LOCATION OF THE CAROTENOID PIGMENTS OF COEYNEBACTERIUM

SPECIES STRAIN 7E1C

APPROVED:

Major Professor

Minor7Professo

Director of the Department ̂ f Biology

Dean lof the Graduate School

/ ' / /

Wilkinson, Joanne C., Location of the Carotenoid Pigments

of Corynebaaterium Species Strain 7E1C. Master of Science

(Biology), August, 1973, 33 pp., 1 table, 4 figures, bibliog-

graphy, 76 titles.

Carotenoid pigments occur in a large number of red, orange

and yellow nonphotosynthetic microorganisms of the genera

Mycobacterium3 Nocardia and Micrococcus. The chromogenic

organism tentatively named Corynebaaterium species strain

7E1C is closely related taxonomically to Nocardia and Myco-

bacterium. This strain produces carotenoid pigments which

are xanthophylls rather than carotenes. In most of the

organisms investigated so far, the carotenoids are found in

the cell membrane where they perform an unknown role, although

several functions have been hypothesized.

The purpose of this investigation was to determine the

site of the carotenoid pigments in C. spp. strain 7E1C as a

step towards resolving the role of the pigment in the cell.

Initially, protoplast formation of C. spp. strain 7E1C

was attempted by a number of procedures used successfully with

other microorganisms. It was reasoned that production of pig-

mented protoplasts would indicate that the carotenoids were

located in the cell membrane or cytoplasm. On the other hand,

pigmentless protoplasts would indicate that the cell wall was

the site of carotenoid pigments. All attempts to produce

protoplasts were unsuccessful.

As an alternate approach to the determination of the

site of the carotenoids, a pure cell wall fraction was pre-

pared by differential centrifugation and chemical treatment

of broken cells. The purity of the cell wall preparation

was verified by electron microscopy.

Pigment assays of the individual fractions were estimated

1 ̂ using an extinction coefficient CEf ) of 2S00 at 483 nm. 6 v 1 cnr

Approximately eighteen times as much pigment was located in

the cell membrane as in the cell wall. The small amount of

pigment found in the cell wall may be due to trace cell mem-

brane contamination of the cell wall. Since function is

presumably coincident with location, carotenoid pigments of

C. spp. strain 7E1C may serve some purpose in stabilization or

protection at the membrane level.

Carotenoids of C. spp. strain 7E1C cannot be extracted

completely with the usual solvents but only when these solvents

are used in conjunction with a substance that specifically

solubilizes protein. These results indicate that the pig-

ments may be bound in some way. This linkage may be similar

to the carotenoid-glycosidic bond demonstrated in Sarcina

morrhuae and as such could serve as a means of stabilizing the

membrane.

LOCATION OF THE CAROTENOID PIGMENTS OF COEYNEBACTERIUM

SPECIES STRAIN 7E1C

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Joanne C. Wilkinson, B. S.

Denton, Texas

August, 1973

TABLE OF CONTENTS

Page

LIST OF TABLES iv

LIST OF ILLUSTRATIONS v

INTRODUCTION 1

MATERIALS AND METHODS 6

RESULTS AND DISCUSSION 11

CONCLUSION 24

SUMMARY 26

REFERENCES 27

i n

LIST OF TABLES

Table Page

I. Pigment Recovery from Differential Centrifugation 19

IV

LIST OF ILLUSTRATIONS

Figure Page

1. Purification of Cell Walls 13

2. Whole Cells of Corynebaotevium Species Strain 7E1C 15

3. Cells of Corynebaoterium Species Strain 7E1C After Disruption in the MSK Cell Homogenizer. 17

4. Purified. Cell Walls of Corynebacterium Species Strain 7E1C 18

INTRODUCTION

"Carotenoids are a class of hydrocarbons (carotenes)

