746

Proteomic assessment of an established

technique for carboxysome enrichment from
Synechococcus PCC7942
Ben M. Long, G. Dean Price, and Murray R. Badger

Abstract: Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme
for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). Sequencing of
cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes.
Cyanobacteria containing form 1A Rubisco possess -carboxysomes, while those with form 1B Rubisco possess carboxysomes. Given the central importance of carboxysomes in the CO2-concentrating mechanism of cyanobacteria,
understanding the nature and composition of these structures is of considerable importance. In an effort to develop
techniques for the characterization of the structure of -carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the PercollMg2+ carboxysome enrichment technique using the freshwater
cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorptionionization time of flight mass
spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used
to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using
MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis
on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of
38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL
and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.
Key words: CO2-concentrating mechanism, cyanobacteria, carboxysomes, proteomics.
Rsum : Les carboxysomes sont des corps polyhdriques lis des protines, quon retrouve chez les cyanobactries;
ils contiennent une enzyme cl pour la photosynthse, la carboxylaseoxygnase du ribulose-1,5-bisphosphate (Rubisco). Le squenage des gnomes a rvl que les cyanobactries possdent lun ou lautre de deux types de carboxysomes. Les cyanobactries qui contiennent la Forme 1A de la Rubisco possdent des -carboxysomes, alors que
celles qui contiennent la Forme 1B de la Rubisco possdent des $-carboxysomes. Compte tenu du rle important des
carboxysomes dans le mcanisme de concentration du CO2 des cyanobactries, une comprhension de la nature et de la
composition de ces structures revt une importance considrable. Afin de dvelopper des techniques pour caractriser la
structure des $-carboxysomes, en particulier lenveloppe protinique externe, les auteurs ont conduit une valuation
protomique de la technique denrichissement en carboxysomes par le PercollMg2+, en utilisant la cyanobactrie deau
douce Synechococcus sp. PCC7942. Pour dterminer la teneur en protines dune nouvelle fraction enrichie en carboxysomes, les auteurs ont utilis deux mthodes, soit le temps de dsorption matriciel assist au laser par spectroscopie de
masse (MALDI-TOF MS), ou soit lidentification multidimensionnelle des protines (MuDPIT). Ils ont identifi un total de 17 protines en utilisant la spectroscopie de masse MALDI-TOF, partir de fractions enrichies en carboxysomes,
alors quis ont reconnu 122 protines en analyant le mme matriel par analyses MuDPIT. La protine carboxysomique
CcmM a t identifie par ultidimensionnelle des protines (MuDPIT). Ils ont identifi un total de 17 protines en utilisant la spectroscopie de masse MALDI-TOF comme constitue de deux protines de 38 et 58 kDa respectivement.
Les seules autres protines carboxysomiques identifies sont les petite et la grande sous-units de la Rubisco (RbcL et
RbcS). Les auteurs discutent les raisons pour lesquelles on a pas obtenu une vue plus complte de lensemble des protines carboxysomiques, ainsi que les directions futures envisages.
Mots cls : mcanisme de concentration du CO2, cyanobactrie, carboxysomes, protomique.
[Traduit par la Rdaction]

Long et al.

757

Received 25 August 2004. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 26 July 2005.
B.M. Long, G.D. Price,1 and M.R. Badger. Molecular Plant Physiology Group, Research School of Biological Sciences,
Australian National University, P.O. Box 475, Canberra ACT 2601, Australia.
1

Corresponding author (e-mail: dean.price@anu.edu.au.).

Can. J. Bot. 83: 746757 (2005)

doi: 10.1139/B05-058

2005 NRC Canada

Long et al.

Introduction
Cyanobacteria represent an ancient group of oxygenic
photoautotrophs whose evolution has coincided with dramatic changes in the earth atmospheric gas composition over
2.7 billion years (Buick 1992). During this time, the atmospheric composition of CO2 has decreased while O2 has increased. As a consequence of these changes, cyanobacteria
have needed to develop a means by which they can effectively fix lower concentrations of atmospheric CO2 in the
presence of higher concentrations of O2. However, because
of the relatively low ratio of carboxylase activity to coexisting oxygenase activity of the CO2-fixing enzyme ribulose
1,5-bisphosphate carboxylaseoxygenase (Rubisco), CO2
fixation has theoretically become increasingly difficult over
time. In response to this evolutionary pressure, cyanobacteria have evolved an effective CO2-concentrating mechanism (CCM) to overcome the relatively high substrate
requirements for the carboxylase activity of Rubisco and, as
a result, they remain successful CO2-fixing photoautotrophs
in a wide range of ecological niches.
Cyanobacterial CCM consist of a number of adapted processes whose roles are to both elevate cytosolic HCO3 concentrations and then to elevate CO2 in close proximity to
Rubisco. These processes include HCO3 and CO2 transport
systems, located on the cytoplasmic and thylakoid membranes, and carboxysomes; polyhedral, protein-bound
microbodies that contain Rubisco. The carboxysomes are
characteristic of, and central to, the CCM of cyanobacteria.
Carboxysomes serve to concentrate CO2 in close physical
proximity to Rubisco to maintain relatively high carboxylase
activity while keeping oxygenase activity to a minimum.
This process is thought to be achieved by the presence of a
carboxysomal carbonic anhydrase which converts HCO3,
accumulated within the cytosol, to CO2 within the
carboxysome (Kaplan and Reinhold 1999; Badger et al.
2002; Price et al. 2002; So et al. 2004), while properties of
the carboxysomal protein coat minimize CO2 efflux. The
CO2 pump also plays a critical role in recycling leaked CO2,
thereby minimizing CO2 loss from the carboxysome (Price
et al. 2002). In this manner, CO2 fixation can occur at relatively high rates, even at very low external inorganic carbon
(Ci).
While carboxysomes have been found in all cyanobacteria
characterized to date, they and homologous polyhedra are
also present in a number of chemoautotrophic bacteria (Cannon et al. 2001). At the gene level, carboxysomes from all
groups share degrees of similarity, although there are distinct
differences that separate carboxysomes into two groups.
Based on their Rubisco phylogenies, carboxysomes that contain form 1A Rubisco form one group, while those with
form 1B Rubisco form another (Badger et al. 2002). Phylogenetic analysis of the genomes of these two groups has revealed that there are also distinct differences between their
carboxysome genes, prompting Badger et al. (2002) to propose that carboxysomes containing form 1A Rubisco and
those containing form 1B Rubisco should be termed - and
-carboxysomes, respectively. Under this scheme, the
cyanobacteria fall into two groups: - and -cyanobacteria
producing - and -carboxysomes, respectively. Those with
-carboxysomes share carboxysome protein homologies

