THE BIOLOGICAL OXIDATION
OF NICOTINE
1; NICO‘I‘INE DEGRADATION BY
NICOTIANA RUSTICA

H. NICOTINE DEGRADATION BY
AN ARTHROBACTER SPECIES

 

 

 

Thesis Icr IIm Degree 0% pk. D.
MICHIGAN STATE UNIVERSITY

Gail Denise Griffith
1961

 

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LIBRARY ‘"
Michigan Stab:
University

    
    
  

I I IailCIIICis‘a'I SUITE FIIVERSITY
17361

Of AGRICULILTTIE. HID AWLIED SCIENCE

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EAST LANSING, MICHIGAN

ABSTRACT

THE BIOLOGICAL OXIDATION OF NICOTINE
I. DEGRADATION OF NICOTINE BY NICOTIANA RUSTICA
II. DEGRADATION OF NICOTINE BY AN ARTHROBACTER SPECIES

 

 

by Gail Denise Griffith

Metabolism of nicotine in the tobacco plant has remained largely
unexplored, although it has been demonstrated that the alkaloid is not
an inert plant constituent but can be converted to other materials.

In the present study nicotine-C“ was supplied to tobacco plants and
nicotine, nicotinic acid, and cotinine were isolated after periods of
metabolism of 4, 7, or 14 days. Approximately 60 to 80% of the

total radioactivity fed to the plants was recovered as nicotine dipicrate
in all experiments.

Nicotinic acid, isolated as the hydrochloride, contained a
significant amount of isotope. Calculated dilutions of specific activity
in going from nicotine to nicotinic acid were low enough to support
the hypothesis that nicotinic acid was formed from nicotine in the plants
by oxidation of the pyrrolidine ring of nicotine. The essential meta-
bolic role of nicotinic acid in living organisms has been well estab-
lished; its production from nicotine in the tobacco plant suggests that
the alkaloid may serve as a reserve source of this compound.

Cotinine, isolated from the plants as the perchlorate, was also
radioactive but evidence indicated that the incorporation of C“ into
the molecule was the result of a nonmetabolic process.

Microorganisms were observed in nutrient medium in which
tobacco plants had been grown which had the ability to catalyze the

oxidation of nicotine to a product having an ultraviolet absorption

 

Gail Deni. s e Griffith

spectrum markedly different from that of the parent compound.
A microorganism was isolated from this source that proved to be a

species of Arthrobacter resembling Arthrobacter oxydans in cultural

 

 

and morphological characteristics. The bacteria catalyzed the
production of 6-hydroxynicotine in approximately 50% yield from
nicotine in a medium containing nicotine, inorganic salts and a small
amount of yeast extract. The identity of the product was established
by its melting point, the melting point of the picrate derivative,
ultraviolet and infrared absorption spectra, molecular extinction
values, elementary analysis, and specific rotation.

The significance and implications of these findings are dis-

cussed.

THE BIOLOGICAL OXIDATION OF NICOTINE
I. NICOTINE DEGRADATION BY NICOTIANA RUSTICA

 

II. NICOTINE DEGRADATION BY AN ARTHROBACTER SPECIES

 

BY

Gail Denis e Griffith

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1961

ACKNOWLEDGM ENTS

The author expresses her appreciation to Dr. Richard U.
Byerrum for his guidance and encouragement during the course
of this study. The author is also indebted to Dr. Willis A. Wood,
Dr. Richard L. Anderson, and Dr. Edward D. Devereux for
their interest and direction in the isolation and identification of
the Arthrobacter species. Dr. Sydney C. Rittenberg of the
University of Southern California kindly supplied a sample of
6—hydroxynicotine and helpful discussion of the bacterial oxidation

 

of nicotine.

Thanks are also due to the National Institutes of Health for
a Predoctoral Fellowship and other funds provided in support of
this work.

:',< >§< >§< >§< >§< 3:: :k :1: >:< >{< 3:: 3::

ii

TABLE OF CONTENTS

 

 

Page

INTRODUCTION........................ 1

EXPERIMENTAL AND RESULTS . . . . . . . . . . . . . . . 5

I. Nicotine Degradation by Nicotiana rustica . . . . . . . 5
Preparation of nicotine-Cl4 and its administration

to the plants. 5

Control eXperiments . . . . . . 6

Isolation of nicotinic acid hydrochloride. . . . . . . 7

Determination of endogenous nicotinic acid

concentration . . . . . . . . . . . . . . . . . . . 8

Isolation of nicotine . . . . . . . . . . . . . . . . . 11

Isolation of cotinine . . . . . . . . . . . . . . . . . 11

II. Nicotine Degradation by an Arthrobacter species. . . . 12

Isolation and characterization of the microorganism 12

Nicotine oxidation . . . . . . . . . . . . . . . 16

Isolation and identification of 6- -hydroxynicotine . . 16

DISCUSSION.......................... 21

I. Nicotine Degradation by Nicotiana rustica . . . . . . . 21

 

II. Nicotine Degradation by an Arthrobacter species. . . . 24

 

SUMMARY 26

LITERATURECITED..................... 27

iii

LIST OF TABLES

TABLE Page

I. Radioactivity of nicotinic acid hydrochloride
isolated from tobacco plants . . . . . . . . . . . . . . 10

II. Radioactivity of nicotine dipicrate isolated from
tobaccoplants.....................10

III. Physical properties of 6-hydroxynicotine . . . . . . . 20

iv

LIST OF FIGURES

FIGURE Page

1. Proposed pathways for the bacterial oxidation of
nicotine......................... 2

2. Ultraviolet absorption spectra of samples of
Arthrobacter cultures during oxidation of nicotine
in aerated liquid medium . . . . . . . . . . . . . . . 17

 

3. Relative concentrations of nicotine and 6-hydroxy—
nicotine in Arthrobacter cultures during oxidation of
nicotine in aerated liquid medium . . . . . . . . . . . 18

 

INTRODUCTION

Nicotine, which has a variety of physiological effects in
animals, is the major alkaloid of several plant species in the genus

Nicotiana. Its presence in these plants is of unknown significance.

