Plant Cell Physiol. 38(3): 274-281 (1997)
JSPP © 1997
Pigment Composition of a Novel Oxygenic Photosynthetic Prokaryote
Containing Chlorophyll d as the Major Chlorophyll
Hideaki Miyashita', Kyoko Adachi 2 , Norihide Kurano 1 , Hisato Ikemoto 1 , Mitsuo Chihara 13 and
Shigetoh Miyachi 4
1
2
3
4
Marine Biotechnology Institute, Kamabhi Laboratories, Heita, Kamaishi, Iwate, 026 Japan
Marine Biotechnology Institute, Shimizu Laboratories, Sodeshi, Shimizu, Shizuoka, 424-19 Japan
Japanese Red Cross College of Nursing, Shibuya-ku, Tokyo, 150 Japan
Marine Biotechnology Institute, Bunkyo-ku, Tokyo, 113 Japan
The principal pigment found in the majority of oxygenic photosynthetic organisms is known to be chlorophyll a.
However, we isolated a new oxygenic photosynthetic prokaryote that contained chlorophyll (/ as a predominant
pigment with chlorophyll a being a minor pigment. Chlorophyll d had previously been noted but its natural occurrence and function remained unclear. Cells of the new
prokaryote had an absorption maximum at red region of
714-718 nm due to chlorophyll d absorption, but no characteristic absorption peak of chlorophyll a around 680 nm
was observed. Chlorophyll d of the new organism was identified spectrophotometrically in several solvents and its
chemical structure was confirmed by NMR and FABMS
analysis. The cell also contained a chlorophyll c-like pigment, zeaxanthin and a-carotene but not chlorophyll b and
/^-carotene. The content of chlorophyll d accounted for
more than 2% of the cell dry weight, while the content of
chlorophyll a was less than 0.1%. The chlorophyll a/d
ratio remained between 0.03 and 0.09 under different culture conditions. The light absorption characteristics and
the high content of chlorophyll d along with the small content of chlorophyll a indicated the existence of a new light
utilization mechanism involving chlorophyll d.
Key word: Chlorophyll d — In vivo absorption — NMR —
Oxygenic photosynthesis — Pigment composition — Prokaryote.
Chlorophyll a is an essential pigment for oxygenic photosynthesis. Therefore it is contained as a predominant pigment in all oxygenic photosynthetic organisms (Lee 1989).
Oxygenic phototrophs, consequently, show a characteristic
absorption maximum of red light around 680 nm due to a
bathochromic shift of chlorophyll a bound to the photosynthetic apparatus (French et al. 1972). Chlorophyll d was
first reported as a minor accessory pigment in various species of red macroalgae (Manning and Strain 1943). However, the evidence for its existence was inconsistent. And it
was suggested that chlorophyll d could be an artifact produced by the extraction process of the pigment, since one
of the oxidation derivatives of chlorophyll a gave an ab274
sorption spectrum identical with that of chlorophyll d
(Holt and Morley 1959, Holt 1961). The in vivo occurrence
of chlorophyll d as well as its photosynthetic function has
been unclear for longer than half a century.
Recently, we isolated a new oxygenic photosynthetic
prokaryote containing chlorophyll d as a major green pigment (Miyashita et al. 1996). In this report, pigment composition of the organism was characterized and the in vivo
occurrence and the chemical structure of chlorophyll d
were confirmed. Also the possibility of a new light-utilization system using chlorophyll d was discussed.
Materials and Methods
Isolation and cultivation of cells—The new oxygenic photosynthetic prokaryote was isolated from a suspension of algae
squeezed out of Lissoclinum patella, a colonial ascidian, collected
in 1993 from the coast of Palau in the western Pacific Ocean. Cells
of the algal suspension were enriched in K medium (Keller et al.
1987). Clonal culture of the new organism was established by
serial single-colony transfer on agar plates (0.7%). The cells were
grown in K+ESM medium (Miyashita et al. 1995) with a gentle
bubbling of air. The culture was kept under white fluorescent light
(~80/imol photons nT 2 s~') with a 12/12 h light/dark cycle at
28°C.
