JOURNAL OF BACTERIOLOGY, Aug. 1974, p. 527-533 Vol. 119, No.

2
Copyright 0 1974 American Society for Microbiology Printed in U.SA.

Biogenesis of ,B-Carotene in Mycobacterium kansasii

HUGO L. DAVID
Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, U.S.
Department of Health, Education, and Welfare, Atlanta, Georgia 30333
Received for publication 1 April 1974

The biogenesis of #-carotene in the photochromogen Mycobacterium kansasii

consists of two reactioxis. The first reaction is photochemical, and is dependent
on the wavelength of the incident light and on oxygen but is independent of
temperature. The second reaction does not require illumination, and is depend-
ent on the temperature and on oxygen. The latter, or dark reaction,
requires the synthesis of new protein, and was shown to have the characteristics
of an inducible system. Carotenogenesis was stimulated by incident light of
wavelengths of 420, 540, and 650 nm. Immediately after illumination there was
an increase in the synthesis of ribonucleic acid and ,-carotene accumulation
started after a lag of 8 to 10 min. The synthesis of a-carotene exhibited

Downloaded from jb.asm.org by on February 23, 2008

temperature dependence with an optimum of about 36 C.

Mycobacterium kansasii is a photochromogen Bacteria and growth conditions. M. kansasii