 

Investigators have used three major methods of attack to
discover how nicotine is metabolized or detoxified. The first,
bacterial oxidation, using organisms that can utilize nicotine or its
derivatives as their sole carbon source, has been most extensively
investigated. The organisms are usually isolated from soil by
enrichment culture techniques.

Several bacterial oxidation products have been isolated and
identified, including 6-hydroxynicotine, 6-hydroxypseudooxynicotine,
pseudooxynicotine, and a group of derivatives in which the pyrrolidine
ring of nicotine is opened and degraded. These compounds and
proposed pathways for their formation are shown in Figure 1.

Hochstein and Rittenberg (1-4) have obtained cell-free
preparations of a soil bacterium, designated as P934, which catalyze
the oxidation of nicotine successively to 6-hydroxynicotine, 6-hydroxy-
pseudooxynicotine, and 2, 6-dihydroxypseudooxynicotine. Wada and

co-workers (5,6) reported that Pseudomonas species also isolated

 

from soil can oxidize nicotine to some pyridine and 6-hydroxypyridine
derivatives. Frankenberg and Vaitekunas (7) have described similar
nicotine metabolites produced by unidentified organisms derived from
the surface of tobacco seeds. Wadsworth (8) has shown that a

Corynebacterium species can convert nicotine to a compound resembl-

 

ing 6-hydroxynicotine in some respects. Sguros (9) has described

the cultural and morphological characteristics of Arthrobacter oxydans,

 

an organism obtained from air and tobacco leaves, that has the ability

\ ‘
I u‘3

/
/ N
2.7) nicotine (5.7)
\ N
/

. C- CH -NH "CH
i 11H3 J \ ‘6 ( 2); 3
Ho N /

6-hydroxynicotine

(3) 7-methylaminopropyl(3-pyridyl)
J, ketone
\ 3‘(C“2)§NH‘C_H5 (pseudooxynicotine)
I l (5, 7)
“o H CH
6-hydroxypseudooxynicotine \ i-<C 2)2 3
(4) I N/
r H -N\\'CH
m (C a?) 3 3-PyridY1pr0pyl ketone
/ +
H H \ 96‘6“»; COOH
2, 6-dihydroxypseudooxynicotine I
/
: 'y-keto-y-(3-pyridyl)—butyric acid
: (4) (3-nicotinoylpropionic acid)
I
1

fl!) \, <6. 7)

v -cu -(cuz)-cooI-I
a glutaconic acid derivative 3 fl 2.
u I(

I 3-pyridylmethyl 3-succinoyl-6-
' (4)
l

ketone hydroxypyridine
v 1(7)
blue pigment \ 60H :(7)
I
I I
n (7) / 4’
l nicotinic acid a glutaconic
4/ ,L (7) acid derivative
OOH
QHs-NI-I‘2 + NH5 4— com-I \ I
“"H <— - I / I (6)
<7) .
‘1' $00“ ‘1' $°°H 6-hydroxynicotinic acid I
_______________________ 1
cu, (“Dz é
C°°H AooH

Figure 1. Proposed pathways for the bacterial oxidation of
nicotine. Pertinent references are shown by the numbers in
parentheses.

 

to catalyze the oxidation of nicotine. The identity of the oxidation
products was not established, however.

The present study presents evidence for the formation of
6-hydroxynicotine as a metabolite of nicotine in approximately 50%

yield produced by a Species of Arthrobacter present on the roots of

 

tobacco plants.

The second method of study of nicotine metabolism, using
animals as experimental organisms, has shown that the major sites
of nicotine detoxication are liver, kidneys, and lungs (10). McKennis
and co-workers (11-13) have isolated and identified 7-(3-pyridyl)-y-
methylaminobutyric acid, cotinine, demethylcotinine, and hydroxy—
cotinine from the urine of dogs that had been given nicotine or
cotinine intravenously. Hucker, (it a}. (14), using a rabbit liver
microsome preparation, found cotinine as a nicotine metabolite.

The most significant difference in mode of nicotine metabolism
by bacteria and animals is the site of primary oxidation. ‘ In bacteria
oxidation first occurs at the 2 carbon of the pyrrolidine ring or the
6 carbon of the pyridine ring, whereas in animals oxidation occurs
first at the 5 carbon of the pyrrolidine ring.

Nicotine metabolism in higher plants has remained largely
unexplored. This alkaloid is a natural plant product and is present
in concentrations as great as 2 to 4% of the dry weight of tobacco plant
tissue. These considerations raise the question of what part nicotine
might play in the metabolic economy of the plant. Speculation has
assigned nicotine roles ranging from that of an endogenous insecticide
to that of a metabolic waste product. These explanations are
inadequate and difficult to prove, however. The availability of iso-
topic tracers has provided a tool with which to investigate this question
in a more definitive fashion.

It has been demonstrated that nicotine is not an inert plant

constituent but that it can be converted to other plant materials.

T50 and Jeffrey (15) have shown the interconversion of Cl4 and N15
labeled tobacco alkaloids within tobacco plants; Leete and Bell (16)
have demonstrated that the C14 of administered nicotine-2, 5-Cl4
gradually disappears from the molecule when fed to tobacco plants.
They also showed that the isotope of nicotine-methyl--C14 appears in
choline methyl groups.

