 0 0 0 2 5
5 o _0 V o o o 5
0 0 0 0 0 0 5
26 Hours DIrSerine ug/ml
120 90 60 50 10 0
L-Glutamine ug/ml 120 5 5 7 10 10 710
90 5 5 7 10 10 >10
60 3 5 5 8 10 >10
30 0 l 5 6 9 >10
15 0 0 0 4 8 710
10 0 o 0 3 7 >10
5 0 0 0 5 6 710
0 0 0 0 0 5 >10
40 Hours DID-Serine ug/ml
r 120 90 60 30 10 0
L-Glutamine ug/ml 120 j>10 ‘>10 710 710 ,>10 >10

90 ‘>10 >10 >10 1710 >10 710
60 710 710 710 710 >10 >10

 

50 710 710 710 710 710 710

15 9 10 >10 >10 >10 >10

10 1 9 >10 >10 >10 >10

5 o 5 10 >10 >10 >10

0 o o 5 10 >10 710
2 Hours Growth in all tubes

-85..

n
‘l' '

 

Effect of asparagine and serine on growth of

12 Hours

L-Asparagne ug/ml

26 Hours

L-Asparagine ug/ml

1+0 Hours

L-As paragine ug/ml

2 Hours

TABLE

52

Salmonella gallinarum

DL~Serine u g/ml

 

 

 

 

 

120 90 6O 5O 10 O

120 o o 0 o 2 6

10 0 0 0 0 5

0 O O 0 0 0 5
DL‘Serine ug/ml

120 90 60 50 10 0

120 0 0 0 H 7 >10

10 0 0 O O 5 '710

0 0 0 0 0 5 710
DL‘Serine ug/ml

120 90 60 50 10 0

120 1 10 >10 2710 I710 '510

10 0 0 1 >10 7 10 710

0 0 0 5 10 710 710

 

Growth in all tubes

-86..

Effect of histidine and serine on growth of Salmonella gallinarum

2’+ Hours

L-Histidine mg/ml

§8 Hours

LsHistidine rug/ml

l+8 Hours

L-Histidine mg/ml

2.0
1.0

0.5

2.0

1.0

0.5

2.0
1.0

0.5

TABIE 55

 

 

 

‘:.L"“1 $19,137
..
i
2
..m. ' e)

r51" ' .._-‘ '.

 

 

 

 

DL~Serine ug/ ml

400 200 100 50 0

O O 1 5 5

O 0 1 7 7

0 0 1 '7 10

O 0 0 5 5
DL‘Serine ug/ ml

1+00 200 100 50 0

8 >10 7 10 710 7 10

0 710 710 710 >10

0 O > 10 7 10 ‘7 10

0 0 10 P 10 710
DL‘Serine ug/ml

[+00 200 100 50 0

7 10 P 10 7 10 > 10 7 10

0 >10 7 l0 >10 >10

3 5 710 7 10 '7 10

0 0 7 10 "P 10 7 10

 

TABLE 54

Effect of proline and serine on growth of Salmonella gallinarum

18 Hours

L-Proline rug/ml

24 Hours

L~Proline mg/ml

l+8 Hours

L ~Proline mg/ml

52 Hours

L-Proline mg/ml

DL~Serine ug/ml

 

 

 

 

 

 

 

800 400 200 100 50 25 12.5 0
2 . o o o o 1 2 1+ 6 8
1.0 0 O 0 1 2 5 5 7
0.5 0 O 0 0 1 3 5 7
0.25 0 0 0 0 1 3 5 6
0.125 0 0 0 0 1 3 5 5
O 0 0 0 0 0 l 5 5
DL-Serine ug/ m1
800 #00 200 100 50 25 12.5 o
2.0 3 3 3 5 7 9 710 710
1.0 3 3 3 5 6 9 >10 '710
0.5 0 l 3 3 6 7 9 710
0.25 O O l 3 5 7 9 ‘710
0.125 0 0 1 3 5 7 9 10
0 0 0 0 0 0 3 9 9
DL- Serine ug/ml
800 400 200 100 50 25 12.5 0
2.0 >10 5 >10 >10 >10 ;>10 >10 >10
1. 0 3 5 >10 >10 >10 >10 2 10 >10
0.5 l 3 >10 >10 >10 >10 >10 ,>10
0.25 l 3 5 >10 710 710 > 10 >10
0.125 3 5 >10 > 10 >10 >10 >10 210
0 0 0 O 7 710 >10 710 '710
DL‘Serine ug/ml
800 400 200 100 50 25 12.5 0
2 . 0 >10 >10 710 >10 710 >10 > 10 >10
1.0 .10 >10 >10 >10 >10 >10 >10 >10
00 5 7 10 >10 3 10 710 710 >10 >10
025» 5 9 >10 )10 >10 >10 >10 >10
0 15 10 >10 > 10 710 710 7 10 7 10 7 10
0 0 0 3 >10 '710 >10 >10 >10