Spectrophotometry—Absorption spectrum of a cell suspension was made spectrophotometrically (DU-640, Beckman,
U.S.A.). Prior to the measurement, cell aggregation was dispersed
by ultrasonication of the cells for a few seconds. This absorption
spectrum was compared with those of a cyanophycean alga,
Synechocystis sp. ATCC27266 and a green alga, Nannochloris
maculata CCAP251/3.
Electron microscopy—Cells were prefixed with glutaraldehyde [1% in 50 mM phosphate buffer (pH 7.2)] for 2 h and postfixed with osmic acid (2% in the same phosphate buffer) for 2 h.
The fixed cells were harvested by centrifugation, washed with
water and then dehydrated by a series of alcohol washes. For the
observation of thylakoids and plasmalemma, cells were initially
fixed with glutaraldehyde (as above), and then with potassium permanganate (1% in the phosphate buffer) for 2 h. The fixed cells
were washed with water and dehydrated by a series of acetone extractions. Fixed cell samples were mounted in Spurr's resin (Spurr
1969). Ultrathin sections were double-stained with uranyl acetate
and lead citrate (Reynolds 1963).
Pigment analysis—Cells were harvested by centrifugation
and washed with filtered seawater. Pigments were extracted with
cold methanol (4°C) for a few minutes. After centrifugation, the
supernatant was immediately injected into the HPLC system with
Chlorophyll d as a predominant pigment
a reversed-phase column, TSKgel ODS-80Ts (15 cm x 4.6 mm, 5
fttn particle size; Tosoh, Japan). The pigments were eluted with
methanol: water (9 : 1) for 0.5 min, with a linear gradient to
100% methanol for 4.5 min, and then with 100% methanol. The
initial flow rate, 1 ml min~', was changed to 2 ml min" 1 at 9 min
after the sample injection. Eluted pigments were detected with a
UV-visible detector (UV8010, Tosoh, Japan) at 440 nm and a photodiode array detector (SPD-M6A, Shimadzu, Japan). Absorption spectra of the methanol extract and of the pigments fractionated from HPLC stream were made with a spectrophotometer
(DU-640, Beckman, U.S.A.). Pigments were identified by comparing the retention times and absorption spectra with those of
known pigments extracted from Prochloron didemni (collected at
Palau) containing chlorophylls a and b, zeaxanthin, Mg 2,4-divinyl pheoporphyrin a5 monomethyl ester (MgDVP), or from Prochlorococcus sp. SB strain (Shimada et al. 1995) containing divinyl-chlorophylls (DVChl.) a and b, zeaxanthin and a-carotene.
Comparison was also made to existing data (Rowan 1989).
Identification of a major green pigment—A major green
pigment of the new organism was fractionated from the stream of
the HPLC with a reversed phase column (TSKgel, ODS-80TM,
4.7 mm x 30 cm, Tosoh, Japan) and purified by fractionation with
the HPLC several times. Absorption peaks of the pigment in several solvents were compared with those of the published data
(Rowan 1989). Acidification product of the pigment (phaeophytin) was prepared by addition of a few drops of 0.5 M HC1.
Since there was no available authentic chlorophyll d nor a
reference algal strain which contained chlorophyll d, chemical
structure of the major pigment was confirmed by nuclear magnetic
resonance (NMR) spectroscopy and fast atom bombardment mass
spectroscopy (FABMS), in comparison with the data obtained
from authentic chlorophyll a. The NMR spectra of the pigment in
acetone-rf6 were recorded on a Varian Unity 500 NMR Spectrometer. The FABMS was measured with a JEOL JMS-SX102
mass spectrometer.
Quantification of pigments—Each pigment was quantified
based on the peak area of HPLC analysis using the method of
Mantoura and Llewellyn (1983). A tentative extinction coefficient
for chlorophyll d, E 1 ^ (440 nm)=400, obtained from the measurement of the relationship between the dry weight of purified chlorophyll d injected into HPLC and the peak area with our system was
applied for the quantification of the pigment. For calculation of
the a-carotene content, the extinction coefficient for /?-carotene,
E1% (440nm) = 2,200 (Mantoura and Llewellyn 1983), was used,
since there was no appropriate data for the a-carotene quantification.