In the present study nicotine-Cl4 was administered to intact
tobacco plants and nicotinic acid and nicotine were isolated from the
plants 4, 7, and 14 days later. The nicotinic acid contained a
significant amount of radioactivity, demonstrating the conversion of
nicotine to this metabolically important compound. . Cotinine was
also isolated from the plants and proved to be radioactive, but
evidence indicated that the incorporation of C14 into this compound
was probably not of metabolic significance. Approximately 60 to 80%
of the total radioactivity fed to the plants was recoverable as nicotine

dipicrate.

EXPERIMENTAL AND RESULTS

I. Nicotine Degradation by Nicotiana rustica

 

Preparation of nicotine-C14 and its administration

 

5‘ the plant 3

 

Radioactive nicotine was obtained by administration of sodium
acetate-Z-Cl4 or sodium prOpionate-Z-C”, both purchased from
Volk Radiochemical Company, Chicago, to tobacco plants. Nicotine
was isolated as the dipicrate with a specific activity of 2. 86 x 106
c.p.m, /mmole from plants fed acetate-Z-Cl4 and l. 46 x 106 c.p.m. /
mmole from plants fed propionate-Z-CM‘. Nicotine prepared in this
manner had 50% of its C14 in the pyridine ring and 2 carbon of the
pyrrolidine ring (17, 18). Nicotine was recovered from the dipicrate
by distillation through a Widmer column from an alkaline aqueous
solution into dilute hydrochloric acid, yielding nicotine hydrochloride.
Water and excess acid were removed from the distillate. The hydro-
chloride was taken up in water to obtain a solution containing
approximately 2 mg nicotine per ml which was adjusted to pH 7 with
sodium hydroxide.

Plants used in these experiments were Nicotiana rustica, var.

 

humilis and were 4 to 6 inches high. About two weeks before
administration of nicotine-C14 the plants were brought into the
laboratory from the greenhouse and their roots were removed to a
length of approximately 1 cm.’ Plants were placed in an inorganic
nutrient solution (19) to allow new rOOts .to grow; iEach plant was given
1 m1 of the nicotine-C14 solution described above by a procedure in
which the solution was taken up by the roots in 3 to 5 hours (20).

It was assumed that the rapid absorption of nicotine-C14 would

minimize any microbial action on nicotine. The plants were then
allowed to grow in inorganic nutrient solution for 4, 7, or 14 days.
Nicotine from plants fed acetate-Z-Cl4 was used in experiment

1 and that from plants fed propionate-Z-C14 in experiment 2.

Control experiments

 

To determine whether there was any non-metabolic degradation
of nicotine during the isolation procedures, control experiments in
which nicotine and nicotinic acid were added to the plant homogenates
and both then immediately reisolated were performed as follows.

In both experiments two plants were homogenized in 80% ethanol solu-
tion to which carrier nicotinic acid and 2 ml of the previously used
nicotine-—C14 solution had been added. Carrier cotinine was added
later to fractions from which it was reisolated. Nicotine dipicrate,
nicotinic acid hydrochloride, and cotinine perchlorate were recovered
from the plant extracts by the methods described below. The results
of these experiments are reported along with data for the compounds
isolated from plants fed nicotine-CM.

Gram negative bacilli were noted in stained preparations of the
sediment of nutrient solution in which plants had been growing.

(They will be described in a later section.) To determine whether
these bacteria could effect the conversion of nicotine to nicotinic acid,
the roots from three plants were placed in nutrient solution containing
2 ml of the previously described nicotine-Cl4 solution. The mixture
was shaken for 1 hour, after which the roots were removed to
eliminate any possible effect of metabolism of nicotine by the root
tissue itself. The solution was allowed to stand for 24 hours at room
temperature. Ten mg of non-radioactive nicotinic acid was added to

the solution as a carrier and was subsequently reisolated.

The nicotinic acid hydrochloride contained no significant C14,
indicating that the bacteria were not able to convert nicotine to
nicotinic acid under these conditions.

It was also determined that the bacteria were inhibited by the
presence of aureomycin. In experiment 2, 1 ml of a 0. 5% aqueous
solution of aureomycin solution was added to the nutrient solution
in which each plant was growing. There was no appreciable difference
in incorporation of C14 from nicotine into nicotinic acid between

experiments 1 and 2.

Isolation of nicotinic acid hydrochloride

 

In preliminary experiments a number of plants were allowed to
metabolize nicotine-Cl4 for 2 to 7 days and 80% ethanol extracts of
the soluble plant material were chromatographed on paper in several
solvents. Pyridine alkaloids were located on the chromatograms by
spraying the papers with 0. 5% E-aminobenzoic acid solution in 95%
ethanol, drying, and placing them in a sealed jar containing a small
amount of solid cyanogen bromide. Most pyridine alkaloids appear
as yellow to red spots with this reagent (21). It was possible to
detect at least nine colored spots on chromatograms developed in
the organic phase of a n-butanolzacetonezwater mixture (45:5:50) (22).
The areas containing the alkaloids were eluted with water and the
eluates re-chromatographed successively in the organic phase of a
n-butanol:95% ethanol:0. 5 N ammonium hydroxide mixture (80:20:20)
(12), and the organic phase of a 50:50 mixture of t-amyl alcohol and
0. 2 N acetate buffer, pH 5. 6 (23). By comparison with the Rf values
of authentic samples (for nicotine 0. 80, 0. 91, and 0. 26, for cotinine
0. 70, 0. 73, and 0.75, and for nicotinic acid 0. 23, 0. 27, and 0. 51 in
the three solvents, reSpectively), their colors with the reagent used

(nicotine and cotinine were rose-red, nicotinic acid red-orange), and

co-chromatography with authentic material it was possible to identify
nicotine, cotinine, and nicotinic acid in the plant extracts. Samples
of the isolated materials proved to be radioactive in each case.