747

with a number of chemoautotrophic proteobacteria, while carboxysomes are, to date, confined to the -cyanobacteria
(Badger and Price 2003).
An understanding of the protein composition of
carboxysomes, and details of their assembly, is essential to
determining the structure and function of these microbodies
and their role in CCMs. To date, carboxysomal protein composition has been studied in greatest detail in the carboxysomes, with many studies concentrated on the
chemoautotrophic
proteobacterium
Halothiobacillus
neapolitanus (Cannon and Shively 1983; Holthuijzen et al.
1986; Cannon et al. 1991; English et al. 1994; Shively et al.
1996, 1998; Baker et al. 1998, 1999, 2000; So et al. 2004).
The polypeptides making up the protein coat of carboxysomes from H. neapolitanus are coded for by genes
of the cso type (csoS1A, csoS1B, csoS1C, csoS2, csoS3,
orfA, and orfB) as described by Cannon et al. (2002), while
the genes cbbL and cbbS code for Rubisco large and small
subunits, respectively (Fig. 1). Together, these genes form a
putative carboxysome operon (Cannon et al. 2003). Of the
proteins residing in the carboxysomal coat of
H. neapolitanus, CsoS3 has recently been characterized as
an -class carbonic anhydrase (CA) (So et al. 2004), while
the CsoS1 proteins are major constituents of the carboxysome coat (Cannon and Shively 1983). Two coat
polypeptides encoded by csoS2 (namely CsoS2A and
CsoS2B) differ in their degree (or perhaps type) of posttranslational glycosylation (Baker et al. 1999).
Among the cyanobacteria, little information has yet been
gathered to describe the protein composition of either - or
-carboxysomes. Based on genetic homology comparison,
the -cyanobacteria are likely to share several of the
carboxysomal proteins so far described from proteobacteria
(Badger et al. 2002). However, the genetic structure of carboxysomes suggests several unique proteins constitute
this type of carboxysome (Fig. 1). Unlike -carboxysomes,
-carboxysome proteins are coded for primarily by a cluster
of genes of the ccm type (ccmKLMNO), along with the
genes for the large and small subunits of Rubisco (rbcL and
rbcS). In addition, a carboxysomal CA (icfA or ccaA) and
sometimes additional CcmK homologues are coded for elsewhere on the genome (Fig. 1). Based upon homologies between several - and -carboxysomal proteins (namely:
CcmK/O and CsoS1; and CcmL and proteins A/B) it is assumed that these proteins will have common functional and
(or) structural roles in both carboxysome types (Cannon et
al. 2002). Noteworthy, however, CcmM and CcmN have no
sequence homologues in -carboxysomes, and CsoS2 and
CsoS3 have no homologues in -carboxysomes (Badger et
al. 2002; Cannon et al. 2002). Nevertheless, it is suggested
that CcmM and CcmN may be functionally similar to CsoS2
and CsoS3 in -carboxysomes (Cannon et al. 2002). It is
also interesting to note that while CsoS3 has now been characterized as an -CA (So et al. 2004) and that CcmM has a
-CA domain (Ludwig et al. 2000), -carboxysomal CA activity is currently attributed to CcaA (Price et al. 1992; Yu et
al. 1992; Badger et al. 2002). Interestingly, some cyanobacteria lack any recognizable CA apart from the CA of CcmM (Badger and Price 2003). Perhaps in these
species, CcmM functions similarly to CsoS3 as a means to
convert HCO3 to CO2 in the carboxysome.
2005 NRC Canada

748
Fig. 1. Comparison of carboxysomal genes from both - and carboxysomes. The arrows represent individual carboxysome
genes, with homologous genes identified by the same patterning.
The -carboxysomes are here represented by the proteobacterium
Halothiobacillus neapolitanus and by the -cyanobacterium
Synechococcus WH8102. The -carboxysomes are represented by
the cyanobacterium Synechococcus PCC7942. Many of the
carboxysomal genes from Synechococcus PCC7942 are coded for
by a single gene cluster, close to the Rubisco large and small
subunit genes (rbcL and rbcS) although the carboxysomal carbonic anhydrase gene (icfA) from this species and several CcmK
homologues (CcmK3 and CcmK4) are coded for elsewhere on
the genome. CcmK3 and CcmK4 are unlikely to play a part in
carboxysome structure and function, since inactivation of ccmK1
alone leads to high CO2 requirement for growth (Price et al.
1993). The diagram is not to scale.

Can. J. Bot. Vol. 83, 2005

carboxysomal CA activity in preparations enriched in this

manner and later provided evidence that both CcmM (existing as both 38-kDa and 58-kDa proteins) and CcaA copurify in the same preparations (Price et al. 1998). However,
what is expected to be a full complement of -carboxysomal
proteins has yet to be described from PercollMg2+ preparations.
With the rapid development of proteomic technologies in
recent years, and with the growing list of completed
cyanobacterial genome sequences, we are now well placed
to apply these technologies to the characterization of discrete proteomes from cyanobacteria. In an effort to develop
techniques to characterize the complete protein complement
of -carboxysomes from cyanobacteria, herein we report results from a proteomic assessment of a carboxysomesenriched fraction from the -cyanobacterium Synechococcus
PCC7942 using the PercollMg2+ method.

Materials and methods

In the -cyanobacterium Synechococcus PCC7942, there

are at least eight genes coding for carboxysome-related proteins including rbcL and rbcS (Fig. 1). The identity of these
genes has become apparent through analysis of high CO2 requiring (HCR) mutants and those with aberrant
carboxysome structure: ccmK1 (Price et al. 1993); ccmL
(Price and Badger 1989a, 1991; Price et al. 1993); ccmM
(Price et al. 1993); ccmN (Schwarz et al. 1988; Friedberg et
al. 1989); ccmO (Friedberg et al. 1989; Martnez et al.
1997); icfA (ccaA) (Price and Badger 1989a, Fukuzawa et al.
1992; Yu et al. 1992). In addition, these genes, along with
rbcL, are concomitantly induced in response to Ci-limitation
(Woodger et al. 2003). The ccmKLMNO gene cluster (Fig. 1)
putatively codes for CcmK1 (10.9 kDa), CcmL (11 kDa),
CcmM (57.9 kDa), CcmN (16.3 kDa), and CcmO (29.3 kDa)
proteins. The gene icfA encodes the putative carboxysomal
CA (CcaA, 30.2 kDa), while RbcL (52.4 kDa) and RbcS
(13.3 kDa) are believed, for the most part, to complete the
protein
complement
of
Synechococcus
PCC7942
carboxysomes. The additional CcmK homologues are not
likely to play a part in carboxysome structure and function,
since inactivation of ccmK1 alone results in high CO2 requirement for growth (Price et al. 1993).
There are several reports of carboxysomes successfully
enriched from -cyanobacteria using the method of Percoll
Mg2+ precipitation (Price et al. 1992, 1993; Ors et al. 1995;
Satoh et al. 1997; So et al. 2002). Price et al. (1992) confirmed the presence RbcS and RbcL and associated