(16, 17) in which the mechanism of carotenoid
synthesis has not been investigated before. In
other photochromogens the mechanism of ca-
rotenoid synthesis consists of two main reac-
tions. The first is photochemical, and its action
spectrum seems characteristic of each orga-
nism; the second is independent of light (dark
reaction) and has the characteristics of induci-
ble systems. The subject was recently reviewed
by Batra (1).
In M. kansasii the pigment that accumulates
after illumination was previously identified as
,-carotene; other carotenoids identified to date
are phytoene, phytofluene, lycopene and a-
carotene (6, 7, 15).
The purpose of this report is to describe the
main features of the photochromogenic response
and the results of experiments into the mecha-
nism of carotenoid synthesis in M. kansasii.
MATERIALS ANI) METHODS
Materials. Commercially available organic sol-
vents, analytical grade, were used without further
purification. Chloramphenicol was obtained from
Sigma Chemical Co., St. Louis, Mo., and rifampin
was from Dow Chemical Co., Indianapolis, Ind. Di-
phenylamine was purchased from Fisher Scientific
Co., N.J. Aluminum oxide, acid washed, was pur-
chased from Merck Co., Rahway, N.J. [2-"4C]DL-
mevalonic lactone (specific activity 135 uCi/mg),
[1-'_4Cacetate (690 pCi/mg) and [2-14Clacetate (667
pCi/mg) were purchased from Amersham Searle,
Arlington Heights, ill. [6-sHIuracil (27.6 Ci/mmol),
Aquafluor, and Liquifluor were purchased from New
England Nuclear Corp., Boston, Mass.
527
strain no. 37 from the Mycobacteriology Section,
Center for Disease Control, was used in all experi-
ments. The bacteria were grown in a chemically
defined medium containing in grams per liter the
following: ferric ammonium citrate, 0.05; ammonium
chloride, 2.0; casitone, 0.3; glycerol, 20.0; citric acid,
2.0; potassium sulfate, 0.6; dipotassium phosphate,
4.0; sodium glutamate, 1.75; magnesium chloride, 1.2.
When appropriate, the medium was made solid by
the addition of 1.5% (wt/vol) of agar.
Extraction of the carotenoids. The wet cell mass
was extracted with chloroform-methanol (1:1, voV
vol) until no more pigment was visible. The extracts
were evaporated to dryness and saponified in 4%
potassium hydroxide in methanol for 1 h at room
temperature. The unsaponifiable lipids were then
extracted into petroleum ether.
Column and thin-layer chromatography. Col-
umn chromatography was performed on aluminum
oxide columns (2.5 by 14.0 cm). The carotenoids were
eluted by the stepwise addition of 50 ml each of
petroleum ether; 25, 50, and 75% diethyl ether in
petroleum ether; and finally diethyl ether. Usually,
7.5-ml fractions were collected.
For thin-layer chromatography (TLC), Silica Gel
plastic coats without fluorescent indicator (obtained
from Eastman Kodak Co., Rochester, N.Y.) and TLC
plates ALOX 25-22 (obtained from Brinkman Instru-
ments, Inc., N.Y.) were used. The plates were devel-
oped in hexane or in hexane-acetone.
Assay of radioactivity. [6-'Hjuracil-labeled cells
were sampled into cold 10% trichloroacetic acid. The
cell suspensions were kept at 4 C ovemight and were
then filtered through membrane filters (0.8 pm pore
size; Millipore Corp.) and washed twice with cold 10%
trichloroacetic acid. The filters were then transferred
to scintillation vials and the radioactivity measured
in Aquafluor.
528 DAVID
The lipid extracts were counted in Liquifluor di-
luted in toluene, as recommended by the manufac-
turer.
The radioactivity was measured in a Nuclear Chi-
cago liquid scintillation spectrometer, Isocap 300
model.
Assay of carotenoids. The carotenoid content in
the extracts was determined as recommended by
Liaaen-Jensen and Jensen (10) at 450 nm in a
Beckman DU spectrophotometer. In all assays, the
pigments were dissolved in petroleum ether, and this
same solvent was used in the blank.
Identification of,-carotene. ,8-carotene was puri-
fied by a combination of column chromatography and
TLC. The pigment on the TLC plates was scraped
and dissolved in acetone. The acetone extracts were
evaporated to dryness in vacuum, redissolved in
petroleum ether, and ,8-carotene was then identified
by its characteristic spectrum.
RESULTS
Main features of the photochemical
reaction. As shown in Fig. 1, the photochemical
reaction depends on the wavelength of the
incident light. The action spectrum exhibits
three maxima, at 420, 540, and 650 nm, and
minima at 360, 470, 600, and 700 nm. Table 1
shows that pigment accumulated in cells illumi-
nated at 4 C and then incubated in darkness at
36 C. When illumination was done in an atmos-
phere of nitrogen and incubation in darkness at
36 C took place in an atmosphere of either
c
0 perature of incubation. The study of the tem-
-Q
o, 0. 1-
I)
-Q
II
0 - -r..
-30 IrI .
l-
300 400 500 600
Wavelength, nm
FIG. 1. Action spectrum of photochromogenecity.
The cell mass on the surface of solid medium was
illuminated for 1 h with a high intensity monochroma-
tor. After illumination the cultures were shielded and
the covers were kept loose. After overnight incubation
the pigments were extracted. The relative content of
pigment was estimated by measuring the absorbancy
at 450 nm.
J. BACTERIOL.
TABLE 1. Temperature and oxygen dependencies of
the photochromogenic reactiona
Expt conditions ug of Pigment per
100 mg of cells
Light Dark dry weight
4C 36 C 20
36C 4C <5
36C 36C 25
02 N2 <1
N2 02 4
02 02 30
N2 N2 <1
a
In these experiments the cells were illuminated for
1 h and incubated in darkness overnight, then the
pigments were extracted and assayed. Except where
indicated, incubation in darkness was at 36 C.
oxygen or nitrogen, only small amounts of
pigment accumulated. Thus the photochemical
Downloaded from jb.asm.org by on February 23, 2008
reaction is dependent on the wavelength of the
incident light and on oxygen but not on temper-
ature.
Main features of the dark reaction. When
the cells were illuminated for increasing inter-
vals of time, the concentration of pigments
increased exponentially for about 60 min.
Thereafter, the concentration of pigment in-
creased by a constant factor for about 4 days of
continuous light exposure, and it leveled off at
day 5 (Fig. 2). The daily microscope observation
of the cell mass revealed the presence of pig-
ment crystals at day 7, when the accumulation
had already stopped.
The data in Table 1 show that the dark
reaction is dependent on oxygen and the tem-
perature dependence showed that the optimal
temperature for pigment synthesis was about
36 C (Fig. 3).
Incorporation of carbon- 14 into the
carotenoids. Attempts to incorporate car-
bon-14 from mevalonic [2-"4C ]acid were unsuc-
cessful. However, carbon-14 from [81-"Clace-
tate and [2- "C ]acetate were rapidly incor-
porated into the petroleum ether extracts. The
results showed that [2-'4C]acetate was a most
suitable substrate, probably because less car-
bon is lost as respiratory "4CO2 (Table 2).
Identification of,-carotene. Figure 4 shows
the elution profile of the radioactivity in the
petroleum ether epiphase from the extracts of
the cells grown in darkness and after illumi-
nation. The data showed that most of the
radioactivity in the extracts from the bacteria
grown in darkness eluted with petroleum ether;
three other radioactive fractions were in trace
VOL. 119, 1974
1-CAROTENE BIOGENESIS IN M. KANSASII 529

4-
40-

-4--

-C

u'
0 20 3.