and their oxygenated derivatives (xanthophylls) consisting of

eight isoprenoid units joined in such a manner that the arrange-

ment of the isoprenoid units is reversed at the center of the

molecules so that the two central methyl groups are in a 1,5

positional relationship" (25). These compounds are chemically

related to lycopene, the tomato pigment, and are widely dis-

tributed in nature (74). In photosynthetic organisms,

carotenoids participate in light absorption for photosynthesis,

protection of chlorophylls from light destruction and in some

way contribute to the complex infrared spectrum of bacterio-

chlorophyll (12,28,59,74). Several fungi (10,20,21,26,27)

and many yellow, red and orange pigmented nonphotosynthetic

bacteria (1,4,11,30,31,34,37,42,60,63,65,63) also contain

carotenoid pigments. The presence of carotenoid pigments in

microorganisms seems to have taxonomic significance only in

the algae (10).

An integral part in the determination of the relation-

ship of carotenoid pigments of nonphotosynthetic bacteria to

cell functions is the localization of the carotenoids in the

cell. In Savoina lutea, for example, the carotenoid pigments

are located in the cell membrane (45) and are thought to

prevent photodynamic killing by either internal or external

photosensitizers (44) . Micrococcus lyeodeikticus (54),

Sarcina flava (66), Staphylococcus aureus (31), Halobacterium

halobium and Halobacterium salinarium (61) also contain

carotenoids which seem to be associated with the cell membrane

rather than with the cell wall. On the other hand, carotenoids

are located in the cell wall of Micrococcus radiodurans (76)

and in the "knallgas" bacterium 12/60/x (15) . Work and

Griffith (76) showed that the site of carotenoid pigments

was the middle layer of the three-layered cell wall of M.

radiodurans.

Salton and Freer (55) considered carotenoid pigments to

be localized almost exclusively (at least 951) in the cell

membrane of microorganisms and suggested that carotenoids

could be used as "marker molecules" in the fractionation of

these membranes. Salton and Ehtisham-ud-Din (54) postulated

that, although the carotenoids are not indispensable, they

contribute to membrane stability in S. lutea and M. lysodeik-

ticus. Additional theories of carotenoid function in the

bacterial cell include protection from oxidizing agents (26)

and protection from photo-oxidation by visible light (37,43).

Studies utilizing Mycobacterium marinum showed that the

efficiency of photoprotection was directly proportional to

the amount of carotenoid pigment per cell (42) . Since carot-

enoids are generally not produced in cultures maintained in

the dark but are produced in response to light stimulus (3,63),

a photoprotective function may be a valid hypothesis for these

organisms.

Formation of a spheroplast or protoplast of a chromogenic

organism is useful in the determination of the site of pigment

location (45). The term "protoplast" is used to denote the

structure remaining when the cell wall is removed from the

bacterial cell (48). "Spheroplasts" are osmotically fragile,

spherical cells but still retain part of the cell wall (48).

If the pigments are released into the media when a protoplast

is formed, presumably the pigments are localized in the cell

wall. On the other hand, if protoplast formation is complete

and all cell wall material is removed, but the cells en masse

are pigmented, then the carotenoids are said to be in the

cell membrane or the cytoplasm. Lysis of the protoplast by

osmotic shock can then be used to determine the precise

location of the pigments.

Protoplasts can be formed by a variety of methods. One

of the most commonly used agents for the production of proto-

plasts is lysozyme which causes the lysis of many gram positive

bacteria. Lysozyme catalyzes the hydrolysis of the l-*-4 gly-

cosidic linkages in the polymer peptidoglycan, N-acetyl

glucosamine —N-acetyl muramic acid (57) . Pencillin has also

been used for protoplast production, especially with gram

negative microorganisms (40) . This antibiotic inhibits the

terminal transpeptidation reaction in which the linear pep-

tidoglycan becomes cross-linked to another strand (62). Once

this linkage is established it cannot be reversed by penicillin.