Growth and harvesting cyanobacteria and carboxysome

enrichment
Synechococcus PCC7942 was grown in modified BG-11
medium (Price and Badger 1989b) containing 20 mmolL1
HEPESKOH, pH 8.0 at 30 C and approximately 80 mol
photonsm2s1. Cultures were grown in either 1- or 3-L
flattened bottles (Roux bottles) and sparged with 1% CO2 in
air until 24 h prior to harvesting, at which time cultures were
sparged with air only. Cells were harvested by centrifugation
at room temperature (2700g, 15 min) when Chl concentration had reached approximately 78 gmL1. Carboxysomes
were enriched using the PercollMg2+ method of Price et al.
(1992) as outlined in Fig. 2. During this procedure, it is believed that carboxysomes undergo a Mg2+-dependent aggregation with Percoll beads, allowing them to be enriched
using differential centrifugation techniques (Price et al.
1992). Further clean-up of the preparation can be carried out
through washing steps using Triton X-100 and EDTA, and
final collection of EDTA-washed carboxysomes is achieved
by Mg2+ precipitation (Price et al. 1992). Extracts were prepared either in the presence of the protease inhibitor PMSF
or using a protease inhibitor cocktail for bacterial lysates
(Sigma Chemical Corp., St. Louis, Missouri). The fractions
collected during the enrichment procedure are identified using the following scheme: PM supernatant and pellet
(PercollMg2+ treated); TP supernatant and pellet (Triton X100 Percoll treated); EE supernatant and pellet (EPPS (N(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid))
EDTA treated); and EEM supernatant and pellet (EPPS
EDTAMg2+ treated). The previously undescribed EE pellet
fraction was used for proteomic analysis. Rubisco activity in
enriched fractions was confirmed as described previously
(Badger and Price 1989), except that assays were carried out
at room temperature in a total volume of 250 L. Specific
activities were not determined because of difficulties encountered in determining accurate protein concentrations in
the presence of Percoll. Relative protein content of enriched
fractions was determined from Coomassie-stained gels,
which had been scanned using bright white light in transmittance mode. Gel images were analysed in a manner similar
to that described by Cannon and Shively (1983) using
2005 NRC Canada

Long et al.

749

Fig. 2. A flow chart for the enrichment of carboxysomes using

the PercollMg2+ method of Price et al. (1992). The percent values to the right of each fraction name are the percentage of recovery of Rubisco activity in that fraction relative to that found
in the clarified lysate. Rubisco activity was determined as described previously (Badger and Price 1989), except that assays
were carried out at room temperature in a total volume of
250 L. Total Rubisco activity in the clarified lysate fraction was
3.53 molmin1. Abbreviations: 1 EM buffer, 40 mmolL1
EPPSNaOH, pH 8.0, 27 mmolL1 MgSO4; EE buffer,
40 mmolL1 EPPSNaOH, pH 8.0, 10 mmolL1 EDTA; PM,
supernatant and pellet (PercollMg2+ treated); TP, supernatant
and pellet (Triton X-100 Percoll treated); EE, supernatant and
pellet (EPPSEDTA treated); and EEM, supernatant and pellet
(EPPSEDTAMg2+ treated).
Whole cells

Lysozyme / French Press (70 MPa )

Centrifuge (12 000g, 10 min)

Clarified lysate

100%

Dilute with 3 volumes 1 EM buffer

containing 20% Percoll and 0.133% Triton
X-100
Centrifuge (12 000g, 15 min)

PM
supernatant

13%

PM pellet

83%

Wash pellet in 0.75 EM buffer and 1%

Triton X -100
Wash pellet in 0.75 EM buffer

TP
supernatant

0%

TP pellet

88%

Resuspend pellet in EE buffer

Centrifuge (10 000g, 5 min)

EE
supernatant

14%

EE pellet

40%

Add MgSO4 to final concentration of 40 mmolL1

Centrifuge (10 000g, 5 min)

EEM
supernatant

7%

EEM pellet

7%

ImageQuant gel analysis software. Approximate molar quantities were estimated after accounting for protein molecular
mass. Variation in staining of the Rubisco small and large
subunits was corrected for using the molar ratio of
RbcS:RbcL in the PM pellet fraction as a reference.
Transmission electron microscopy
Enriched carboxysomes from the EE pellet fraction were
fixed with 2.5% glutaraldehyde and 4% formaldehyde in
0.75 EM buffer (30 mmolL1 EPPSNaOH, pH 8.0,
20 mmolL1 MgSO4) for 2 h. Fixed material was washed
three times in 0.75 EM buffer and postfixed in 1% OsO4
in 0.75 EM buffer for 1 h. Samples were dehydrated
through a graded ethanol series, followed by 1:1 ethanol
propylene oxide for 30 min, and finally 100% propylene oxide for 30 min. Samples were subsequently infiltrated with
Epon Araldite, and then embedded in LR white resin, and
cured overnight at 60 C. Sections were cut using a Leica