Extraction of water-soluble pigment—Water-soluble pigments
were extracted by hard grinding of the cells with quartz sand in 20
mM acetate buffer, pH 5.2. After precipitating at 2,000xg for 5
min at 4°C, the supernatant was centrifuged again at 20,000xg
for 2 h at 4°C. The absorption spectrum of the resulting supernatant was compared with the water-soluble fraction obtained from
Anacystis nidulans R2 IAM200 (University of Tokyo) by the same
procedures.
Photosynthetic activity measurement—Photosynthetic activity was measured using the oxygen electrode system obtained from
Rank Brothers Inc. (Cambridge, U.K.) under a halogen lamp
(JCD100V, Iwasaki-denki, Japan) at 28°C. Irradiation strength
was measured with a LI-1000 multichannel dataloger (RI-COR,
U.S.A.) with a LI-192SA sensor (RI-COR, U.S.A.). Before the
measurement, aggregates of the cells were dissociated by ultrasonication for a few seconds, then washed arid resuspended in
fresh culture medium.
275
Results
Morphological characteristics of the new isolate—The
cell of the new phototrophic organism was unicellular and
spheroidal or ellipsoidal, 1.5-2.0 pm in diameter and 2.03.0nm in length (Fig. 1A). Electron microscopy revealed
the prokaryotic nature of the new organism (Fig. IB, C).
Its genomic DNA was dispersed cytoplasmically and not
enclosed in a nuclear envelope (Fig. IB). No organized chromosomes, mitochondria or other membrane-limited organ elles were visible. Layers of thylakoid-like structures were
appressed peripherally (Fig. 1C). No protein entities similar
to phycobilisomes could be observed on the thylakoid-like
membranes.
In vivo absorption—The cell suspension was green
and had an absorption maximum in the red region at 716
nm (Fig. 2). Cell suspensions of Synechocystis and Nannochloris have absorption maxima at 675 nm and 685 nm, respectively. The absorption maximum of the new prokaryote was located at least 30 nm longer wavelength than
those of Synechocystis and Nannochloris. The new prokaryote has an absorption minimum at 678 nm where absorption peaks are normally found in known oxygenic photoautotrophs.
Pigment analysis—Methanol extract of the cells had
Fig. 1A-C Micrographs of the new oxygenic photosynthetic
prokaryote cells. (A) Photomicrograph. Scale bar=5 fim. (B) Electron micrograph of a longitudinal section showing the cytoplasmic space (glutaraldehyde/osmic acid fixation), c: carboxysomelike structure, n: nucleoide. t: thylakoid-like membranes. Scale
bar=0.3//m. (C) Electron micrograph of the transverse section
showing thylakoid-like membranes and plasmalemma (glutaraldehyde/potassium permanganate fixation). Arrowheads: thylakoidlike membranes. Arrows: plasmalemma. Scale bar=0.5/im.
276
Chlorophyll d as a predominant pigment
•v prokaryote
Synechocystis sp.
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350 400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 2 Absorption spectrum of the new oxygenic photosynthetic
prokaryote suspended in fresh culture medium in comparison
with those of Synechocystis sp. (Cyanophyta) and Nannochloris
(Chlorophyta).
an absorption maximum at 697 nm (Fig. 3), but not at 665
nm, the characteristic absorption maximum of chlorophyll
a in methanol. Analysis by HPLC revealed that the cell contained five detectable pigments (Fig.4A). The first band
had retention time similar to MgDVP extracted from Proeft loron (Fig. 4B). The absorption spectrum of this pigment
showed a large absorption peak in blue region and a
350 400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 3 Absorption spectrum of methanol extract from the cells
of the new oxygenic photosynthetic prokaryote.