 

..88-

 

Effect of methionine and serine on growth of

2% Hours

DL~Methionine mg/ml

28 Hours

DL -I-1et hionine mg/ ml

l+0 Hours

DL-Met hionine rug/ml

1+8 Hours

DL‘Met hionine mg/ml

TABLE 55

Salmonella gallinarum

DL-Serine ug/ml

 

 

 

 

 

 

 

800 #00 200 100 50 25 12.5 o
2.0 o o o 3 5 6 8 10
1.0 o 0 o o 5 6 8 10
0.5 0 o o 0 5 6 8 9
0.25 o o o o 5 6 8 9
0.125 0 o 0 3 6 7 8 9
o o o o o 3 5 7 9
DL~Serine ug/ml
800 400 200 100 50 25 12.5 o
2.0 1 1+ 6 7 9 710 >10 710
1.0 1 1+ 5 6 9 10 >10 710
0.5 1 4 5 6 8 10 >10 710
0.25 1 1+ 5 7 8 10 >10 r10
0.125 1 a 5 7 10 10 >10 >10
0 0 o 4 1 7 8 >10 >10
DL—Serine ug/ml
800 #00 200 100 50 25 12.5 o
2.0 >10 >10 >10 710 ~>1o >10 >10 >10
1.0 10 710 >10 >10 >10 710 >10 >10
0.5 9 >10 >10 >10 >10 >10 >10 >10
0.25 7 >10 710 >10 >10 710 710 >10
0.125 5 >10 >10 >10 >10 710 >10 >10
0 0 0 >10 >10 710 .10 >10 >10
DL-Serine ug/ml
800 400 200 100 50 25 12.5 o
2.0 710 >10 >10 >10 >10 >10 >10 :10
1.0 >10 >10 >10 >10 >10 >10 >10 >10
0.5 >10 >10 >10 710 710 >10 >10 >10
0.25 ~>1o >10 ,10 >10 >10 >10 .10 >10
0.125 9 >10 >10 >10 >10 >10 >10 710
o o 0 >10 >10 >10 >10 >10 >10

 

89"

.. _ mimatw II Hui-3' “
l E

_»-gc-g- nun-r

TABLE 56

Effect of urea and serine on growth of Salmonella gallinarum

 

 

 

 

 

 

20 Hours DLrSerine ug/ml
800 400 200 100 50 25 0
Urea mg/ml 2.0 o o 4 6 6 6 6 “'1
1.0 o o o 5 6 6 6 3
0.5 0 0 0 1 4 5 6 '
0.25 o o o o 9 4 6
0.125 O O O O O k 7
o o o o o o 6 7
0 Hours DIrSerine ug/ml 1:3
800 #00 200 100 50 25 o
Urea mg/ml 2.0 0 5 9 10 10 10 9
1.0 0 0 8 10 10 710 >10
0.5 0 0 5 9 10 710 >10
0.25 0 0 0 5 9 710 710
0.125 0 0 0 5 8 >10 >10
0 0 0 0 1 7 >10 >10
#5 Hours DL-Serine ug/ml

800 #00 200 100 50 25 0

 

 

Urea mg/ml 2.0 0 710 >10 1>10 >10 >10 710
1.0 0 5 710 >10 710 '710 >10
0.5 0 5 9 710 710 1>10 >10
0.25 0 5 5 710 >10 710 710
0.125 0 0 5 >10 '710 ‘710 ‘710
O 0 0 5 10 710 710 '710
55 Hours DL'Serine ug/ml

800 400 200 100 50 25 0

 

Urea mg/ml 2.0 7 >10 >10 >10 >10 >10 >10
1.0 5 9 >10 >10 710 >10 >10
0-5 # 8 >10 >10 >10 >10 ,10
0.25 3 8 9 v10 >10 ,10 >10
0.125 4 5 7 710 710 >10 >10
0 3 3 10 510 710 710 -710

 

Effect of cysteine and serine on growth of Salmonella gallinarum

18 Hours

L-Cysteine

22 Hours
L ‘Cysteine

30 Hours
L-Cysteine

#2 Hours

L- Cysteine

Hours

L~Cysteine

mg/ml

mg/ml

TABLE 57

 

DL~Serine ug/ml

 

 

 

 

 

 

 

 

 