ultracut microtome and stained with 6% uranyl acetate for

20 min followed by lead citrate for 15 min. Stained sections
were viewed using an Hitachi H-7100FA transmission electron microscope at 75 kV.
Proteomic analysis
Proteins in carboxysome-enriched preparations were analysed using both peptide mass fingerprinting (PMF) and peptide fragmentation fingerprinting (PFF) techniques
(Chamrad et al. 2003). PMF utilized matrix-assisted laser
desorptionionization time-of-flight mass spectrometry
(MALDI-TOF MS) to determine the mass fingerprint of
trypsin-digested proteins from SDSPAGE gels. Multiple dimension protein identification technology (MuDPIT;
Washburn et al. 2001), which utilizes tandem liquid chromatography (LC) tandem MS, was used for analysis of tryptic
peptides and their fragments from digested soluble protein
samples (PFF). This method has advantages over traditional
2D gel electrophoresis-based proteomics, in that it enables
high throughput analysis of complex protein mixtures and is
relatively unbiased in its ability to identify proteins that are
typically not found using 2D gel techniques (e.g., low abundance proteins, those of extreme pI and molecular mass, and
membrane proteins; Washburn et al. 2001).
For MALDI-TOF MS analysis of proteins associated with
the EE pellet fraction, approximately 50 g of protein was
separated by SDSPAGE using precast 10% acrylamide BisTris gels (Invitrogen, Carlsbad, California). Gels were
stained with colloidal Coomassie blue G-250 for visualization of protein bands, and bands were excised using a clean,
sharp scalpel blade. Excised bands were transferred to 500L low-bind Eppendorf tubes and destained by washing
three to four times in 50150 L 50% acetonitrile,
25 mmolL1 ammonium bicarbonate, then dried at 50 C for
20 min. Dried gel pieces were rehydrated in 15 L digestion
solution (12.5 gmL1 bovine trypsin (Roche Diagnostics,
Mannheim, Baden-Wrttemberg, Germany), 25 mmolL1
ammonium bicarbonate) on ice for 15 min and then transferred to 37 C for protein digestion overnight. Tryptic peptides were extracted twice from gel pieces using 75% (v/v)
methanol, dried in a vacuum centrifuge and redissolved in
1020 L of 50% acetonitrile 0.1% trifluoroacetic acid.
Peptide mass fingerprints from each digested protein were
determined using a Bruker Daltonics OmniFlex MALDITOF MS. The matrix (-cyano-4-hydroxycinnamic acid) was
prepared for use with a Bruker AnchorChip target as described by the manufacturer, and 12 L of peptide extract
was spotted onto the target along with an equal volume of
matrix and 0.10.5 L peptide standards diluted 1:100
(PepMix1, LaserBio Labs, Sophia-Antipolis, France) for onspot mass calibration. Peptide masses were determined in
positive ion, reflectron mode within the range 8004000 m/z.
Resulting spectra were annotated using X-MASS software,
and peak reports were analysed using MASCOT database
search engine software (Matrix Science, London) on an inhouse server. Data were searched against the complete
Synechococcus PCC7942 protein database assuming up to
two missed trypsin cleavages and a mass error of 75 ppm.
Where significant matches were found, any unmatched
masses from that data set were researched using the same
criteria as above to examine the likely presence of multiple
2005 NRC Canada

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Can. J. Bot. Vol. 83, 2005

Fig. 3. Transmission electron micrographs of embedded and sectioned carboxysomes from the EE pellet fraction prepared using the
PercollMg2+ technique. In both images carboxysomes are indicated by arrowheads: (a) enriched intact carboxysomes;
(b) carboxysomes and associated cell debris carried over during enrichment. Scale bars = 500 nm.

proteins in each band. For positive identification of proteins,

probability-based MOWSE scores had to be significant with
P < 0.05.
For MuDPIT analysis of carboxysome preparations,
whole samples were dissolved in 8 molL1 urea,
100 mmolL1 Tris-HCl, pH 8.5 to a final protein concentration of 5 mgmL1. Prior to tryptic digestion, cysteines were
reduced with 1 mmolL1 dithiothreitol for 20 min at 37 C
followed by alkylation with 10 mmolL1 iodoacetamide for
30 min in the dark at room temperature. Reduced and
alkylated proteins were diluted to 2 molL1 urea with
100 mmolL1 Tris-HCl, pH 8.5, and bovine trypsin was
added such that the ratio trypsinprotein was 1:50. Protein
digestion was allowed to proceed overnight at 37 C. The resulting peptide mixture was dried in a vacuum centrifuge
and redissolved in 20 L 50% acetonitrile, 5% formic acid.
Samples were clarified by centrifugation prior to analysis.
MuDPIT analysis was performed on a Thermo Finnigan
ProteomeX workstation running XCalibur software. This
system is equipped with two Surveyor LC pumps to supply
mobile phases for each dimension of LC separation and a
10-port switching valve, which enables high throughput, sequential loading and elution of peptides from two reversedphase columns. The MS pump supplied mobile phases A
(0.1% formic acid) and B (acetonitrile, 0.1% formic acid) to
the reversed-phase columns (BioBasic-C18, 300 D (1 D =
0.1 nm) pore size; 0.18 mm 100 mm, Thermo HypersilKeystone, Thermo Electron, San Jose, California) fitted directly to the 10-port valve. The sample pump supplied mobile phases C (5% acetonitrile, 0.1% formic acid) and D (1
molL1 NH4Cl 5% acetonitrile, 0.1% formic acid) to a

strong cation exchange column (BioBasic-SCX; 300 D pore

size; 0.32 mm 100 mm, Thermo Hypersil-Keystone). The
flow rate on both pumps was set to 200 Lmin1 and was
reduced to 3 Lmin1 prior to the columns using a flow
splitter. Peptides were analysed from 10 L of sample, injected via a Surveyor auto-sampler using no-waste mode. In
the first dimension, peptides were separated based on their
relative charge on the strong cation exchange column equilibrated with 100% C. Peptides were eluted from the SCX
column in a series of 25-min steps with 0, 50, 100, 150, 200,
and 800 mmolL1 NH4Cl using mobile phase D. As they
eluted from the SCX column, peptides passed directly onto
one of the two reversed-phase columns to perform separation in the second dimension. The reversed-phase columns
were equilibrated with 5% A : 95% B, and peptides eluted
with a linear gradient from 5% to 65% acetonitrile in 0.1%
formic acid, using mobile phase B, over 30 min, followed by
10 min at 80% acetonitrile and re-equilibration to 5% A :
95% B. While peptides were eluted from one reversed-phase
column, the next salt-step elution from the SCX was being
loaded onto the second reversed-phase column. Eluted peptides were introduced directly into the electrospray ionization (ESI) source of a Thermo Finnigan LCQ DecaXP Plus
ion trap MS (Thermo Electron) via a 30-m internal diameter metal spray needle set orthogonally to the MS. The spray
voltage was set to 2.6 kV, the capillary temperature was set
to 170 C, and the sheath gas flow rate was set to 5 units.
For MSMS analysis, the normalized collision energy was
set at 35%, and the ion trap was set to carry out one full
scan followed by three MSMS scans of the most intense
ions. Dynamic exclusion was enabled with the following set 2005 NRC Canada

Long et al.

751

Fig. 4. Comparison of two carboxysome enrichment preparations using Coomassie-stained SDSPAGE gels. (a) Fractions collected
during the enrichment procedure, indicating the relative enrichment of carboxysome proteins. Approximately 25 g of protein from
each fraction was loaded onto the gel. The positions of RbcL (58 kDa), RbcS (10 kDa), 5-kDa and 38-kDa proteins, and lysozyme
(14 kDa) are marked by arrowheads to the right of lane 7. Note the enrichment of the carboxysomal proteins specifically in lane 5 (EE
pellet fraction) and relatively high concentration of lysozyme in other fractions. See Fig. 2 for an explanation of abbreviations. Lane 1,
molecular mass markers; lane 2, PM pellet; lane 3, TP pellet; lane 4, EE supernatant; lane 5, EE pellet; lane 6, EEM supernatant; lane
7, EEM pellet. (b) Fractions collected from a different enrichment preparation to be used for proteomic analysis. Approximately 50 g
of protein from each fraction was loaded onto the gel. The protein bands excised from lane 13 only (EE pellet fraction) for peptide
mass fingerprinting by matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF MS) are indicated by
the numbered arrowheads to the right of this lane. These numbers correspond with those presented in Table 1. Lane 8, molecular mass
markers; lane 9, clarified lysate; lane 10, PM pellet; lane 11, TP pellet; lane 12, EE supernatant; lane 13, EE pellet.