smaller one in the red region (Fig. 5A). Although it was
similar to that of MgDVP, it was difficult to confirm the pigment as MgDVP from these results, since some of pigment
resembling chlorophyll c, the so-called chlorophyll c-like
pigments, have a similar retention time and absorption
spectra (Jeffrey 1989). The second pigment showed a retention time identical with those of zeaxanthin from Prochloron and Prochlorococcus (Fig. 4). Since the absorption
spectrum (Fig. 5B) was also identical to the compound
Table 1 Maxima of the absorption spectra of the major green pigment in the new oxygenic photosynthetic prokaryote
and those of its acidification product as compared with the respective values of chlorophyll d and pheophytin d
Solvent
Absorption maxima (nm)
Chlorophyll d
The major pigment
Red
Blue
Red
Blue
References
Diethyl ether
390, 445
686
445
447
445
686
688
686
1
2
3
Acetone
392, 447
688
445
693
688
4
5
Methanol
400, 455
697
456
696
696
1
5
Ethanol
397, 451
695
696
5
Dioxane
394, 449
685
684
5
Acidification product
Diethyl ether
392, 421
692
Pheophytin d
421
421
692
692
2
6
1, Manning and Strain 1943; 2, Smith and Benitez 1955; 3, Holt and Morley 1959; 4, Jensen and Aasmundrud 1963; 5, Lu et al. 1983; 6,
Goedheer 1966.
Chlorophyll d as a predominant pigment
A
c
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A
CO
B
o
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277
Q
A
1
•
—
|
20
10
Retention time (min)
30
Fig. 4 HPLC profile of pigments extracted with methanol from
the cells of the new oxygenic photosynthetic prokaryote (A), Prochloron sp. (B) and Prochlorococcus sp. (C).
from the two reference strains, the pigment was identified
as zeaxanthin. The third pigment was the major green pigment of the cell. It had similar retention time to that of
chlorophyll b or divinyl-chlorophyll b, but the absorption
spectrum (Fig. 5C) was obviously different from those of
the chlorophyll pigments. Chlorophyll b was not detected.
The fourth pigment was identified as chlorophyll a (Fig. 4,
Fig. 5D). The fifth pigment was identical to a-carotene
from Prochlorococcus in both retention time and absorption spectrum (Fig. 4, Fig. 5E). /J-Carotene was not detected.
Identification of chlorophyll d—The absorption spectra of the major green pigment measured in several solvents
were the same as those reported for chlorophyll d (Table
1). Absorption peaks of its acidification product in dimethyl ether was identical to phaeophytin d (Table 1). The
'H-NMR spectrum of the pigment (Table 2) was very
similar to that of chlorophyll a, the differences being in the
absence of a vinyl proton signal and the presence of a formyl signal. The assignments of the macrocycle and short
side-chains of chlorophyll d were straightforward using correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC) and heteronuclear
multiple-bond multiple quantum coherence (HMBC), on
the basis of known 'H and 13C chemical shifts of chlorophyll a (Lotjonen et al. 1987). The complete assignments of
350 400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 5 Absorption spectra of pigments fractionated at each peak
from the stream of HPLC. The number in each parenthesis corresponds to the peak number of pigment in Fig. 4A. For identification of the peaks, see Table 3.
protons of the phytyl side-chain were made by the agreement of the chemical shifts between chlorophyll d and chlorophyll a, which was confirmed by HSQC and HMBC.
