Journal of’General Microbiology (1985), 131, 2497-2500.
Printed in Great Britain
2497
S H O R T COMMUNICATION
Immunoelectronmicroscopic Localization of Calvin Cycle Enzymes in
Chlorogloeopsisfvitschii
By A . M . H A W T H O R N T H W A I T E , T . LANARAST A N D G . A . C O D D *
Department of’Biologica1 Sciences, University of’Dundee, Dundee D D l 4HN, UK
(Receitted 23 May 198.5)
Antisera against the Calvin cycle enzymes D-ribulose-l,5-bisphosphatecarboxylase/oxygenase
(RuBisCO) and phosphoribulokinase (PRK) have been used in immunogold electronmicroscopy studies on cell sections of the cyanobacterium Chlorogloeopsis fritschii. RuBisCO
antiserum consistently labelled the carboxysomes (polyhedral bodies) and the cytoplasm. PRK
antiserum-labelling occurred in the cytoplasm but not in the carboxysomes. The data agree with
in vitro enzyme localization studies, and confirm that both enzymes occur in the cytoplasm and
that RuBisCO, but not PRK, also occurs in the carboxysomes of C. jritschii.
INTRODUCTION
We have isolated carboxysomes (polyhedral bodies) from the cyanobacterium Chlorogloeopsis
jritschii and have obtained evidence in ttitro that these inclusion bodies contain the C0,-fixing
enzyme of the Calvin cycle, ribulose-l,5-bisphosphatecarboxylase/oxygenase (RuBisCO)
(Lanaras & Codd, 1981 a, 1982). The association of RuBisCO with carboxysomes has also been
demonstrated for other autotrophs, including colourless sulphur-, nitrite- and ammoniaoxidizing bacteria (Codd & Marsden, 1984). The possibility was raised that other Calvin cycle
enzymes may also be located in carboxysomes and evidence for, and against, this concept has
been obtained for Thiobacillus neapolitanus (Beudeker & Kuenen, 1981 ; Beudeker et al., 1981 ;
Cannon & Shively, 1983). Our interest in the location of the second enzyme essential for and
unique to the Calvin cycle, phosphoribulokinase (PRK), was prompted by the presence in
dissociated C .jritschii carboxysomes of a cryptic 39 kDal polypeptide (Lanaras & Codd, 1981a)
with almost the same molecular mass as the subunits of purified C. fritschii PRK (40 kDal;
Marsden & Codd, 1984).
PRK activity and enzyme protein were almost entirely in the soluble (cytoplasmic) fraction of
C. fritschii cells, broken under conditions which favour carboxysome recovery, and no
association of the enzyme with carboxysomes was found (Marsden et al., 1984). Nevertheless,
the difficulties of carboxysome isolation make desirable a complementary approach to
subcellular enzyme localization. Here, we have used immunocytochemical labelling, involving
colloidal gold with RuBisCO and PRK antisera, to examine the distribution of Calvin cycle
enzymes in thin sections of C. fritschii cells.
METHODS
Organism and growth. Chlorogloeopsis jritschii 1411/16, from the Culture Centre of Algae and Protozoa,
Cambridge, UK, was grown as detailed before (Codd & Stewart, 1973), except that cells were harvested during
early stationary phase.
Celljxation. Cells were harvested by centrifugation at 2000g for 10 min and fixed in phosphate buffer (0.1 M,
pH 7.2) containing 2% (v/v)glutaraldehyde for 2-6 h at room temperature. Excess glutaraldehyde was removed by
t Present address: Department of Botany, Aristotle University of Thessaloniki, GR-540 06 Thessaloniki,
Greece.
Abbreviations : RuBisCO, ribulose-l,5-bisphosphate carboxylase/oxygenase ; PRK, phosphoribulokinase.
0001-2716 0 1985 SGM
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Short cornmunicat ion
Fig. 1. Localization of RuBisCO and PRK in C..fritschii. Cell sections were labelled with 20 nm
colloidal gold particles after treatment with RuBisCO antiserum (a) and P R K antiserum (b). CA,
carboxysome. Bar markers represent 1 pm.
washing twice for 15 min in the same buffer. The cells were dehydrated in a graded ethanol series (50%, 2 x 15
min, followed by 15 min each of 70, 90, 95 and loo%, v/v, ethanol), and then embedded in LR White Resin
(London Resin, Basingstoke, Hants, UK) for 2 x 30 min, followed by an 18 h incubation. Samples were then
polymerized at 60 "C for 20 h in glycerine moulds.