200
116.3
97.4
66.3

b
66.3
55.4

55.4

36.5
31

36.5
31
21.5

21.5
14.4

6
3.5

tings: repeat count, 2; repeat duration, 0.5 min; exclusion

duration, 3 min; and exclusion mass width, 2 Da.
MuDPIT data were analysed using BioWorks v3.1 software (Thermo Finnigan) using the SEQUEST algorithm
(Eng et al. 1994) and the same protein sequence file as used
for PMF analysis. The database was searched assuming up
to two missed trypsin cleavages, with methionine oxidation,
carboxyamidomethylation of cysteine, and carbamylation of
lysine as optional modifications. Output data were filtered
according to charge state, cross-correlation (Xcorr) and
delta-correlation (Cn) parameter values used by Peng et al.
(2003), which have been determined to limit false positive
identifications to below 1%. Thus, only fully tryptic peptides
with charge states of 1+, 2+, and 3+ and Xcorr values of
>2.0; >1.5, and >3.3, respectively, and Cn > 0.08 were accepted as significant matches. Partially tryptic peptides required Xcorr values of >3.0 and >4.0 for charge states of 2+
and 3+, respectively. Where only one or two peptides were
identified from a protein, manual interpretation of MSMS
spectra was carried out to confirm identity.

Results
Electron microscopy (Fig. 3) and recovery of Rubisco activity (Fig. 2) of PercollMg2+ preparations from
Synechococcus PCC7942 indicate that carboxysomes had
been enriched into several fractions using this method. SDS
PAGE analysis of fractions collected during the enrichment
procedure indicated a relative enrichment of the proteins

14.4

6
3.5

10

11

12

13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

with approximate molecular masses of 10, 38, 53, and

58 kDa specifically in the EDTA-treated pellet fraction (EE
pellet; Fig. 4). This is a novel fraction not previously described from the PercollMg2+ technique. These protein
bands correspond to CcmM (38 and 58 kDa) and Rubisco
small (10 kDa) and large (53 kDa) subunit proteins previously described for carboxysome preparations from this
strain (Price et al. 1992, 1998); on NuPAGE gels, the small
subunit consistently runs at approximately 10 kDa rather
than 13.3 kDa. The EE pellet fraction also contained a comparatively low concentration of lysozyme (Fig. 4). The remaining fractions enriched in Rubisco activity contained
relatively high concentrations of lysozyme carried over from
the cell lysis procedure and appeared to contain relatively
large numbers of proteins (Fig. 4). For some unknown reason the EDTA treatment leads to a loss of net assayable
Rubisco activity in the EE pellet (Fig. 2). It should be noted,
however, that aggregation of protein in this fraction seems to
occur readily. Nonetheless, a high yield of Rubisco protein
was detected in the EE pellet (Fig. 4). Because of the high
enrichment factor in the EE pellet, this fraction was chosen
for subsequent proteomic analysis. However, a considerable
carry-over of cellular debris was observed in all pellet fractions including the EE pellet (Fig. 3b), as has been described
previously for the TP pellet alone (Price et al. 1992).
SDSPAGE analysis of the EE pellet fraction from one
preparation (Fig. 4b) indicated at least 19 major bands that
would be suitable for PMF. A total of 17 proteins were positively identified as significant matches to proteins in the
2005 NRC Canada

123
110
87
87
87
73
73
58
53
38
35
29
29
26
22
20
17
17
12
10
9
7
4

1
2
3a
3b
3c
4a
4b
5
6
7
8
9a
9b
10
11
12
13
14
15
16
17
18
19

143.8

97.8
90.1
80.0
78.8
76.6
57.8
52.4
57.8 (35.2d)
35.2
28.4
30.3
27.7
23.2
19.3
17.1
17.7
14.3
13.3
9.5
9.2
3.9

Sequence mass
(kDa)

MOWSE
score
36

90
61
59
213
59
89
207
52
35
103
51
147
68
45
50
66
54
53
101
50
51

Description
RNA polymerasec
Unidentifiedc
Aminopeptidase N
Superfamily I DNA and RNA helicase
Catalaseperoxidase
Phycobilisome linker polypeptide
Outer membrane protein protective antigen
CO2-concentrating mechanism protein M
Rubisco large subunit
CO2-concentrating mechanism protein M
DnaJ-class molecular chaperonec
30S ribosomal protein S2
30K phycocyanin rodrod linker protein
30S ribosomal protein S3
50S ribosomal protein L4
30S ribosomal protein S5c
50S ribosomal protein L13
30S ribosomal protein S7
Hen egg white lysozyme
Rubisco small subunit
30S ribosomal protein S16
Superfamily I DNA and RNA helicasec
No homology
10

16
11
9
27
10
9
21
6
3
16
8
11
7
4
5
7
5
5
8
11
4

Matched
masses

Multiple dimension protein identification technology.

Molar ratios in this table were determined from lane 13 of the gel in Fig 4b.
c
Indicates proteins that have not been significantly identified at the P < 0.05 level. Closest matches to the database are listed in these cases.
d
The sequence mass in parentheses listed for band 7 is based on predicted sequence mass after N-terminal sequencing.

Apparent mass
(kDa)

Band No.
11

28
20
19
45
14
30
37
15
14
43
26
48
49
44
39
53
60
31
60
16
58

Sequence
coverage (%)
+

+
+
+
+
+

+
+
+

+
+
+
+

Also found by
MuDPIT analysisa

0.12
0.16
0.13

0.31

0.42
1.00
0.70
0.37
1.04

1.18
1.35
1.60
1.34
3.16
4.49
1.00
1.38
4.19
14.98

mol:mol
RbcL ratiob

Table 1. Proteins from enriched PercollMg2+ carboxysome preparation from Synechococcus PCC7942 determined by peptide mass fingerprinting using matrix-assisted laser
desorptionionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (band numbers refer to proteins indicated in Fig. 4b).

752
Can. J. Bot. Vol. 83, 2005

2005 NRC Canada

Long et al.