By way of contrast, the mode of protoplast formation by

D-cycloserine is, theroretically, interference with the bio-

synthesis of the uredine nucleotide precursors (62) . Glycine

induction of protoplasts is presumably due to reversal of the

terminal crosslinking reaction in cell wall synthesis (62).

Enzymes that solubilize lipid (16) or protein outer layers of

the wall (1,36), or chelating agents (48) that bind ions

essential for the synthesis of the cell wall and for its

stability, have also been used in conjunction with lysozyme,

glycine or penicillin to produce protoplasts.

In organisms that do not form protoplasts, disruption

of bacterial cells by mechanical means and preparation of a

pure cell wall fraction by differential centrifugation has

been used as a means of determining the location of caroten-

oids (45) . Assay of total pigment at every step of the

process is necessary for determining the cell fraction in

which the pigment is located.

Corynebacterium spp. strain 7E1C has a cell wall com-

position similar to those of Mycobacterium species and

Nocardia species. All of these genera have a high content

of lipid in the cell wall. Preparation of pure cell walls of

these organisms is difficult because the lipid apparently

enhances entrapment of cell membrane fragments (53) . According

to Salton (53), electron microscopy is the best single method

of validating the purity of cell wall material.

The taxonomic position of the organism tentatively named

Corynebaaterium species strain 7E1C (33), one of the "strains

in search of a genus" (19), is still uncertain (5,30,39,41,68,

72,73). This strain is a chromogenic organism whose photo-

inducible carotenoid pigments have been partially characterized.

The carotenoid pigments are xanthophylls rather than carotenes

(30); six hypophasic and three epiphasic pigments have been

detected so far. Cardini and Jurtshuk (9) have reported

difficulty with contamination of enzymatically active fractions,

presumably of membrane origin, of C. spp. strain 7E1C with a

carotenoid pigment that absorbs at 450 nm. Their investigation

did not determine whether the carotenoid is located in the cell

membrane, or is an absorbed contaminant. Also, they did not

report whether all of the carotenoid or just one of the nine

pigments is the cause of their difficulty.

This investigation attempts to show the location of the

carotenoid as a step towards resolving the role of the pigment

in the cell but makes no attempt to prove the function of the

carotenoid pigments to Corynebacterium spp. strain 7E1C.

MATERIALS AND METHODS

The organism used in this investigation, Corynebacterium

species strain 7E1C, was originally isolated from soil by

Kester and Foster (33) on mineral salts media enriched with

propane as the sole carbon and energy source. The organism

was grown in "L-salts" media, according to the formula of

Leadbetter (38), supplemented with 1% dextrose. Flasks were

incubated at room temperature (22-25 C) on an Eberbach rotary

shaker at 140 revolutions per minute at a rotation diameter of

1.2 inches. Two per cent agar was added to the L-salts medium

for slants on which stock cultures were maintained. Inocula

for liquid cultures were prepared from logarithmic phase cells

grown in L-salts media. The cell density was adjusted to an

optical density of 0.30 at 580 nm by dilution with sterile

L-salts. One part of this suspension was added to 99 parts

of L-salts media. Purity of inoculum was determined by streak

plates on L-salts agar and by microscopic examination.

Protoplast Production —Procedures used for protoplast

formation were those of Lederberg (40) (penicillin G), Jeynes

(29) (glycine), Hines et al. (23) (glycine and penicillin),

Gooder (17) (lysozyme), Heppel (22) (lysozyme and ethylene-

diaminetetraacetic acid (EDTA)), Noller and Hartsell (49)

(lysozyme, trypsin and butanol), Warren et al. (71) (polymyxin),

Kohn (36) (freeze-thaw and lysozyme), Barker and Thorne (2)

(trypsin, lysozyme and EDTA), Ghosh and Murray (16) (lipase,

bile salts and lysozyme) and Mattman (46) (D-cycloserine).