The position of the formyl group was confirmed by nuclear Overhauser effect (NOE) spectroscopy, as shown in
Fig. 6A. The results showed that the chemical structure of
the pigment is 2-desvinyl-2-formyl chlorophyll a (3-desvinyl-3-formyl chlorophyll a by current nomenclature under
International Union of Pure and Applied Chemistry;
IUPAC) (Fig. 6B) which was identical with the chemical
structure for chlorophyll d proposed in 1959 (Holt and
Morley 1959). The molecular formula of the pigment was
278
Chlorophyll d as a predominant pigment
Table 2
karyote
'H and " C NMR assignments for the predominant green pigment in the new oxygenic photosynthetic pro-
Position
Chemical shift (ppm)
'H S (multiplicity, J in Hz)
10
147.33
3.68 (s)
11.40 (s)
10.20 (s)
3.33 (s)
3.86 (q, 7.6)
1.73 (t,7.6)
<).8 (s)
3.65 (s)
13 2
<5.28 (s)
17'
n17'2
18
18'
19
20
PI
P2
P3
P3'
P4
P5
P6
P7
P7 1
P8
P9
P10
Pll
Pll1
P12
P13
P14
P15
P15 1
P16
1,2,3
3,4
3,6,7
136.31
11.39
145.13
20.12
18.12
148.64
6,7,8
7 , 8 , 9, 82
8, 8
107.8
8,9 , 11 , 12
150.35
136.63
12.89
11, 12, 13
133.04
13'
14
15
16
17
11.75
135.12
189.54
146.36
104.41
152.55
11
12
12'
13
13'
i34
Cd
151.81
1
2
2'
3
3'
4
5
6
7
7'
8
8'
82
9
Carbons showing
HMBC correlation
3
:3.83 (s)
1.25 (m)
:2.63, 2.46 (m)
2.48, 2.21 (m)
:
t.63 (qd, 7.1, 1.8)
1.82 (d, 7.1)
i$.81 (s)
<•t.36, 4.26 (dd, 12.9, 7.1)
5.04 (t, 7.1)
1.54 (s)
1.85 (m)
1.30 (m)
1.02, 1.20 (m)
1.34 (m)
().79 (d, 6.6)
1.23, 1.04 (m)
1.23 (m)
1.23, 1.04 (m)
1.34 (m)
().81 (d, 6.6)
1.23, 1.04 (m)
.30 (m)
.13 (m)
.51 (m)
0.85 (d, 6.7)
0.85 (d, 6.7)
° The I3C assignment of P71 and P l l 1 are interchangeable.
* The 13C assignment of P81, P10 and P12 are interchangeable.
c
The I3C assignment of P151 and P16 are interchangeable.
190.50
66.47
171.33
52.84
162.68
107.04
158.60
52.05
30.64
31.37
173.45
49.80
24.25
168.34
95.36
61.67
119.53
142.70
16.40
40.46
25.86
37.40
33.47
20.19"
38.21*
25.25
38.26*
33.65
20.23"
2
13', 13 , 14, 15
13'
2
16, 17', 17 , 18'
16, 17'
17, 17', 17'
16, 17, 17', 18', 19
17, 18, 19
1,2 , 18
17', P2,, P3
P 3 \ , P4
P2, P3, P4
P7, P8
P10, P l l , P12
38.14*
25.64
40.27
28.85
23.15C
23.07 c
P12, P13, P15, P15', P16
P14, P15, P16
P14, P15, P15'
Chlorophyll d as a predominant pigment
279
Table 3 Content of pigments in the new oxygenic photosynthetic prokaryote
CO 2 Phy
17 3
Pigment
Content
(mg(g dry cells)"1)
1
2
Chlorophyll c-hke pigment
Zeaxanthin
N.D.*
3
Chlorophyll d
21
4
Chlorophyll a
0.73
5
a-Carotene
2.1
2.0
° Each number corresponds that of pigment peak in HPLC chromatogram shown in Fig.4A.
* Not determined.
I
OCH 3
134
(Fig. 2) did not appear in the spectrum of a methanol
extract (Fig. 3). Also a pale-blue solution having an absorption maximum at 614 nm could be obtained as the watersoluble fraction of the cells (data not shown). The absorption maximum was located at a wavelength slightly shorter
than that of Anacystis nidulans R2, but the fluorescent
characteristics were similar to each other (data not shown).
These results indicated the existence of a small amount of a
phycobiliprotein-like pigment and that the light absorption
in yellow-orange light by the intact cells was due to the presence of this pigment.