753

Fig. 5. Sequence coverage maps of the 58-kDa (a) and 38-kDa

(b) proteins identified as CcmM by matrix-assisted laser
desorptionionization time-of-flight mass spectrometry (MALDITOF MS) analysis of excised gel bands. Bold letters indicate
peptide sequences whose masses were found in tryptic digests of
excised protein bands. No tryptic peptides were identified from
the N-terminal region of the parent protein in the 38-kDa protein, suggesting that either this part of the full-length CcmM is
either removed post-translationally or the C-terminal region is
translated independently. Amino-terminal sequencing of the 38kDa protein indicates an amino-terminal start site at Val-216
(boxed) and a predicted mass of 35.2 kDa.
A

1
51
101
151
2 01
251
301
351
401
451
501

MPSPTTVPVA TAGR LAEPYI DPAAQVHAIA SIIGDVR IAA GVRVAAGVSI

RADEGAPFQV GKESILQEGA VIHGLEYGRV LGDDQADYSV WIGQR
VAITH
KALIHGPAYL GDDCFVGFRS TVFNARVGAG SVIMMHALVQ DVEIPPGR
YV
PSGAIITTQQ QADRLPEVRP EDR EFARHII GSPPVIVRST PAATADFHST
PTPSPLRPSS SEATTVSAYN GQGRLSSEVI TQVRSLLNQG YRIGTEHADK
RRFRTSSWQP CAPIQSTNER QVLSELENCL SEHEGEYVRL LGIDTNTRSR
VFEALIQRPD GSVPESLGSQ PVAVASGGGR
QSSYASVSGN LSAEVVNKVR
NLLAQGYRIG TEHADKRRFR TSSWQSCAPI QSSNER
QVLA ELENCLSEHE
G EYVRLLGID TASRSRVFEA LIQDPQGPVG SAK AAAAPVS SATPSSHSYT
SNGSSSSDVA GQVRGLLAQG YRISAEVADK RRFQTSSWQS LPALSGQSEA
TVLPALESIL QEHKGKYVRL IGIDPAARRR VAELLIQKP

1
51
101
151
2 01
251
301
351
401
451
501

MPSPTTVPVA
RADEGAPFQV
KALIHGPAYL
PSGAIITTQQ
PTPSPLRPSS
RRFRTSSWQP
VFEALIQRPD
NLLAQGYRIG
GEYVRLLGID
SNGSSSSDVA
TVLPALESIL

TAGRLAEPYI DPAAQVHAIA SIIGDVRIAA GVRVAAGVSI

GKESILQEGA VIHGLEYGRV LGDDQADYSV WIGQRVAITH
GDDCFVGFRS TVFNARVGAG SVIMMHALVQ DVEIPPGRYV
QADRLPEVRP EDREFARHII GSPPVIVRST PAATADFHST
SEATT VSAYN GQGR LSSEVI TQVR SLLNQG YRIGTEHADK
CAPIQSTNER QVLSELENCL SEHEGEYVRL LGIDTNTR
SR
GSVPESLGSQ PVAVASGGGR QSSYASVSGN LSAEVVNKVR
TEHADKRRFR TSSWQSCAPI QSSNERQVLA ELENCLSEHE
TASR SRVFEA LIQDPQGPVG SAK AAAAPVS SATPSSHSYT
GQVRGLLAQG YRISAEVADK RRFQTSSWQS LPALSGQSEA
QEHKGKYVRL IGIDPAARRR VAELLIQKP

Synechococcus PCC7942 database from 13 of the gel bands

(Table 1). Of the remaining 6 bands, band 15 was confirmed
to be hen egg white lysozyme (carried over from the cell
lysis procedure) after searching against the NCBI
nonredundant database using MASCOT, while bands 1, 2, 8,
12, and 18 could not be identified at the P < 0.05 significance level (Table 1). The carboxysomal proteins RbcL,
RbcS, and CcmM were positively identified, with CcmM
represented as two bands at 58 and 38 kDa (bands 5 and 7
respectively, Table 1). Sequence coverage maps from these
two protein bands indicate that the N-terminal region of the
full-length protein is absent from the 38-kDa protein
(Fig. 5). Six of the positively identified proteins were of ribosomal origin, while a number of other unrelated proteins
were identified (Table 1).
Analysis of EE pellet preparations by MuDPIT identified
122 proteins from 161 unique peptides. For simplicity, the
identified proteins were divided into categories based upon
functional relationships (Table 2). Of the proteins identified,
27 were 30S or 50S ribosomal proteins, while an additional
21 proteins were nucleic acid binding (Table 2). Eight transferases were identified, as were four ABC-type transporter
system components and three proteins classed as proteases
or peptidases. Another 23 proteins represent a group of, as
yet uncharacterized or hypothetical proteins, while the remaining 34 proteins are made up of a wide variety of proteins including membrane proteins, kinases, and ATPases.
Three carboxysomal proteins, namely CcmM, RbcL, and
RbcS, were successfully identified. Since MuDPIT analysis
does not provide information regarding the complete mass of

Table 2. Protein categories identified by multiple dimension protein identification technology (MuDPIT) analysis of
carboxysome-enriched preparations from Synechococcus
PCC7942.
Protein category

No. of identified
proteins

Carboxysomal proteinsa
Ribosomal proteins
Other nucleic acid binding proteins
Transferases
Proteasespeptidases
ABC-type transport components
Hypothetical or uncharacterized proteins
Other
Total

3
27
21
8
3
4
23
34
122

a
The carboxysome proteins identified by MuDPIT were RbcL, RbcS,
and CcmM.

the parent protein, it was not possible to distinguish if the

peptides identified from CcmM represented the 38- or 58kDa bands found by PMF analysis. Nonetheless, all peptides
identified from this protein using MuDPIT were represented
within the shorter C-terminal sequence noted in Fig. 5. Notably, however, CcmM was not identified by MuDPIT analysis in EE pellets prepared using PMSF as the only protease
inhibitor. Common to both the PMF and MuDPIT analyses
were 14 proteins, including CcmM, RbcL, RbcS, many of
the ribosomal proteins, and aminopeptidase N (Table 1).
Repeated extraction and enrichment procedures resulted in
some variability in relative protein content of fractions
(Fig. 4). However, the ratios of the four carboxysomal proteins (RbcL, RbcS, and both forms of CcmM) remained similar within fractions from different preparations (Tables 1
and 3; Fig. 4). As measured by image analysis of
Coomassie-stained gels, many of the contaminant proteins
identified by PMF analysis of gel bands were of greater or
equal molar quantity to RbcL in the particular extract analysed (lane 13 in Fig. 4b; Table 1). Comparison of protein
content in fractions collected during the enrichment procedure indicated an enrichment of the four carboxysomal proteins, identified by both MuDPIT and PMF analysis (namely
RbcL, RbcS, and both forms of CcmM), in the EE pellet
fraction (Fig. 4a). Notably, the 58-kDa full-length CcmM
protein band is almost undetectable in all other fractions
(Fig. 4a). Image analysis of Coomassie-stained gels indicates changes in the relative molar quantities of these proteins throughout the enrichment procedure, indicative of the
effects of various steps on carboxysomes during enrichment
(Table 3). This same analysis also revealed an approximately
fourfold enrichment of Rubisco into the EE pellet fractions,
whereas the enrichment factor for other fractions was comparatively low (Table 3). In fractions collected early in the
enrichment procedure, the ratio of the 38-kDa CcmM band
with Rubisco proteins is roughly 1:1, while the 58-kDa protein is present at a ratio of approximately 0.4:1 with Rubisco
(Table 3).