0
a)
0)

U20 3 0 9 2
0~~~~ E
0
10~ ~ ~ ~ ~~ 5 i 0

a)

0
a)
0 30 60 9 10

Downloaded from jb.asm.org by on February 23, 2008

0

Minutes 2..

FIG. 2. Dependence of carotenogenesis on the du-

ration of illumination. In this experiment the cells
were illuminated for the indicated times, were incu-
bated at 36 C in darkness overnight, and the pigments
were then extracted and assayed. In the experiment
shown in the inset, the pigments were extracted and 36 40
assayed at the indicated times (the time scale in the
inset is in days) of continuous illumination at 36 C.
Temperature, 0C
amounts. The elution profile of illuminated FIG. 3. Dependence of carotenogenesis on temper-
cells showed the disappearance of the above ature. The cells were illuminated for 2 h, were
major fraction and the appearance of newer incubated and in darkness at the indicated temperatures
ones.
overnight, the pigments were then extracted and
Fraction I was not further analyzed. TLC of assayed.
fraction II on Alox 25-22 developed with 1%
acetone in hexane showed a single yellow spot,
Rt 0.52, that gave the spectrum characteristic of TABLE 2. Distribution of carbon-14 from acetate in
d-carotene; TLC of fraction III in the same the cells and respiratory CO2a
system showed six distinct pigments (Rt 0.0, Radio-
yellow; R1 0.06, red; Rf 0.10, yellow; R, 0.14,
red; Rt 0.32, brown; Rr 0.53, yellow). The pig- Substrate
Substrate Radioactivity inivi- % 1'CO"D
in cells respi- %1c2
ratory
ment with Rt 0.14 exhibited the spectrum char-
acteristic of lycopene and the pigment with Rt co,
0.53 had the spectrum of a-carotene (Fig. 5). [1-14C ]acetate 103,884 17,590 14.4
The remaining pigments were not identified. [2-14C Jacetate 174,820 4,230 2.3
Table 3 shows that ,-carotene was a small
a The cell suspensions (1.0 ml) were transferred to a
fraction of the total radioactivity in the lipid
extracts. ,-carotene purified by preparative screwcap test tube (16 by 125 cm) containing an inner
TLC was shown to chromatograph as a single vial (0.5 by 5.0 cm); 500 Aliters of concentrated potas-
spot in various systems. sium hydroxide was added. The vials were then illumi-
nated for 1 h, the substrate was added, and the caps
Time course of carotenoid synthesis. When were tightened. After 1 h of incubation, in the pres-
the cells were illuminated for 1 h and shifted to ence of substrate, samples were taken and the radio-
darkness and the pigments were extracted and activity in the cells and the potassium hydroxide was
assayed colorimetrically at regular intervals measured.
during dark incubation, the curve shown in Fig. b Percentage of total reactivity.
530 DAVID J. BACTERIOL.

12- lates during growth in darkness disappeared

soon after illumination and that the chromatog-
raphy profiles changed in a manner suggestive
of a sequential synthesis of the d-carotene inter-
mediate precursors. (-carotene was synthesized
within 8 to 10 min (Fig. 8 and 9).
Time course of carotenoid and RNA
synthesis. Figure 7 shows that the rate of
synthesis of RNA increases almost immediately
after illumination and returns to a normal rate
about 40 min after the cells are shifted back to
darkness. Changes in the rate of incorporation
ro0 of carbon-14 were apparent after 20 min of
x
illumination. When the cells were sampled into
rifampin or chloramphenicol, the synthesis of
E the carotenoids was interrupted (Fig. 10). Ca-
0
rotenoid synthesis was blocked until the 120th
minute by rifampin and until the 160th minute
by chloramphenicol.