Since the amount of nicotinic acid present was very small, a
weighed amount of carrier nicotinic acid was added to each plant
extract. In a typical experiment 3 plants that had been fed nicotine-C14
were homogenized in 80% ethanol containing 10 mg of carrier nicotinic
acid. The homogenate was heated on a steam bath for 10 minutes and
filtered. The insoluble residue was washed with 80% ethanol and
discarded. Most of the ethanol was removed from the filtrate under
reduced pressure at which point a dark-green precipitate of water-
insoluble material was formed. This precipitate was removed by
centrifugation at 00. The total amount of radioactivity present in the
precipitate plus that in the insoluble residue was less than 3% of the
Cl4 fed to the plants. The yellow supernatant solution was adjusted to
pH 3. 3 with hydrochloric acid and extracted for 48 hours with diethyl
ether in a liquid-liquid extractor. Under these conditions nicotinic
acid is soluble in diethyl ether (24). The aqueous solution was retained
for isolation of nicotine and cotinine.

Ether was removed from the extract and the dry residue was
taken up in acetone. The acetone solution was saturated with hydro-
chloric acid gas to precipitate nicotinic acid hydrochloride. The hydro-
chloride was dried, sublimed at 110-1200 at 1 mm Hg and its radio-
activity determined. The yield was 7. 0 mg of nicotinic acid hydrochloride.
An elementary analysis of non- radioactive nicotinic acid hydrochloride
prepared by this method corresponded with the calculated values for

nicotinic acid hydrochloride .

Determination of endogenous nicotinic acid concentration

 

The amount of free nicotinic acid in the plants was determined by

the isotOpe dilution technique. Radioactive nicotinic acid hydrochloride

for this experiment was obtained by oxidation of nicotine-CM using
neutral permanganate (25) and conversion of the acid to the hydro-
chloride. Two groups of 3 plants each were fed 1 ml of a solution
containing 2 mg of non- radioactive nicotine. The plants were harvested
after 4 days and homogenized in 80% ethanol solution containing 10 mg
of radioactive nicotinic acid hydrochloride. Diluted nicotinic acid
hydrochloride was isolated from the plants and its radioactivity
determined after complete combustion to carbon dioxide and precipi-
tation as barium carbonate. Samples of the original nicotinic acid
hydrochloride were also submitted to combustion and counted as barium
carbonate.

Calculations based on the difference in radioactivity between
the original and diluted nicotinic acid showed that the average concen-
tration of free nicotinic acid in the plants was 30 ug/gm wet weight of
plant material.

This value was used to calculate the amount of endogenous
nicotinic acid in each plant sample. By assuming that the free nicotinic
acid concentration in the plants remained constant throughout the
experimental period it was possible to correct the specific activity of
the nicotinic acid hydrochloride isolated from the plants for dilution
by carrier.

Nicotine-C14 fed to the plants contained 50% of its C14 in the
pyridine ring and 2 carbon of the pyrrolidine ring. If the nicotinic
acid were derived directly from nicotine and there was no dilution by
the free nicotinic acid pool, its specific activity should be 50% of that
of the nicotine. Dilution of radioactivity between nicotine and nicotinic
acid was calculated by dividing the specific activity of the correspond-
ing nicotine dipicrate sample (see Table II) by the Specific activity of
nicotinic acid hydrochloride (corrected for dilution by carrier) and
multiplying by 0. 5. The results of these experiments and calculations

are shown in Table I.

10

Table I. Radioactivity of nicotinic acid hydrochloride isolated from
tobacco plants.

 

 

 

 

 

Period of Nicotinic Acid
Metabolism Specific Activity
of Nicotine c. p. m. /mmole x 10"3 - Dilution
(Days) Expt. 1 Expt. 2 Expt. 1 ' Expt. 2
0 0 0 — -
4 44 6. 0 18 38
7 7. 5 5. l 88 24
14 8. 4 3. 2 74 66

 

Table II. Radioactivity of nicotine dipicrate isolated from tobacco

 

 

 

 

plants.
Period of Nicotine Dipicrate Recovery of C14
Metabolism Specific Activity in Nicotine*
of Nicotine c. p. m. /mmole x 10'6 (Percent)
(Days) Expt. 1 Expt. 2 Expt. 1 Expt. 2
0 2. 1 0. 58 100 108
4 l. 6 0. 46 70 72
7 1. 3 0. 34 72 67
14 l. 3 0. 41 78 75

 

*Calculated by dividing the total radioactivity of the isolated nicotine
dipicrate plus that of the residual nutrient solution (12% in 4 days,
7% in 7 days, 0% in 14 days) by the total radioactivity fed and multi-
plying by 100.

11

Isolation of nicotine

 

The aqueous solution after extraction of nicotinic acid was adjusted
to pH 11. 0 with sodium hydroxide and extracted with diethyl ether for
24 hours in a liquid-liquid extractor. The ether phase containing
nicotine was evaporated to dryness and the nicotine distilled through
a Widmer column from an alkaline aqueous solution into dilute hydro-
chloric acid. Nicotine was isolated as the dipicrate and recrystallized
from hot water (19). The yield in a typical experiment was 44 mg, m. p.
226-2270 (uncorrected). The radioactivity of each sample was determined
and the results are shown in Table II, along with a calculation of percent

recovery of isotope.