Discussion
The aim of this study was to identify the proteins associ 2005 NRC Canada

754

Can. J. Bot. Vol. 83, 2005

Table 3. Apparent ratios of Coomassie-stained bands of carboxysomal proteins in different fractions during the PercollMg2+ enrichment procedure and relative RbcL content of each fraction.
mol:mol RbcL ratioa
Enrichment procedure fraction

Relative RbcL contentb

CcmM (58 kDa)

RbcL

CcmM (38 kDa)

RbcS

PM pellet
TP pellet
EE supernatant
EE pellet
EEM supernatant
EEM pellet

1
1.4
0.8
4.2
1.7
0.6

0.4
0.4
NDc
0.5
NDc
0.6

1
1
1
1
1
1

0.9
1.1
1.4
0.7
1.4
1.4

1.0
1.1
1.3
1.0
1.6
0.6

Note: PM, PercollMg2+ treated; TP, Triton X-100 Percoll treated; EE, EPPSEDTA treated; EEM, EPPSEDTAMg2+ treated.
a
Molar ratios in this table were determined from the Coomassie-stained gel in Fig. 4a and have been corrected as outlined in the Materials and methods
section.
b
Relative RbcL content is a proportion of the total Coomassie stain in the relevant SDSPAGE gel lanes (Fig 4a) and has been normalized to that measured in the PM pellet.
c
Protein was not detected in fraction.

ated with PercollMg2+ enriched -carboxysomes from

Synechococcus PCC7942 (Price et al. 1992) and therefore
assess the efficacy of this enrichment technique. To our
knowledge, this is the first report of a proteomic analysis of
a carboxysome-enriched preparation from cyanobacteria (either or ). Both peptide PMF and PFF techniques were
successful in identifying a wide range of proteins (Tables 1
and 2) from a previously undescribed fraction (the EE pellet). From this fraction, the carboxysomal proteins CcmM,
RbcL, and RbcS were successfully identified using both
techniques, and two forms of CcmM were identified using
PMF. These results confirm existing evidence for the presence of these proteins in carboxysome-enriched preparations
from -cyanobacteria (Price et al. 1992, 1998). Reverse genetics provides evidence for the involvement of CcmK1,
CcmL, CcmM, CcmN, CcmO, and CcaA in carboxysome
formation (Price et al. 1993, 1998, 2002; Kaplan and
Reinhold 1999; Ludwig et al. 2000; So et al. 2002; Badger
and Price 2003). However, it is clear from the results that a
full complement of what are expected to be carboxysomal
proteins (in particular CcmK1, CcmL, CcmN, CcmO, and
CcaA) have not been identified from the PercollMg2+ preparations of carboxysomes. This is despite microscopic evidence that carboxysomes are present in these preparations
(Price et al. 1992; Ors et al. 1995; Fig. 3). These data suggest that the unconfirmed carboxysomal structural proteins
are of very low abundance since, by analogy to carboxysomes from Halothiobacillus, we expect them to be
found in preparations from -cyanobacteria. In the case of
CcaA, however, there is physical evidence that this protein
is present in -carboxysomes (Price et al. 1998; So et al.
2002).
The carboxysomal protein CcmM was identified as two
forms (38 and 58 kDa) by PMF (Fig. 5; Table 1). It is worth
noting that this is the first time that the 58-kDa protein has
been seen as a visibly distinct and predominant band on
Coomassie-stained gels in our hands, indicating the relative
enrichment of this protein into the EE pellet fraction. Both
forms have been identified previously from western blots
(Price et al. 1998), and aminoterminal sequencing data indicate that the smaller form of CcmM begins at Val-216 (M.
Ludwig and G.D. Price, unpublished data). In addition, analysis of protein sequence coverage maps of both forms suggests that the aminoterminal sequence of the full-length

polypeptide is absent from the 38-kDa protein (Fig. 5). This

missing N-terminal region of CcmM corresponds with the CA-like domain of the full-length protein described by Ludwig et al. (2000). The remaining carboxy-terminal sequence
corresponds with a region of three RbcS-like repeats (Ludwig et al. 2000), which have been suggested to play a role in
targeting RbcL octamers to the carboxysome during its formation (Price et al. 1993). It is also noted that the proposed
aminoterminal valine (Val-216) suggests this protein
should have a molecular mass of 35 kDa, raising the possibility that the 38-kDa protein may be post-translationally
modified (e.g., glycosylated), as has been found for the carboxysomal coat proteins CsoS2A and Csos2B (Baker et
al. 1999). Since the full-length CcmM protein appears as its
expected mass under SDSPAGE, one possibility would be
that only the smaller polypeptide would be modified. These
findings raise questions about the role of the -CA domain
and the potential processing of CcmM to carry out bifunctional roles in the carboxysome. Since the use of protease inhibitor cocktails during enrichment does not alter the
composition of the 38-kDa form relative to the 58-kDa form,
it is likely that the 38-kDa form is specifically processed.
Further work is continuing on the characterization of these
two forms of CcmM.
In PercollMg2+ carboxysome preparations, there is copurification of a large number of ribosomal and nucleic acid
binding proteins. A likely source of this and other contaminant protein is the cell debris carried over during enrichment
(Fig. 3b). The putative carboxysomal proteins CcmN,
CcmO, and CcaA have masses in the same range as many of
the ribosomal proteins identified (1730 kDa). Assuming
CcmN, CcmO, and CcaA are not post-translationally modified, the presence of abundant ribosomal proteins is likely to
make identification of these proteins difficult using PMF
techniques. Any relationship between carboxysomes and ribosomal proteins should not be considered too strongly at
this stage. Many of the ribosomal proteins identified have
extremely high pI values (1012), suggesting a net positive
charge under the extraction conditions (see Price et al.
1992). This could be interpreted as a charge interaction association that has led to co-purification. While twodimensional gel electrophoresis would be advantageous in
this instance, this does not enable clear definition of which
proteins form a close association with a carboxysome and
2005 NRC Canada