Since C. spp. strain 7E1C has a long generation time,

organisms for protoplast production were grown in L-salts

media for four days at room temperature at which time they

were in early logarithmic phase. Organisms were incubated

for six days, at which time plate counts yielded about 1.2 x

Q

10 cells per ml, for techniques requiring stationary phase

cultures. Cells were grown in the presence of 1 mM magnesium

chloride in experiments utilizing EDTA for protoplast form-

ation, since magnesium ion limitation negates the effect of

EDTA.

Cells were examined by phase-contrast microscopy for

protoplast formation. They were also checked for osmotic

fragility by dilution of treated and untreated samples in

equal volumes of either distilled water or 1.41 M sucrose.

The dilutions were measured spectrophotometrically and any

decrease in optical density was recorded.

Cell Wall Preparation —Cells used as a source of pure

cell walls were grown in L-salts for five days to yield a

late logarithmic phase culture. The organisms were collected

by continuous flow centrifugation at 23,500 x g in a Sorvall

Model SS-3 centrifuge. After one wash in 0.85% NaCl (pH 7.0),

the cells were centrifuged and the sedimented cells resuspended

in 0.851 NaCl to yield a sediment with the consistency of

heavy cream. In order to facilitate disruption, this bac-

terial suspension was frozen at -20 C and then thawed to room

temperature in warm water. Glass beads of 0.10 to 0.11 mm in

diameter were added to give a ratio of one part beads to five

parts cell suspension. The bacteria-bead mixture was agitated

in an MSK cell homogenizer at 2000 rpm for two minutes under a

constant stream of liquid carbon dioxide to prevent excessive

heating. The suspension was observed by phase-contrast micro-

scopy to ascertain the degree of breakage. When no whole cells

could be observed, the broken cells were subjected to differ-

ential centrifugation in a Sorvall Model SS-3 centrifuge

according to the method of Takeya and Hisatsune (64). The

crude cell wall material was washed once with 2% Triton X-100

as recommended by Schnaitman (57,58). Specimens from each step

of the differential centrifugation procedure were lyophilized

for subsequent preparation for electron microscopy.

Assay of Total Pigment —Carotenoid pigments were obtained

from each fraction by one extraction with a 1:5 (v/v) solution

of carbon disulfide in methanol followed by six subsequent

extractions with carbon disulfide alone. Since carbon disul-

fide has a density greater than that of the whole cells and

cell fragments, the utilization of carbon disulfide was some-

what more inconvenient. However, it was the preferred solvent

because it gave more complete extraction of total cell pigments

The pigment extracts were pooled and evaporated to dryness at

40 C in a Buchler flash evaporator. In order to remove residual

carbon disulfide and methanol, the dried pigment was solubilized

in chloroform, re-evaporated and finally taken up in a small

amount of chloroform.

Estimation of the total pigment in a sample was obtained

1 9-

by using an extinction coefficient (E^) of 2500 at the wave-

length of maximum absorption, 483 nm in chloroform. If "X"

grams of carotenoid dissolved in "y" nil of solvent give an

extinction coefficient of "E" at its wavelength of maximum

absorption, then:

Ey X = (13).

1% E x 100 1 cm

Spectrophotometry assays were performed on a Bausch and Lomb

Spectronic 20.

Electron Microscopy —Specimens for electron microscopy

were fixed by a method similar to that of Cagle et al. (8).