800 #00 200 100 50 25 12.5 0
2.0 0 0 0 0 0 O 0 O
1.0 0 0 O 0 0 O 2 h
0.5 0 0 0 O O 0 4 #
0.25 0 0 0 0 0 0 0 6
0.125 0 0 0 O 0 2 5 7
0 0 O 0 0 l # 6 7
DL‘Seri ne ug/ml
800 400 200 100 450 25 12.5 0
2.0 0 0 0 0 0 0 0 l
1.0 O 0 0 0 2 5 8 8
0.5 0 O 0 0 0 l 10 10
0.25 O 0 0 O l 6 6 710
0.125 0. 0 0 0 5 7 10 ‘>10
0 0 0 0 l l 8 10 >10
DlrSerine ug/ml
800 400 200 100 50 25 12.5 0
2.0 0 0 0 0 0 0 5 5
1. 0 0 0 0 O 9 > 10 > 10 >10
0.5 0 O 0 0 3 '5 >10 710
0. 25 0 0 0 0 7 >10 >10 710
0.125 0 0 0 0 9 >10 710 710
0 0 0 0 43 >10 '>10 710 710
DL~Serine ug/ml
800 400 200 100 50 25 12.5 0
2.0 10 5 5 l 1 . 0 10 10
l. 0 7 10 >10 710 9 >10 710 >10 >10
0. 5 0 0 l 9 >10 7 10 2 10 >10
0.25 0 0 0 7 >10 :>10 ‘>10 >10
0. 125 0 0 0 7 710 710 710 >10
0 0 0 0 10 210 > 10 > 10 >10
DIrSerine ug/ml
800 400 200 100 #50 25 12.5 0
2.0 ~10 >10 >10 >10 >10 8 >10 >10
1.0 le 710 710 P10 710 >10 >10 >10
0&5 7 7 8 >10 >10 >10 ->10 >10
0.25 7 7 8 >10 >10 >10 710 >10
0.125 7 7 7 >10 >10 710 710 ,10
0 6 6 7 510 ‘710 710 >10 210

 

 

TABLE 58

Effect dfglutathione aniserine on growth of Salmonella gallinarum

18 Hours

Glutathione
mg/ml

24 Hours

Glutathione
mg/ml

42 Hours

Glutathione
mg/ml

2 Hours

Glutathione
mg/ml

68 Hours

Glutathione
mg/ml

2.0
1.0
0.5
0.25
0.125

2.0
1.0
0.5
0.25
0.125

2.0
1.0
0.5
0.25
0. 125

2. O
1.0
0.5
0.25
O. 125

2.0
1.0
0.5
0.25
0.125

 

DL~Serine ug/ml

 

 

 

 

 

 

 

 

 

 

800 #00 200 100 50 25 12.5 0

0 0 O 0 O 0 0 0

0 0 0 0 O 0 0 O

O 0 O 0 O O O O

0 0 0 0 O 1 3 6

O O O 0 0 l 3 7

O 0 O O 0 0 3 5
DL-Serine ug/ml

800 400 200 100 50 25 12.5 0

O 0 0 0 0 0 O 0

0 0 0 0 O O 0 l

0 0 0 O 0 0 l 7

O 0 0 0 O 3 7 10

0 0 0 0 3 7 9 10

0 0 0 0 0 3 6 9
DIrSerine ug/ml

800 #00 200 100 450 25 12.5 0

3 3 3 3 6 8 10 10

0 0 0 0 5 7 10 >10

0 0 O 0 10 10 >10 >10

0 0 O 10 7 >10 >10 >10

0 0 3 10 >10 >10 )10 >10

0 0 O 5 _2 10 > 10 > 10 710
DL~Serine ug/ml

800 #00 200 100 50 25 12.5 0

9 9 9 >10 >10 >10 >10 >10

3 5 5 7 '>10 >10 ~>10 >10

0 0 2 7 >10 >10 >10 >10

0 3 9 >10 >10 >10 >10 >10

3 3 5 >10 >10 >10 > 10 > 10

0 0 1 >10 7 10 7 10 >10 >10
DL—Serine ug/ ml

800 #00 200 100 50 25 12.5 0

>10 >10 >10 >10 >10 >10 >10 >10

10 710 >10 >10 >10 plO >10 >10

5 7 >10 >10 :10 >10 >10 >10

10 >10 >10 ->10 >10 >10 -710 >10

>10 >10 >10 >10 >10 710 '710 >10

>10 710 <7 >10 >10 ~710 >10 >10

 

-92-

~ . 5')-
\ ‘}-

ROOM USE ONLY

 

MICHIGAN STATE UNIVERSITY

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3 1293 3082 033

 

mm“