Long et al.

which form part of it. Indeed, two-dimensional gel electrophoresis combined with N-terminal sequencing has been
carried out on PercollMg2+ preparations, but no clear evidence for CcmK1, CcmL, CcmN, CcmO, and CcaA
carboxysomal proteins has been found (M. Ludwig and G.D
Price, unpublished data); RbcL, RbcS, and CcmM (38 kDa)
were identified. Clearly, elimination of contaminant proteins
prior to PMF analysis is required to confirm carboxysomal
protein composition. We are currently exploring new approaches to the isolation of carboxysomes, which we hope
will allow us to obtain pure preparations.
Several peptidases were also found during proteomic analysis of the PercollMg2+ enriched preparation (Tables 1 and
2). More specifically, aminopeptidase N, which, as the name
suggests, cleaves amino acids from the aminoterminal of
peptides, was identified both by PMF and PFF techniques.
The presence of peptidases in the carboxysome-enriched
preparation suggests a likelihood of peptide degradation during extraction and prior to proteomic analysis. For PMF
analysis of excised gel bands, this is not as critical, as there
is a denaturing and spatial separation of proteins prior to
analysis. However, the trypsin digestion procedure required
for MuDPIT analysis provides what are likely to be ideal
conditions for high peptidase activity. It is probable that during trypsin digestion, many resulting peptides are further digested by any peptidases present in the protein mixture,
resulting in poor coverage of the protein content of the enriched carboxysome preparation. This is supported by the
finding that CcmM was not identified by MuDPIT in extracts prepared in the presence of PMSF (a serine protease
inhibitor) alone, but was identified when a protease inhibitor
cocktail containing the aminopeptidase inhibitor bestatin
was used. Whether this is a consequence of the inclusion of
bestatin in the extraction procedure or due to variation between preparations is not yet clear. In addition, many of the
protein identifications using MuDPIT were based on the
presence of single peptides from proteins (requiring manual
analysis of MSMS spectra for confirmation of identity),
possibly indicative of some peptide degradation. RbcL, for
example, was identified by seven unique peptides during
MuDPIT analysis, but 21 unique peptides from the protein
were identified by MALDI-TOF analysis. In addition, 8
polypeptides clearly identified by PMF were not found by
MuDPIT analysis (Table 1). This is suggestive of peptide
loss prior to MuDPIT analysis, especially given the relative
sensitivity and good sequence coverage afforded by LCMS
analysis of protein digests (Lim et al. 2003) and unbiased
coverage of low abundance proteins (Washburn et al. 2001).
Nonetheless, lack of evidence for the remaining
carboxysomal proteins (CcmK1, CcmL, CcmN, CcmO, and
CcaA) suggests that they are not present in the extracts examined at concentrations significant for detection by
MuDPIT (i.e., approximately <1 nmolL1).
Assuming that the PM pellet (the initial PercollMg2+ enriched Rubisco fraction) contains predominantly intact
carboxysomes, then the ratios of carboxysomal proteins in
this fraction can be used as a reference to determine the fate
of carboxysomes in subsequent steps in the enrichment procedure. Comparison of the PM pellet with the TP pellet indicates relatively little change in carboxysome protein ratios
during this step (Table 3). Addition of EDTA to the TP pel-

755

let, however, results in a relative increase of the 38-kDa protein (along with some RbcS) into the subsequent supernatant
and a corresponding small loss of this protein from the pellet. At the same time, there is an approximately four-fold enrichment of RbcL and the 38-kDa protein into the EDTAtreated pellet, suggesting that a large number of whole
carboxysomes have been carried through into this fraction.
This is supported by electron microscopy of this fraction
(Fig. 3). However, the EDTA-induced relative loss of the
38-kDa protein from the EE pellet is indicative of some
carboxysome breakage, while the loss of assayable Rubisco
activity in this fraction could result from the formation of
protein aggregates. This EDTA wash treatment is clearly
useful in enriching carboxysomes, although further work
should be directed at stabilizing carboxysomes during this
washing step (e.g., chemical cross-linking), minimizing protein aggregation (e.g., salt treatment), and removing large
fragments of cellular debris (e.g., filtration and (or) density
gradient centrifugation). Traditionally, the TP pellet fraction
was further enriched by washing with EDTA, followed by
precipitation of the resulting supernatant with Mg2+ (Price et
al. 1992, 1993; Ors et al. 1995; So et al. 2002; referred to
here as the EEM pellet fraction). The quality of the EEM
pellet fraction, however, varies considerably between preparations and may depend upon the relative EDTA and protein
concentrations used to clean up the TP pellet fraction. In addition, it now appears clear that the EEM pellet fraction is
not greatly enriched in Rubisco (Fig. 2; Table 3). As such,
use of the EE pellet fraction is a novel and effective way of
further enriching carboxysomes from the TP pellet fraction.
Thus, the TP pellet fraction remains a useful starting point
for further development.
The PercollMg2+ method has been used on a number of
occasions for the enrichment of carboxysomes from cyanobacteria and their subsequent study (Price et al. 1992, 1993;
Ors et al. 1995; So et al. 2002). However, in the light of the
evidence presented here, it is clear that this method requires
further optimization to obtain high purity carboxysomes.
Certainly, for the determination of protein composition and
structure analysis of carboxysomes, this is essential. Several
new refinements are now being developed, which we hope
will lead to pure carboxysomes from -cyanobacteria, although the use of EDTA shows some potential. It is also
clear that to confirm the complete protein complement of carboxysomes, a scale-up of the procedure presented here
may also be required, since CcmK1, CcmL, CcmN, CcmO,
and CcaA proteins do not exist in the preparations reported
here in sufficient quantities to be detected. Attempts have
also been made to utilize techniques that have been successful in the purification of -carboxysomes, but these have not
been successful with -carboxysomes (B.M. Long, G.D.
Price, and M.R. Badger, unpublished data). The
carboxysomes of -cyanobacteria seem not to be amenable
to purification techniques used for other polyhedra, which
may be indicative of a relatively unstable structure or different surface properties.

Acknowledgements
We thank Loraine Tucker for maintaining Synechococcus
PCC7942 cultures and Lily Shen (Australian National Uni 2005 NRC Canada

756

versity Electron Microscopy Unit) for assistance with sample preparation and transmission electron microscope
analysis. This project is supported by funds from the Australian Research Council (to M.R.B. and G.D.P.).

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