Initial fixation was performed in 0.151 ruthenium red (w/v)

in 0.1 M sodium cacodylate buffer (pH 7.2) for thirty minutes

followed by fixation in 41 glutaraldehyde in 0.133 M cacody-

late buffer and 0.05% (w/v) ruthenium red for one hour. After

one wash in cacodylate buffer, the specimens were further fixed

in 1.331 osmium tetraoxide and 0.051 (w/v) ruthenium red in

0.133 M cacodylate buffer for one and one-half hours. A final

10

wash in 0.2 M cacodylate buffer was followed by dehydration in

ethyl alcohol. The dehydrated specimens were embedded in Epon

812 and polymerized at 60 C for 24-48 hours. Thin sections

were cut with a Sorvall Porter-Blum MT-2 ultramicrotome with

glass knives. The thin sections were stained with a saturated

uranyl acetate stain in 30% ethanol for three minutes and then

with lead citrate (47) for five minutes. After carbon coating

(32), the sections were examined in an RCA EMU-3G electron

microscope at an accelerating voltage of 50 kv. Photographs

were taken on Kodak 2 inch by 10 inch projector slide plates

of medium contrast and fine grain film.

RESULTS AND DISCUSSION

Initially, protoplast production was considered neces-

sary in order to determine the location of the carotenoid

pigments. It was reasoned that if a pigmented protoplast

resulted from these preparations, the site of pigment would

be the cell membrane or the cytoplasm. A nonpigmented proto-

plast would indicate that the cell wall was the location of the

carotenoids. Preparations of protoplasts were attempted by

using a broad range of concentrations of penicillin (40),

glycine (23,29,51), lysozyme (17), EDTA (22), D-cycloserine

(46) and polymyxin (71) . Freezing and thawing (36) and com-

binations of these chemicals (2,22,23,49) was also investigated.

In addition, the effect of temperature on conversion of cells

to protoplasts by the substances given above was determined.

Stabilization of protoplasts was attempted with sucrose con-

centrations ranging from 0.25 M-2.0 M. Production of proto-

plasts by any of these methods or any adaptations of these

methods was unsuccessful. In light of similar unsuccessful

attempts with related organisms (48), this failure was not

unexpected. Methods utilizing glycine addition to logarithmic

phase cultures in concentrations of 2% in 0.6 M sucrose did

produce spherical cells rather than the typical short bacillus,

but these were not osmotically fragile and thus could not be

considered to be protoplasts. Penicillin concentrations of

11

12

100 units per ml resulted in aggregation of the cells upon

standing but did not alter the shape of the organism even

after incubation for prolonged periods of time. The failure

of lysozyme to effect the cell wall may well be due to a

permeability factor as well as to the hydrophobic nature of

the organism. The waxy outer layer of the organism may

prevent chemicals from reacting with the wall structure. All

attempts to remove the wax layer with detergents, solvents

and enzymes prior to treatment were unsuccessful.

Cell Rupture and Differential Centrifugation — Due to

failure to produce protoplasts, it was necessary to attempt

an alternate approach. Rupture of bacterial cells followed

by differential centrifugation is useful in determining the

location of the pigments (45) . Freezing and thawing the cells

just prior to disruption was necessary for efficient cell

rupture. Plate counts made of the bacterial suspensions imme-

diately before and after rupture revealed a 99.991 reduction

in viable cells. The disrupted suspensions were observed by

phase-contrast microscopy as small amorphous aggregates of

material not recognizable as intact bacterial cells. The pH

of the disrupted suspensions was also checked since some glass

beads release alkali when shaken. No observable pH change was

noted.

Purification of the cell wall was performed according to

the scheme outlined in Figure 1. Trypsin, 0.2% in 0.067 M

FIGURE 1

PURIFICATION OF CELL WALLS

13

Whole Cells

rupture; coarse filter on a sintered glass filter; centrifuge at 2300 x G, 10 minutes, twice

Supernatant I 1 Sediment I

coarse debris and unbroken cells; wash once with 0.851 NaCl and add wash to Supernatant I

Sediment IB

centrifuge at 25,000 x G, 15 minutes

I Supernatant II

cell membrane

Sediment II

cell wall and cell membrane j

wash once in 1 M NaCl; treat with 0.2% trypsin at 37 C for 3 hours; centrifuge at 25,000 x G, 10 minutes

j

Supernatant III

cell membrane fragments

Supernatant IV

Sediment III

crude cell wall

I -wash #1; phosphate buffer wash #2; 21 Triton X-100 wash #3-#5; 1 M NaCl wash #6-#8; distilled water