Photosynthetic activity—The cells evolved oxygen under illumination (Fig. 7). The rate of dark respiration was
6^mol O2 (mg Chi a + d)~l h~'. The rate of oxygen evolution increased with increasing light intensities and was saturated at lOOyumol photons m~2 s~\ The maximum rate of
oxygen evolution reached 71 ^mol O2 (mg Chi a+d)~l h" 1 .
Photoinhibition was not observed even at l,100yumol photons m"" 2 s~'.
B
CO 2 Phy
Peak
No."
I
OCH 3
134
P16
P7
P111
P15'
Fig. 6 (A) NOE correlation of chlorophyll d. (B) Structure and
numbering system of chlorophyll d.
also established as C54H70O6N4Mg (chlorophyll d) by highresolution FABMS [(M + H) + , m/z 894.5160 (calculated
for 894.5146)].
Quantification of pigment—The amount of chlorophyll a was less than 0. \% of the cell dry weight (Table 3),
while the content of chlorophyll d was higher than 2% of
the cell dry weight. Chlorophyll d was the predominant pigment. The ratio of chlorophyll a/d in the cell varied between 0.027 and 0.092 (Table 4).
Phycobiliprotein-like pigment—The peaks ranging
from 580 run to 660 nm in the in vivo absorption spectrum
Table 4 Comparison of chlorophyll ratio in various oxygenic photoautotrophs which contain two different chlorophylls
Organism
New photosynthetic
prokaryote
Prochloron "
Prochlorothrixb
Prochlorococcusc
Chlorophyta d and
Higher plants
Contained
chlorophylls
Chi a/other
Chi
a, d
0.027-0.092
a,b
a,b
DVea, DVb
a, b
2.6-3.4
7-18
0.6-12.5
2-3
' Alberte et al. 1986.
* Burger-Wiersma and Post 1989.
c
Partensky et al. 1993.
" Lee 1989.
' Divinyl-chlorophyll.
280
Chlorophyll d as a predominant pigment
100
200
300 400
Irradiation
photons nrr2 s'1)
500
Fig. 7 Photosynthesis-irradiation curve of the new oxygenic photosynthetic prokaryote.
Discussion
Oxygenic photosynthetic prokaryotes have been divided into two divisions: the Cyanophyta (Cyanobacteria)
characterized by possession of chlorophyll a with phycobiliproteins and the Prochlorophyta (Oxychlorobacteria) characterized by possession of chlorophylls a and b but without
phycobiliproteins. The stacking of thylakoid-like membrane and the absence of phycobilisome-like particles in
the newly isolated photosynthetic prokaryote (Fig. IB, C)
indicate a morphological similarity of this organism to the
Prochlorophyta rather than to the Cyanophyta. But since
the new prokaryote contains chlorophylls a and d and a
trace amount of a phycobiliprotein-like pigment, it cannot
be accommodated in either of the divisions. The taxonomical consideration will be described elsewhere where the
name Acaryochloris marina Miyashita et Chihara gen. et
sp. nov. for the new organism will be proposed (in preparation).
The pigment composition of the new photosynthetic
prokaryote was characterized by the possession of (1) chlorophyll d as a major pigment, (2) chlorophyll a as a minor
pigment, (3) chlorophyll c-like pigment, (4) a-carotene but
not /J-carotene and (5) trace amount of a phycobiliproteinlike pigment.
The most distinctive feature in the pigment composition was the possession of chlorophyll d. The natural occurrence of chlorophyll d had been unclear because no organism had been reported which contained chlorophyll d,
consistently and predominantly. But chlorophyll d was constantly extracted as a major pigment from the new organism grown under various conditions, confirming the
natural occurrence of chlorophyll d. The new organism is a
unique organism in possession of chlorophyll d as a major
photosynthetic pigment.
The chemical structure of chlorophyll d was proposed
by Holt and Morley (1959) based on the detailed comparison between 2-desvinyl-2-formyl chlorophyll a prepared by permanganate oxidation of chlorophyll a and
chlorophyll d extracted from Gigartina papillata (Rhodophyta). To confirm the major pigment as chlorophyll d we
applied NMR techniques. This was the first approach to apply NMR techniques for the chemical structure analysis of
chlorophyll d. And it was confirmed that the major green
pigment in the new organism has the same chemical formulation of that proposed by Holt and Morley.