Is olation of e oti‘nine

V‘—

 

Paper chromatography revealed the presence of a small amount
of cotinine both in the aqueous solutions after ether extraction of nicotine
at pH 11. 0 and in the aqueous solutions remaining after distillation of
nicotine.

Cotinine was isolated from three samples from both of these
fractions; these were: 1) the zero time control samples, 2) the pooled
4, 7, and 14 day samples from experiment 1, and 3) the pooled 4, 7,
and 14 day samples from experiment 2. Since the control samples
represented material from 4 plants whereas the samples from plants
fed nicotine-Cl4 represented 8 plants each, 0. 01 ml of non-radioactive
cotinine was added to the former and 0. 02 ml to the latter to serve as
carrier. Cotinine was isolated as the perchlorate derivative according
to the method described by Hucker, e_t a_._1. (14).

Each sample was evaporated under reduced pressure to a small
volume and extracted with chloroform. The chloroform phase was
washed with several portions of pH 4. 7 citrate buffer and then evaporated

to dryness under reduced pressure. The dry residue was taken up in a

12

small volume of diethyl ‘ether and a 1:1 mixture of 70% perchloric
acid and absolute ethanol was added. Upon cooling in a refrigerator
overnight shiny flat needles of cotinine perchlorate precipitated.

The precipitate was washed several times with dry diethyl ether and
recrystallized from hot absolute ethanol. A typical yield of cotinine
perchlorate from a sample to which 0. 02ml of carrier cotinine had
been added was 25 mg, m.p. 217-218O (uncorrected). The melting
point reported by Hucker, e_t a}. (14) is 218-2190. calculation of the
molecular extinction coefficient of authentic cotinine perchlorate
from its absorbancy at 261 my. and comparison of this value with that
of the isolated cotinine perchlorate samples showed that the isolates
were 98 to 99.4% pure.

However, radioactivity measurements showed that the cotinine
perchlorate isolated from the zero time control samples contained
the same level of isotope as the samples from the plants fed nicotine-C14.
The specific activity of all samples remained constant upon recrystal-
lization. These findings indicate that the incorporation of isotope from
nicotine-C14 into cotinine was probably the result of non-metabolic
oxidation of nicotine during the isolation or purification procedures
and does not represent nicotine oxidation by the plants.

All radioactivity measurements were made on a Tracerlab
prOportional flow counter and a Nuclear-Chicago-model 192x scaler.

. Counts were corrected for self-absorption.

II. Nicotine Degradation by an Arthrobacter Species

 

Isolation and characterization of the microorganism

 

In an earlier section it was noted that bacteria were present in
nutrient solution in which tobacco plants were grown. These organisms
in washings from the plant roots had the ability to transform nicotine

to a product having an ultraviolet absorption Spectrum markedly

13

different from that of the parent compound. 7 The tobacco plants had
been raised from seed in Vermiculite«* in a greenhouse and were
subsequently brought into the laboratory and grown in inorganic
nutrient solution (19). Two microorganisms were observed in gram
stained preparations and under a phase contrast microsc0pe in sedi-
ment of nutrient solution; the first was a non-motile gram negative
organism with darker-staining granules occurring in several morpholo-
gies from coccoid to bacilliform. The second was a gram negative,
motile, Short slender bacillus. This organism was observed later in
mixed cultures with the first organism but was never isolated.

The first organism was isolated using the nicotine-salt-yeast
extract liquid and agar media described by Sguros (9). The liquid

medium had the following composition:

 

 

Component Gm/100 ml
1- NicotineT 0. 4
KZHPO4 0. 2
KCl 0. 5
Yeast extract (Difco) 0. 01
MgSO4- 7HZO 0. 005
FeSO4- 7HZO 0. 005

Tap water was used as a diluent and the final pH of the medium was
adjusted to 6. 8. .For agar plates and slants l. 5% agar was added.
Media were sterilized at 1210 for 20-30 minutes in an autoclave.
The liquid medium was also used for observation of the organ-
ism's life cycle and nicotine degrading activity. All cultures were

incubated at 24- 260 .

 

*
Commercial heat-expanded mica.

1'95% nicotine solution (Eastman Kodak Co.) purified by distillation
under reduced pressure.

14

The organism was isolated in the following manner. Tubes
containing sterile liquid medium were inoculated with sediment
obtained by centrifugation of inorganic nutrient solution in which
tobacco plants had been grown. After 2 to 3 days of growth, during
which the cultures were aerated by shaking, an inoculum from each
culture was applied to an agar plate using the streak dilution technique.
When the organisms had grown sufficiently, isolated colonies were
transferred to liquid medium. After a suitable incubation period
samples of the liquid cultures were examined under a phase contrast
microscope and those cultures containing the short, motile bacillus
were discarded. Three repetitions of the above procedure were

sufficient to obtain pure cultures of the Arthrobacter Species.

 

The organism exhibited a life cycle which was complete in 29
hours in cultures aerated by shaking. The stages of this cycle were

essentially the same as were described for Arthrobacter oxydans (9).

 

Aged cultures were in the coccoid cystite form; within 4 hours after
inoculation into fresh liquid medium the cystites formed one to three
germ tubes emerging at obtuse angles to one another. Two or more
organisms were often seen linked together in a chain-like arrangement.
After 12 hours the organisms appeared to be long bacilli with a number
of dark- staining granules apparent within them. At about 17 hours
the organisms were Shorter and the granules were more diffuse,
ellipsoidal in shape, and less well defined. At 24 hours the granules
again became more oval and distinct. After 29 hours the cells had
assumed a homogeneous cystite morphology and were arranged singly,
in'pairs, or in clusters as observed in stained preparations.