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mE

Fraction Number
FIG. 4. Elution profile on aluminum oxide column
of the petroleum ether epiphase from extracts of M.
kansasii. The elution mixtures were 50.0 ml of pe-
troleum ether; 25, 50, and 75% diethyl ether in
petroleum ether; and 50.0 ml of diethyl ether. Sym-
bols: 0- 0, grown in darkness; *-*, exposed to
light. 0

n)
6 was obtained. The data showed that pigment
accumulation began after a lag of about 1.25 h;
however, it was clear that pigment had accumu-
lated during the 1 h of previous illumination.
To examine the events that occurred during
the lag period, we performed the experiment
depicted in Fig. 7. The data showed that
carbon-14 was incorporated into the petroleum
ether extracts within about 20 min of light
exposure. The high background radioactivity
observed in this experiment suggested that the
net synthesis of other lipids extracted into the
petroleum ether might have masked the initial
steps of carotene synthesis. In other experi- Wavelength, nm
ments, the extracts from samples taken at
regular intervals were applied on either TLC FIG. 5. Spectra of the fractions I, II, and III indi-
cated in Fig. 4. Fraction I, spectrum in ethanol
plates or chromatography columns. The results (O-0, oxidized; 0-----0, reduced with sodium
of a typical experiment are shown in Fig. 8. The borohydride). Fractions II and III spectra in pe-
data showed that the compound that accumu- troleum ether.
VOL. 119, 1974 -CAROTENE BIOGENESIS IN M. KANSASII 531
TABLE 3. Identification of labeled al-carotenea and its substrate has been postulated to be
either a repressor that is rendered nonfunctional
Determination Time of incubation in
darkness (min) or a compound that is converted into an in-
Sample 10 60 2- ,dark light dark - 200
_
Cells dry weight (mg) 850 850

4
.I-

Total carotenoids (sg/100 mg 2 8 U',

of cells) 0
0 1- -100 °
x
Chloroform-methanol ex- 2,412 33,047 x
tracts (counts per min per E E
100 mg of cells) C) C0
Unsaponifiable lipids 2,078 30,000
(counts per min per 100 mg 180 240
of cells) -50 0 60 120
Minutes
,-carotene (counts per min 70 312 FIG. 7. Time course of carotenoid and RNA syn-
per 100 mg of cells) thesis. To a culture grown in darkness, [2-'4C]acetate
#-carotene (sp actb) 0.015 jCi/mg 0.017 $Ci/mg (10 1sCi/100.0 ml) and [2-3HJuracil (10 ,Ci/100.0 ml)
were added. The mixtures were preincubated 30 min,

Downloaded from jb.asm.org by on February 23, 2008

a The unsaponifiable lipids extracted at the indicated
times of incubation in darkness were purified by preparative
TLC. The radioactivity that cochromatographed with authen-
tic ,-carotene used as a carrier in the chromatograms were
measured as indicated in the text.
b In calculating the specific activity, one curie was taken to
and then 1.0-ml samples were taken at 10-min inter-
vals. At each time interval a sample was taken into
1.0 ml of cold 10%o trichloroacetic acid and another
was taken into 1.0 ml of an equal mixture of chloro-
form and methanol. At time 0 the cells were illumi-

be equilavent to 2.22 x 1012 counts/min. nated and at time 60 the cells were shifted back to
dark conditions. Symbols: 0, 3H; 0, 14C.
21
0
39 0-
,-

C =
004 0~~~~~~
O-C
S
0
o
1 0

1.I...0. . .I 1 E
Q

0 60 120 180 240

Minute s
FIG. 6. Time course of pigment synthesis. The cells
were illuminated for 1 h and the pigments were then
extracted at the indicated intervals during incubation
in darkness.

Inhibition of d-carotene synthesis by

diphenylamine. As shown in Fig. 11, di-
phenylamine blocks the synthesis of ,-carotene
in M. kansasii, but contrary to reports in other
microorganisms (1, 5, 13, 14, 16) the inhibition
did not result in the accumulation of phytoene culture incubated in darkness [2-_4C]acetate was
or other earlier precursors.
DISCUSSION
The mechanism of biosynthesis of carotenoids
in photochromogenic microorganisms was re-
cently reviewed by Batra (1). It consists of two
distinct reactions. The first is photooxidative,
Fraction Number
FIG. 8. Time course of carotenoid synthesis. To a
added. The mixture was preincubated 30 min, and a
10.0-ml sample was added to the same volume of
chloroform-methanol. The mixture was then illumi-
nated and 10.0-ml portions were sampled into chloro-
form-methanol at the indicated intervals. The carote-
noids were then extracted and the extracts were
applied on aluminum oxide columns. The radioactiv-
ity in the fractions was measured in Liquifluor.
532 DAVID J. BACTERIOL.