Cell sectioning and labelling. A modification of the method of Beesley et al. (1984) was used. Cells were sectioned
onto uncoated gold grids (400 mesh) using a diamond knife. The grids were incubated on Tris/HCl buffer (50 mM,
pH 7.4) containing 104 (w/v) bovine serum albumin, 1 % (v/v) Tween-20 and 0.2% (w/v) gelatin, before incubation
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T
CA
CY
M
0
N
II
CA
CY
M
25
O
N
0
Fig. 2. Distribution of colloidal gold particles in the localization of RuBisCO and PRK in C.Jiitschii by
immunoelectronmicroscopy. (a) RuBisCO distribution ; 76 individual cells examined ; 9345 gold
particles counted. (b)PRK distribution; 45 individual cells examined; 28853 gold particles counted. In
control experiments, the number of gold particles on cell sections treated with null serum plus gold-goat
anti-rabbit serum, and with gold-goat anti-rabbit serum alone, varied between zero and 0.6% of the
numbers of particles associated with antiserum treated sections. CA, carboxysomes ; C Y , cytoplasm;
M, cytoplasmic membrane; 0, all other discernible inclusion bodies; N, labelling on outer layers
external to cytoplasmic membrane ; marker bars represent SD.
on a suitable concentration of appropriate antiserum for 18 h at 4 "C. Antisera against purified RuBisCO from
Microcystis 7820 and PRK from C. jritschii were raised in rabbits as before and shown to be monospecific
according to rocket immunoelectrophoresis (Lanaras & Codd, 1981b). The grids were then washed gently in tap
water and 0.02% (w/v) KCl for 15 min before incubating for 1 h with 20 nm colloidal gold probe conjugated to goat
anti-rabbit IgG (GAR G20, Jensen Pharmaceutica, Beerse, Belgium) to a dilution of 1/15. Preparations were
dried in air and negatively stained using 0.2% (w/v) osmium vapour, and examined using an Associated Electrical
Industries EM 801 transmission electron microscope.
R E S U L T S A N D DISCUSSION
Typical results of RuBisCO protein localization using RuBisCO antiserum, raised in rabbits,
followed by colloidal gold-conjugated goat anti-rabbit serum, are shown in Fig. 1( a ) .The label
was consistently clustered over the sectioned carboxysomes, but was not significantly associated
with other inclusion bodies. RuBisCO antiserum labelling of the cytoplasm also occurred,
confirming in vitro findings that part of the cyanobacterial cell complement of RuBisCO is
carboxysomal and that the remainder is cytoplasmic (Codd & Stewart, 1976; Lanaras & Codd,
1981 a, b, 1982). Immunocytochemical labelling of the cyanobacterium Anabaena cylindrica
using RuBisCo antiserum has also resulted in labelling of the carboxysomes, and of the
cytoplasm, where label was present in the region of the thylakoids (Cossar et al., 1985).
The distribution of PRK antiserum labelling was markedly different from that of RuBisCO.
Carboxysomes were not significantly labelled (Fig. 1b);most of the gold particles were observed
in the cytoplasm and in the peripheral region inside the cytoplasmic membrane. The percentage
distribution of gold particles in the presence of both antisera, as determined in sections of whole
C.jritschii cells harvested from early stationary phase photoautotrophic batch culture, is given in
Fig. 2.
The present data confirm that the C. jritschii carboxysomes are a site of RuBisCO
accumulation and that the inclusions do not contain PRK (Lanaras & Codd, 1981a ; Marsden et
al., 1984). We have obtained similar results by the immunogold labelling approach in cell
sections of the unicellular cyanobacterium Anacystis nidulans R2 (Synechococcus PCC 7942)
(unpublished data), indicating a similar composition between carboxysomes from filamentous
and unicellular strains, despite considerable variation in carboxysome size, shape and
abundance within cyanobacteria (Codd & Marsden, 1984).
We thank the Nuffield Foundation for financial support and Dr W. J . N . Marsden for help with techniques.
A.M. H . thanks the Science and Engineering Research Council for a postgraduate studentship.
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