The gram reaction of the cells throughout the life cycle was
also very similar to that reported for‘_A. oxydans (9). Cystites were

largely gram positive with very little gram negative material apparent

within the cells. The germ tubes were faintly gram negative.

15

As the cells matured they became more strongly gram negative with
the granules appearing as darker-staining bodies. At about 26 hours
the cells contained distinctly gram positive granules and at 29 hours
had again assumed an almost completely gram positive coccoid form.

The organism produced an unidentified deep blue diffusible
pigment first visible at about 4 hours in aerated liquid cultures. The
intensity of the color increased until at 24 to 30 hours the cultures
appeared blue-black. Upon aging the color became yellow-brown.
The production of a blue pigment has been reported for A. oxydans
as well as for other nicotine-oxidizing bacteria (3, 4, 9).

Sucrose and fructose support growth of the organism but no
acid or gas are produced either in static or aerated cultures.

A. oxydans strains produce acid from these substrates (9). Possibly
then the isolate represents a unique species or strain.

Growth on nicotine-containing agar plates produced small,
raised, gray-white, mucoid and Slightly opalescent colonies. Older
colonies exhibited a dense diffusible blue pigmentation which became
yellow-brown in about 10 days.

Proteolytic activity of the organism was demonstrated by its
ability to liquify gelatin. Liquifaction began at the surface of the
medium and proceeded to about one-half the depth of the tubes in 10
days and proceeded no further. A. oxydans liquifies gelatin in the
same manner (9).

A. oxydans is described as obligately aerobic (9). Nicotine
oxidizing activity of isolate under consideration is greatly diminished
when the cultures are not aerated during incubation, implying a
dependence on molecular oxygen.

Application of 3% hydrogen peroxide solution to colonies of the
organism on nicotine agar plates resulted in immediate vigorous
formation of gas, indicating catalase activity. The catalase reaction

in A. oxydans is reported as strong (9).

16

Nicotine oxidation

 

During the various stages of the life cycle of the organism
samples of the cultures were inactivated by addition' of 10% trichloroe
acetic acid solution and heating in a boiling water bath for 5 minutes.
Insoluble material was removed by centrifugation and aliquots of
the samples were assayed on a Cary Model 11 recording Spectro—
photometer in the ultraviolet region (220 to 340 mu). Distilled water
was used as a blank. The spectra of several samples are shown in
Figure 2.

Nicotine and 6-hydroxynicotine concentrations shown in Figure 3
were calculated from molecular extinction values and absorbancies
at their characteristic absorption maxima (259 mg for nicotine, 295
mu for 6-hydroxynicotine). For nicotine at 295 mu, 6 = 3500; the
value for 6-hydroxynicotine is reported in a later section. The
nicotine concentration remained relatively constant for 13 hours,
after which it rapidly dropped to zero. Formation of 6-hydroxynicotine
began at 13 hours and reached a maximum at 16. 5 hours. The product
concentration then rapidly decreased. The maximum concentration
of 6-hydroxynicotine reached was approximately 50% of the original

nicotine concentration on a molar basis, as Shown in Figure 3.

Isolation and identification of 6-hydroxynicotine

 

The isolation procedure used for 6-hydroxynicotine was essentially
the one described by Hochstein and Rittenberg (2). Two ml of _1_-nicotine
in l. 5 1 of inorganic nutrient solution was inoculated with organisms
and after 4 days of incubation on a shaker the 6—hydroxynicotine concen-
tration had reached a maximum. The mixture was made 0. 1 N in
hydrochloric acid and heated on a steam bath for 15 minutes. The
insoluble material'was removed by filtration. 7 Saturated aqueous

silicotungstic acid solution was added to the filtrate until no more

17

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precipitate formed. The mixture was allowed to stand overnight at
room temperature and the precipitate was removed by filtration.
The silicotungstic acid salt was decomposed with barium hydroxide
and the excess barium was removed by precipitation as barium
carbonate. The filtrate was decolorized with a small amount of
Norite and evaporated to dryness under reduced pressure at 400.
The solid material was extracted with boiling ligroin (b.p. 60-900).
6-Hydroxynicotine was allowed to crystallize from the solution and
was recrystallized several times from this solvent. The melting
point of the isolated 6-hydroxynicotine was 120. 5- 121. 50 (uncorrected).

The picrate derivative was prepared by dissolving about 10 mg
of the compound in 0. 5 ml of water, adding approximately 0. 05 ml of
6 N hydrochloric acid, 1. 5 m1 of absolute methanol, and 2 ml of
saturated methanolic picric acid solution. After standing in a
refrigerator for 2 days a precipitate of the picrate derivative had
formed. The derivative was recrystallized from hot methanol.
The melting point was 164. 5-165O (uncorrected). ~ A mixed melting
point with 6-hydroxinicotine picrate that had been prepared from a
sample of 6-hydroxynicotine produced by the bacterium P-34, which
was kindly supplied by Dr. Sydney C. Rittenberg was not depressed.

An elementary analysis of the isolated 6-hydroxynicotine gave
the following results:*

c,,,}1',.,,0N2 -- Found: 67.44% c, 7.90% H, 15.81% N

Calculated: 67.42% C, 7.86% H, 15. 72%N

Calculation of extinction values in 0. 1 1:1 hydrochloric acid
solution gave 6 = 5440 at 295 mu and c = 11700 at 231 mu.