15- whose kinetics we have examined in somewhat

more detail than has been previously reported.
The use of radioactive substrates showed that
the long lag reported in other bacteria (1-4, 11,
* 12) may be an apparent lag. Indeed, we ob-
10 served a lag in ,B-carotene synthesis of about
c'J 1.25 h when the accumulation of the pigment
0
x
was estimated colorimetrically, but when the
E0. lag was examined by following the incorporation
Q. /- of carbon-14, we found that the induction reac-
tion started within the first 2 min of illumi-
nation. #-carotene was synthesized 8 to 10 min
from the time illumination was started. Within
this time span, intermediate precursors accu-
mulated in a manner suggestive of a sequential
synthesis (8) of the protein enzymes in the
10 20 30 40 50 60 pathway. After the 10th minute, when the
Minutes synthesis of 3-carotene began, the putative
intermediates were no longer found or were
FIG. 9. Time course of fl-carote, ne synthesis. The detectable only in trace amounts, which indi-
experiment was conaucteu as aescritea In rtg. 6. ine

Downloaded from jb.asm.org by on February 23, 2008

fractions were applied in Silica Gel TLC plates and
developed in hexane-acetone. The radioactivity that
cochromatographed with authentic (3-carotene was
then measured in Liquifluor.

201 dark light 02

I x Chl
o'
C

0I 0 1
o(io I
x101
E
4R-X.--x-- Is
C-) I a.
ro
0

x
E
10 16
0

-50 0 60 120 180 240

Minutes
FIG. 10. Inhibition of carotenoid synthesis by
chloramphenicol and rifampin. To a culture grown in
darkness, [2-'4C]acetate (10 uCi/100.0 ml) was added.
The mixture was preincubated 3 min, and then 1.0-ml
portions were sampled into 1.0 ml of chloramphenicol
(20.0 jug) and rifampin (2.0 /lg). At time 0 the cells
were exposed to light.