Supernatant of washes Sediment IV

pure cell wall

14

phosphate buffer (pH 8.0), was incubated with the cell wall-

cell membrane fraction for three hours at 37 C to selectively

solubilize the proteins of the cell membrane. Trypsin treat-

ment has no effect on the cell walls of gram positive bacteria

(64). After subsequent washing with phosphate buffer, the

crude cell wall fraction was treated with 2% Triton X-100 in

phosphate buffer for twenty minutes at 24 C. Triton X-100

specifically solubilizes proteins of the cell membrane (14,57,

58). This was followed by thorough washing with 1 M NaCl and

distilled water to wash away the dissolved cell membrane

material. The remaining sediment, which was virtually non-

pigmented, was verified by electron microscopy as consisting

entirely of cell wall with little or no discernable cell

membrane contamination. Figure 2 is an electron microscope

photograph of whole cells of C. spp. strain 7E1C. Two or more

globose, lipid-like inclusion bodies are readily observed in

this cell. These inclusion bodies are reminiscent of the

"sap-vacuoles" (2), of unknown substances, evident in thin

sections of Mycobacterium tuberculosis var. avium and a

variant of Mycobacterium -phlei. The small, irregularly shaped

bacillus seen in Figure 2 also demonstrates an intact cell

with cell wall and cell membrane continuous around the cell.

One mesosome-like organelle can be seen as an elaboration of

the membrane in the lower portion of the cell. Inclusion

bodies resembling polyphosphate can also be noted as round

electron-dense areas in the cytoplasm. The irregular edge

15

* jk-

Fig. 2. Whole cells of Corynebaoterium species strain 7E1C The cell wall (CW) is separated from the cell membrane (CM) by a periplasmic space (PS) and seems to be fairly typical of walls normally surrounding gram-positive cells. A meso-some (M) is seen in the lower portion of the irregularly-shaped bacillus. Several lipid-like (L) sacs can also be seen in this cell. Magnification is 161,350 x.

16

of the cell wall may well be due to the wax that is formed by

these organisms.

The appearance of cells after disruption in the MSK cell

homogenizer for two minutes is shown in Figure 3. Micelles

of membrane, as well as membrane fragments and extraneous

cytoplasmic material absorbed to the cell wall, can be seen

in the photograph. The gross morphology of the broken cells

is greatly different from that of the intact cell; no lipid-

like sacs are observed intact and the cells have completely

lost their anatomic integrity. The broken cells appear as

empty shells with the cell wall relatively intact. These

cell fragments appear to be contaminated with extraneous

material. The pure wall fraction (shown in Figure 4) demon-

strates the appearance of the broken cells after treatment

with trypsin, Triton X-100 and adequate washing and is in

marked contrast to the cell fragments seen in Figure 3. The

pure cell wall fraction is free of cell membrane and cyto-

plasmic impurities but is itself relatively intact. Thus,

the procedure used resulted in a pure cell wall preparation.

As shown in Table I, the majority of the pigment is in

the cell membrane fraction. In fact, 100-1421 of the total

pigment accounted for in the Supernatant I was solubilized

with the membrane. Oddly enough, a net yield of 111-145% of

the extractable pigment in Supernatant I (the broken cell

fraction) was obtained from the extraction of the individual

fractions. The range of pigment values is used to indicate

17

Fig. 3. Cells of Corynebacterium species strain 7E1C after disruption in the MSK cell homogenizer. Relatively intact cell walls (CW) have adsorbed cytoplasmic material and cell membrane (CM). Magnification is 66,800 x.

18

Fig. 4. Purified cell walls of Corynebactevium species strain 7E1C. Cell walls are apparently uncontaminated with intact cell membrane. Magnification is 37,340 x.