From Optical rotations in 1.5% aqueous solution measured on
a UniversalHighPrecisionPolarimeter, model 169, a specific
rotation of [0.]?‘D5 - 68. 90 was calculated. This value did not change

upon recrystallization of the compound.

 

* _
Performed by Spang Microanalytical Laboratory, Ann Arbor, Michigan.

20

The infrared absorption spectrum of the isolated compound in
carbon tetrachloride solution was identical with that of Dr. Rittenberg's
sample of 6-hydroxynicotine.

A summary of the physical properties of the isolated compound
and a comparison with those reported by Hochstein and Rittenberg 7(2)

are shown in Table 111.

Table III. Physical properties of 6-hydroxynicotine.

 

 

 

 

Property Isolated Product Reported Value (2)

 

M elting Point 3

6-Hydroxynicotine 120. 5-121. 50 120--1220

Picrate Derivative 165. 5-1660 164. 5-1650
Elementary Analysis 67. 44% C, 7. 90% H, 67. 46% C, 8. 00% H

(CIOHMONZ) 15.81% N 15.62% N

Calculated values:
67.42% N, 7.86% H, 15.73% N

M olecular Extinction

(0.1 .11 HCl)
295 mu 5440 5750
232 mu 11700 12250

Specific Rotation -68. 9O ‘ -54. 8O

 

DISCUSSION

1. Nicotine Degradation by Nicotiana rustica

 

The data in Table 11 show that there is some decrease in the
specific activity of nicotine during the first 4 days after feeding
radioactive nicotine but that there is little change after that time.
The zero time experiment which represents an immediate isolation
of radioactive nicotine after its addition to plants, presents an
appraisal of the dilution of Cl‘by endogenous nicotine. The dilution
was 1.4 in experiment 1 and 2. 5 in experiment 2, indicating that
the added nicotine was a significant portion of the total nicotine in
the plants. The decrease in specific activity of nicotine is presum-
ably the result of two influences, the first being dilution of nicotine
already present by newly synthesized nicotine, the second being
metabolism of nicotine by the plants. A calculation of the recovery
of C14 in nicotine allows an assessment of the metabolism of
nicotine. The results of these calculations, also presented in
Table II, Show that during the first 4 days of metabolism about 30%
of the isotope is lost, presumably throughmetaboiic processes, but
following this little loss occurred up to 14 days.

It has been suggested that nicotine is degraded in tobacco roots
and remains metabolically inert after translocation to the upper
parts of the plant (26). This hypothesis would explain the pattern of
nicotine metabolism obtained in these experiments, assuming that
translocation of nicotine fed to the plants occurred within 4 days.

Leete (16) has fed nicotine-2, 5-c‘4 to Nicotiana tabacum for

 

periods of 1 to 7 weeks and was able to recover in reisolated nicotine

only approximately 5 to 8% of the radioactivity fed in any case.

21

22

It was also ascertained that there was no randomization of radio—
active carbon from the 2 and 5 positions to other portions of the
molecule. The large difference in recovery of isotope between the
present experiments and those of Leete (approximately 70% vs. 5
to 8%) may reflect Species and age differences in the plants.

Solt, Dawson, and Christman (27) supplied sterile root cul-
tures of N. glauca with nicotine labeled with C14 in the pyridine or
pyrrolidine ring and after 38 days recovered 60% of the C” in
nicotine and nornicotine with pyridine labeled nicotine and 43% with
pyrrolidine labeled nicotine. They also presented evidence that
the percent of added nicotine disappearing is related to the amount
of alkaloid supplied. - It appears then that the rate of nicotine
metabolism in N. glauca is comparable with the rate in N. rustica,
whereas that in _N tabacum is greater.

Table I shows the radioactivity of nicotinic acid hydrochloride
samples isolated from plants fed radioactive nicotine. The calcu-
lated dilutions of isotope seem low enough to support the hypothesis
that nicotinic acid is formed directly from nicotine in the plants by
oxidation of the pyrrolidine ring. Metabolic degradation of the
pyridine ring and its resynthesis would involve dilution by a number
of metabolite pools, resulting in a high dilution of isotope. . The most
efficient biosynthetic precursors of nicotine investigated to date give
dilutions in the order of 150 (18).

Nicotinic acid derived from nicotine appears to enter the
metabolic pool and presumably is converted to bound forms such as
the pyridine nucleotides, since dilution of isotOpe increases some-
what with time.

Wada and co-workers (28) have demonstrated extensive oxi-
dation of nicotine by air at room temperature. One of the several
products isolated was nicotinic acid. The lack of radioactivity in

nicotinic acid hydrochloride isolated from the zero time control

23

plants indicates that there was no such nonemetabolic oxidation of
nicotine occurring during the isolation and purification procedures
employed in this study.

The C14 recovered in nicotinic acid hydrochloride samples
amounted to less than 0. 1% of the total C14 fed to the plants; that in
the cotinine perchlorate samples was about 0. 01% of the total. The
fate of the remainder of the Cl4 unaccounted for is unknown.

It would be interesting to determine whether the ratio of radio-
activity in the carboxyl carbon and the pyridine ring of the isolated
nicotinic acid is the same as that of the nicotinic acid moiety of the
original nicotine. However, the small amount of nicotinic acid iso—
lated precluded this experiment. Dawson, it a_l. (29) have shown that
the pyridine ring of nicotinic acid is incorporated into nicotine in
sterile tobacco root cultures whereas the carboxyl carbon is not.
Whether the formation of nicotinic acid from nicotine is a reversal
of the biosynthetic route or aunique degradative pathway cannot be
determined by the present study.

The results obtained by isolation of cotinine from the plants do
not eliminate this compound as a possible metabolite of nicotine.