All oxygenic photosynthetic organisms contain chlorophyll a as the predominant pigment ranging from 03% to
3% of dry weight (Lee 1989). However, chlorophyll a in the
new organism was less than 0.1%, while the amount of
chlorophyll d was higher than 2% of the cell dry weight.
The ratio of chlorophyll a/d in the cells was less than 0.05.
The ratio of chlorophyll a/b, for example, is more than
one ( > 1) in all known oxygenic phototrophs which contain
two different chlorophylls (Table 4). The new prokaryote is
an unique oxygenic photoautotroph which contains chlorophyll a as a minor pigment.
The chlorophyll c-like pigment, probably MgDVP,
was also detected in the prochlorophycean algae, Prochloron (Larkum et al. 1994), Prochlorothrix (Goericke and
Repeta 1992) and Prochlorococcus (Goericke and Repeta
1992), but not in the cyanophycean algae. The chlorophyll
c-like pigment detected in Prochloron had been shown to
contribute to photosynthesis (Larkum et al. 1994). The occurrence of the chlorophyll c-like pigment in the new prokaryote suggests some similarities to prochlorophycean
algae in their pigmentation.
a-carotene is distributed in certain taxa of eukaryotic
algae. But, until the discovery of the presence of the pigment in Prochlorococcus marinus, it had been believed that
oxygenic photosynthetic prokaryotes did not produce acarotene (Goodwin and Britton 1988, Liaaen-Jensen 1979,
1985). The new organism is the second photosynthetic prokaryote which contains a certain amount of a-carotene.
The possession of a small amount of phycobiliproteinlike pigment is also notable. Phycobiliproteins are usually
observed as a light-harvesting antenna in the Cyanophyta,
the Rhodophyta and the Cryptophyta. Therefore the phycobiliprotein-like pigment in the new prokaryote possibly contributes to photosynthesis. But the fact that no particles like
phycobilisomes were observed in the new organism by electron microscopy indicated that this pigment may exist in a
form different from that observed in red algae and cyanophytes.
The new photosynthetic prokaryote may have a unique and as yet unknown structure and function in photosynthesis. The organism contains chlorophyll a* as a predominant pigment and the cell has absorption peak of red
Chlorophyll d as a predominant pigment
light at least 35 run longer wavelength than those of known
oxygenic photoautotrophs. But the characteristic in vivo absorption of chlorophyll a around 680 nm could not be observed, indicating that light absorption by chlorophyll a is
at a negligible level. Moreover, if we calculate the maximum photosynthesis and respiration rate of the new organism on the basis of the chlorophyll a content as commonly
done in evaluation of photosynthetic activity, those were
1,760 jrniol O2 (mg Chi a)" 1 h~' and 146^mol O2 (mg Chi
a)~' h" 1 respectively. These values were extremely high in
comparison with those of known oxygenic photosynthetic
prokaryotes as well as eukaryotes. This support the idea
that chlorophyll d mainly contributes for photosynthesis in
this new organism.
We would like to express our gratitude to the government of
the Republic of Palau for giving us an opportunity for scientific
research and collecting biological specimens. We thank the captain and crews of the R/V "Sohgen Maru" for helpful assistance
in thefieldcollections and Y. Kikuchi, H. Urata, M.F.-Hasegawa
and S. Dobashi of MBI Kamaishi Laboratories for technical
assistance. We also thank Dr. Ralph A. Lewin, Scripps Institution
of Oceanography, Dr. Horst Senger, Philipps-Universitat Marburg, and Dr. N.I. Bishop, Oregon State University, for their kind
help in the preparation of the manuscript. This work was performed as a part of The Industrial Science and Technology Frontier
Program supported by New Energy and Industrial Technology Development Organization, Japan.
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(Received October 7, 1996; Accepted December 16, 1996)