19

TABLE I

PIGMENT RECOVERY FROM DIFFERENTIAL CENTRIFUGATION

Total (10"

Carotenoid 3 grams)

Percentage of Original(6)

Fraction Experiment I II

Experiment I II

Supernatant I 0.53 0.39 100 100

loss to E-M 0.06 0.03 11 7

Supernatant II(1) 0.33 0.01 62 0

Supernatant IIIC2) 0.05 0.02 9 4

Supernatant IVC3) 0.04 0.07 8 18

Sediment IV(4) 0.06 0.01 11 3

Supernatant of washes 0.11 0.48 21 120

Carotenoid attributable to membrane(5) 0.53 0.57 100 142

1. cytoplasmic material and cell membrane 2. cell membrane fragments 3. cell membrane 4. pure cell wall 5. Supernatants II, III, IV and Washes 6. total extractable pigment in Supernatant I

20

the differences in recovery of pigment in repeated experiments

More complete membrane removal can be accomplished through

exhaustive washing. However,this involved hours of washing

and centrifugation and is not significant when the additional

8-34% pigment is compared to a total pigment yield of 111-

145%.

Original experimental procedures (64) required high cen-

trifugation speeds. These resulted in a packed cell button

which was difficult to resuspend. Apparently, sufficient

agitation to resuspend the sediment resulted in dissolution

of the cell membrane. Modified procedures were subsequently

followed with reduced, but adequate, centrifugation speeds as

listed in Figure 1. In these procedures, washing was required

to release the cell membrane. This demonstrates that the

carotenoid is not in the cytoplasm, as it appeared to be in

the original experiments, but rather in the cell membrane.

The reason for pigment recovery levels of 111-145% is

explained by the fact that cells and cell fragments that were

not treated with trypsin and Triton X-100 could not be com-

pletely extracted with carbon disulfide alone nor with carbon

disulfide and methanol. Thus, the initial pigment concen-

trations, which was the basis for the calculation, was low.

Trypsin did not extract pigment from whole, unbroken cells.

When whole cells of C. spp. strain 7E1C were washed with 0.5%

Triton X-100 for one minute and then centrifuged, a deep

orange-red supernatant was recovered and the cell button was

21

pale pink. According to Schnaitman (58), Triton X-100 specif-

ically solubilizes proteins of the cell membrane and has no

effect on the cell wall (57,58). DePamphilis and Adler (14)

similarly reported that it does not have an effect on the

L-membrane. Schnaitman further stated (58) that Triton X-100

is, in fact, specific for that portion of the cell membrane

that is closely bound to the cell wall. Apparently, Triton

X-100 can penetrate the porous cell wall, solubilize the cell

membrane and release the carotenoids.

The pure cell wall fraction, after one extraction with

carbon disulfide and methanol, released all of its pigment

into the extract. The broken, untreated cells still retained

a pink color even after ten extractions with carbon disulfide

over a period of seven hours. This suggests that a small

portion of the pigment is bound in such a way, possibly a

carotenoid-protein linkage, that cannot be broken by solvents

but only by reagents that specifically break the bond.

Saperstein and Starr (56) reported that carotenoid pigments

of Corynebacteriat Mycobacteria and Micrococci occur as partic-

ulate protein complexes in the cell. They hypothesized that

the pigment was necessary for the synthesis of the associated

protein. They further stated that conditions which normally

cause isomerization of carotenoids when in solutions of organic

solvents do not affect carotenoids associated with protein,

possibly because of prevention of rotation around ethylene

bonds as a result of protein attachment (56). A carotenoid

22

that was glycosidically bound to a glucose-bound peptide was

reported by Thirkell and Hunter in Saroina morrhuae (67) .

They reported failure in extracting all of the pigment with

solvents and later hypothesized that the bonding had to be

covalent to account for these results. Synthetic detergents

freed these carotenoids from a membrane preparation. A carot-

enoid-protein complex was identified by Howes and Batra (24)

as the photoreceptor in an unidentified Mycobacterium species.