A small amount of Cl4 could have been introduced metabolically into
cotinine from nicotine only to be overshadowed by the Cl4 entering

the molecule by non-metabolic processes. If the normal endogenous
concentration of cotinine were very low this possibility becomes more
conceivable. Further study would be necessary to draw any definite
conclusions about the role of cotinine in nicotine metabolism in the
tobacco plant.

Since no product in the metabolic pathway closer to the parent
compound than nicotinic acid has been shown to be derived from
nicotine in plants, it is impossible to tell at what point in the molecule

the primary oxidation of nicotine occurred. There is one pathway that

24

does not involve primary oxidation of the pyridine ring, however, since
nicotinic acid is produced. This excludes the possibility of oxidation
and subsequent reduction of the pyridine ring.

The metabolically essential character of nicotinic acid has been
well established. Its production from nicotine in the tobacco plant
suggests one possible part nicotine plays in the metabolic economy of

the plant.

II. Nicotine Degradation by an Arthrobacter Species

 

In contrast to the formation of nicotinic acid from nicotine in the
intact tobacco plant, the microorganisms on the plant roots oxidize
the alkaloid to 6-hydroxynicotine.

The ultraviolet absorption spectra presented in-Figure 2 Show
that the characteristic peaks of 6-hydroxynicotine are retained until
the ultraviolet absorption disappears. This seems to indicate that no
other ultraviolet-absorbing compoundwas produced by the organism.
The second product in the metabolic pathway might then be one in
which the pyridone ring of 6-hydroxynicotine is altered, since this
part of the molecule is largely responsible for ultraviolet absorption.
However, since intact cells were used in these experiments no definite
conclusions can be drawn. Perhaps the system catalyzing the reaction
nicotine-)6-hydroxynicotine operates as an exoenzyme and subsequent
steps take place intracellularly. Later products might then have been
removed from the samples along with protein and other trichloroacetic
acid-insoluble and heat-coagulable material.

The enzymatic character of nicotine oxidation in these experi-
ments was demonstrated by the fact that samples which were acidified
and heated immediately after addition of microorganisms showed no

change in the typical nicotine absorption spectrum after 48 hours.

25

Cell-free protein fractions from the bacillus P-34 have been
obtained that catalyze the successive oxidation of nicotine to 6-hydroxy-
nicotine (2), 6-hydroxypseudooxynicotine (3), and 2, 6-dihydroxypseudo-
oxynicotine (4). The next step appears to be the formation of the I
unidentified blue pigment (4). The first step in nicotine oxidation by

the Arthrobacter Species is the same as that of P-34 and the blue pig-

 

ment is also produced, suggesting that the rest of the oxidative pathway

might be the same. Dr. Rittenberg is currently testing the Arthrobacter

 

species to determine whether cells adapted to growth on nicotine can
also utilize 6-hydroxynicotine, 6-hydroxypseudooxynicotine, and

2, 6-dihydroxypseudooxynicotine, and also for the presence of the
enzymes involved in these oxidations.

The rate of nicotine oxidation is greatest in the Arthrobacter

 

species during the fragmenting stage in the life cycle of the organism.
This may reflect the rapid increase in "total cell substance" reported
by Sguros (9) for this stage ofA. oxydans growth; another possibility is
that the enzymes responsible for catalysis of nicotine oxidation are not
elaborated until this stage of maturation is reached. Approximately
50% of the nicotine present is converted to 6-hydroxynicotine by the
organism. Production of the blue pigment, which appears to be a
nicotine metabolite, may account for at least part of the remaining
nicotine.

The presence of these microorganisms on the roots of tobacco
plants is probably the result of an adaptive phenomenon. Their ultimate
source might be soil or air; they could also be carried on toabcco
seeds. The presence of nicotine-degrading bacteria on the surface of
tobacco seeds has been reported (7). Dawson (30) has shown that
sterile tobacco root cultures excrete a large share of the nicotine they
produce into the medium in which they are grown. If intact plants also
excrete nicotine from their root systems the surrounding medium would
be favorable for growth of microorganisms that can adapt to the use of

nicotine as a source of_,-carbon or nitrogen or both.

SUMMARY

1. When tobacco plants (Nicotiana rustica) are supplied with

 

nicotine-CI4 approximately 30% of the radioactivity is lost from
nicotine within 14 days. The greatest loss occurs within 4 days.

2., Nicotinic acid recovered from the plants contains a Signifi-
cant amount of c“ with little dilution of specific activity, indicating
that nicotine can be converted to this metabolically important com-
pound, presumably by oxidation of the pyrrolidine ring of nicotine.

3. Cotinine recovered from the plants is also radioactive, but
evidence indicates that the incorporation of C14 is the result of a
non-metabolic process.

4. There is present on the roots of N. rustica plants grown in
this laboratory a microorganism very Similar in cultural and morpho-

logical characteristics to Arthrobacter oxydans.

 

5. This Arthrobacter species is capable of catalyzing the oxi-

 

dation of nicotine in approximately 50% yield of 6-hydroxynicotine in
an aerated medium containing nicotine, inorganic salts, and a small
amount of yeast extract.

6. 6-Hydroxynicotine is identified by its melting point, the
melting point of the picrate derivative, ultraviolet and infra-red
absorption spectra, molecular extinction values, elementary analysis,
and specific rotation.

7. The significance and implications of these findings are

discussed.

26

10.

11.

12.

13.

14.

15.

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795 (1960).

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.~ E. Wada, Arch. Biochem. and Biophys., _7_2_, 145 (1957).

 

. W. G..Frankenberg and A. A. Vaitekunas, Arch. Biochem. and
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