ducer; the second appears to be an induction

reaction. Our observations indicate that the
mechanism of carotenogenesis in M. kansasii is
fundamentally similar to that described for
other photochromogens. A major difference,
however, was found in the photochemical reac-
tion per se where the action spectrum exhibited
three maxima: 420, 540, and 650 nm. Since the
main features of the photochromogenic reaction
were similar to those described for other bacte-
rial systems, they will not be further discussed.
Instead, we will discuss the induction reaction
Fraction Number
FIG. 11. Inhibition of (3-carotene synthesis by di-
phenylamine. A culture of M. kansasii was illumi-
nated for 1 h and was then divided into two equal
volumes. [2-'4C]acetate was added to both flasks. One
flask was retained as the control and 50 jig of
diphenvlamine per ml was added to the other flask.
The mixtures were then incubated for 1 h when the
pigments were extracted and chromatographed on
aluminum oxide columns.
 V-CAROTENE
VOL. 119, 1974 BIOGENESIS IN M. KANSASII
cated that the intermediates were rapidly con-
verted into the end product.
The time course of induction showed that
messenger ribonucleic acid (mRNA) synthesis
began almost immediately after illumination.
The synthesis of mRNA did not stop at once
after the cells were shifted to dark conditions
but continued for 40 to 50 min. On the other
hand, the synthesis of the carotenoids contin-
ued for several hours after the cells were shifted
to darkness. Inhibition experiments with rifam-
pin and chloramphenicol showed a lag from the
time of induction of about 120 and 160 min,
respectively. These experiments clearly showed
that the continued synthesis of ,-carotene for
several hours after illumination was withdrawn
was due to residual enzyme activity rather than
to a continuing synthesis of the protein en-
zymes. Furthermore, since rifampin acts at the
transcription step and chloramphenicol at the
translation step of protein synthesis, the time
difference between the two inhibitions appears
to represent the residual enzyme synthesis from
mRNA present at the steady state (9).
The above observations explain the typical
time course curve of pigment accumulation.
Initially, pigment accumulates at an increasing
rate that is proportional to the increase in
concentration of mRNA and protein enzymes;
when induction stops, the residual synthesis of
protein and the synthesis of pigment by the
residual protein enzymes reaches a steady state
and, therefore, pigment accumulation becomes
linear in respect to time. Inhibition experiments
with diphenylamine did not show the accumu-
lation of phytoene (5, 13, 14, 16), which suggests
that this was a very early inhibition in the
induced pathway and that, possibly, phytoene
is the first carotenoid to be synthesized on
induction.
LITERATURE CITED
1. Batra, P. P. 1971. Mechanism of light-induced carotenoid
synthesis in nonphotosynthetic plans, p. 47-76. Photo-
physiology, vol. 6. Academic Press Inc., New York.
533
2. Batra, P. P., R. M. Gleason, Jr., and J. Jenkins. 1969.
Mechanism of photoinduced and antimycin A-induced
carotenoid synthesis in Mycobacterium marinum. Re-
quirements for carotenogenesis and further evidence for
protein synthesis following induction. Biochim. Bio-
phys. Acta 177:124-135.
3. Batra, P. P., and H. C. Rilling. 1964. On the mechanism
of photoinduced carotenoid synthesis: aspects of the
photoinductive reaction. Arch. Biochem. Biophys.
107:485-492.
4. Batra, P. P., and L. Storms. 1968. Mechanism of photoin-
duced and Antimycin-A induced carotenoid synthesis
in Mycobacterium marinum. Effect of proflavine. Bio-
chem. Biophys. Res. Commun. 33:820-827.
5. Cohen-Bazire, G., and R. Y. Stainier. 1958. Specific
inhibition of carotenoid synthesis in a photosynthetic
bacterium and its physiological consequences. Nature
(London) 181:250-252.
6. David, H. L. 1973. Response of Mycobacteria to ultravio-
let light radiation. Amer. Rev. Resp. Dis. 108:1175-
1185.
7. Ebina, T., M. Motomiya, K. Munakata, and 0. Satake.
1962. Pigments of unclassified mycobacterial. Amer.
Rev. Resp. Dis. 86:740-743.
8. Harding, R. W., P. C. Haurig, and H. K. Mitchell. 1969.
Downloaded from jb.asm.org by on February 23, 2008
Photochemical studies of the carotenoid biosynthetic
pathway in Neurospora crassa. Arch. Biochem. Bio-
phys. 129:696-707.
9. Kepes, A. 1963. Kinetics of induced enzyme synthesis.
Determination of the mean life of Galactosidase-
specific messenger RNA. Biochim. Biophys. Acta
76:293-309.
10. Liaaen-Jensen, S., and A. Jensen. 1971. Quantitative
determination of carotenoids in photosynthetic tissues,
p. 586. In Methods of enzymology, vol. 23. Academic
Press Inc., New York.
11. Rilling, H. C. 1962. Photoinduction of carotenoid synthe-
sis of a Mycobacterium sp. Biochim. Biophys. Acta
60:548-556.
12. Rilling, H. C. 1964. On the mechanism of photoinduction
of carotenoid synthesis. Biochim. Biophys. Acta
79:464-475.
13. Rilling, H. C. 1965. A study of inhibition of carotenoid
synthesis. Arch. Biochem. Biophys. 110:39-46.
14. Schlegel, H. D. 1959. Conversion of carotenoids to oxy-
carotenoids by Mycobacterium phlei. J. Bacteriol.
77:310-316.
15. Tarnok, I., and Zs. Tarnok. 1971. Carotenes and xanto-
phylls in Mycobacteria. II. Lycopene, a- and ,B-
carotene and xantophyll in mycobacterial pigments.
Tubercle 52:127-135.
16. Turian, G., and F. Haxo. 1952. Further use of diphenyla-
mine for the study of carotenoid biosynthesis in Myco-
bacterium phlei. J. Bacteriol. 63:690-691.
17. Wayne, L. G., and J. R. Doubek. 1964. The role of air in
the photochromogenic behavior of Mycobacterium
kansasii. Amer. J. Clin. Pathol. 42:431-433.