The data thus far provided strongly infer the existence

of a carotenoid-protein moiety in C. spp strain 7E1C. However,

although the evidence is highly suggestive, its existence can

be confirmed only by isolation and characterization of the

complex.

The distribution of the pigment as determined by assay

of the various purification steps is seen in Table I. Only

3-121 of the total carotenoid can be accounted for by the pure

cell wall. This residual carotenoid may be due to a minority

of cell membrane contamination or to absorption of a small

amount of carotenoid by the cell wall. The cell wall may

serve as a reservoir for superflous carotenoid; excess produced

in the membrane may be deposited in the cell wall. It is also

possible that the cell wall is the production or storage site

of one of the nine carotenoid pigments. Even if a portion of

the pigment is found in the cell wall and is not a comtaminant,

the cell membrane still contains approximately eighteen times

as much carotenoid pigment as the cell wall. The carotenoids

23

are most likely situated at the cell site where they function.

The location of the carotenoids on or in the membrane, points

to a role at that level. Whether the role is one of pro-

tection from light or oxidizing agents, an alternate pathway

for electron transport in times of overproduction or a stabil-

izing agent to the membrane structure remains to be determined

CONCLUSION

Liquid cultures of C. spp. strain 7E1C grown in L-salts

media under fluorescent lighting at room temperature accu-

mulate xanthophylls (30) . An attempt to localize the pink-

orange pigment by formation of protoplasts was unsuccessful

since protoplasts could not be formed using known methods of

production. Failure to produce a protoplast may be due to

the impermeability of the wax outer layer of the cell wall to

the usual agents employed in protoplast formation or to an

unusual cell wall structure. As an alternate approach to the

determination of the pigment site in the cell, the organism

was efficiently disrupted in an MSK cell homogenizer and

purified cell walls prepared by differential centrifugation

according to the procedure of Takeya and Hisatsune (64) with

the modification of Schnaitman (58) . Pigment analysis of

fractions obtained by differential centrifugation revealed

that the majority (in fact, nearly all) of the carotenoid was

found in the cell membrane, rather than in the cell wall or

free in the cytoplasm. The small amount of carotenoid found

in the cell wall may be due to contamination with cytoplasmic

membrane or else the cell wall may indeed be a unique site for

a portion of the carotenoid. Since location is usually coin-

cident with function, the carotenoid may serve some purposes

in protection or stability at the membrane level.

24

25

Inability to extract the entire carotenoid pigments from

broken cells that have not been treated with substances which

solubilize protein indicates that a portion of the carotenoid

in the cell may be bound in some fashion. This linkage may

be similar to the carotenoid-glycosidic bond demonstrated in

S. morrhuae and as such could serve as a means of stabilizing

the membrane.

SUMMARY

Carotenoid pigments occur in a large number of red,

orange and yellow nonphotosynthetic bacteria of the genera

Mycobacteriumj Corynebaater-Lum and Nocardia. In most of the

organisms investigated so far, the carotenoids are found in

the cell membrane where they perform an unknown role, although

several functions have been hypothesized. Corynebaatevium spp.

strain 7E1C produces carotenoids which are xanthophylls (30)

rather than carotenes. The majority of these pigments have

been found to be located in the cell membrane. A small portion

of the total carotenoids cannot be extracted with solvents but

can be extracted completely from broken cell fragments after

treatment with chemicals that solubilize protein. These

carotenoids may be similar to those of S. morrhuae in which a

carotenoid-glycoside peptide bond has been found.

Attempts to produce protoplasts of C. spp. strain 7E1C

by many procedures used successfully with other microorganisms

were not successful. Differential centrifugation of broken

cells of the organism yielded a pure cell wall fraction uncon-

taminated with cytoplasmic membrane, as determined by electron

microscopic examination.

26

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