Plant Cell Physiol. 45(10): 1390–1395 (2004)
JSPP © 2004
The rbcX Gene Product Promotes the Production and Assembly of Ribulose1,5-Bisphosphate Carboxylase/Oxygenase of Synechococcus sp. PCC7002 in
Escherichia coli
Takuo Onizuka 1, 4, Sumiyo Endo 1, Hideo Akiyama 1, Shozo Kanai 1, Masahiko Hirano 1, Akiho Yokota 2,
Satoshi Tanaka 3 and Hitoshi Miyasaka 3
1
Biological Science Laboratories, Toray Research Center, Inc., 1111 Tebiro, Kamakura, Kanagawa, 248-8555 Japan
Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan
3
Environmental Research Center, Kansai Electric Power Co., Keihannna-Plaza, 1-7 Seikacho, Sourakugun, Kyoto, 619-0237 Japan
2
;
The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX
and rbcS, in this order. Introduction of translational
frameshift into the rbcX gene resulted in a significant
decrease in the production of large (RbcL) and small
(RbcS) subunits of the Rubisco protein in Synechococcus
sp. PCC7002 and in Escherichia coli. To investigate the
function of the rbcX gene product (RbcX), we constructed
the expression plasmid for the rbcX gene and examined the
effects of RbcX on the recombinant Rubisco production in
Escherichia coli. The coexpression experiments revealed
that RbcX had marked effects on the production of large
and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX
coexpression system provides a novel and useful method for
investigating the Rubisco maturation pathway.
Keywords: Coexpression — Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) — Synechococcus sp.
PCC7002 (Agmenellum quadruplicatum PR-6).
Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/
oxygenase; PBS, phosphate buffered saline; RT, reverse transcription.
Introduction
Oligomeric proteins produced in Escherichia coli cannot
always assemble by themselves (Pelham 1988). In some cases,
molecular chaperones facilitate production of soluble proteins
and enhance the post-translational assembly of these polypeptides into oligomeric structures (Hemmingsen et al. 1988).
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),
which catalyzes the CO2 fixation reaction in photosynthesis, is
composed of eight large (RbcL) and eight small (RbcS) subunits in plants and most photosynthetic prokaryotes (Miziorko
and Lorimer 1983). In higher plants, newly synthesized RbcL
4
associates with Rubisco-binding protein (cpn60) (Barraclough
and Ellis 1980, Ellis and van der Vies 1988), which is involved
in assembly of the hexadecameric (L8S8) structure (Gatenby
and Ellis 1990). The Rubisco-binding protein (cpn60) is homologous to the E. coli 60-kDa GroEL chaperonin (Hemmingsen
et al. 1988), and the GroEL oligomer, like the Rubisco-binding
protein, binds to newly synthesized plant RbcL (Gutteridge and
Gatenby 1995).
Rubisco from cyanobacteria is an L8S8-form enzyme and
its genes are in the rbc operon in which one possible open
reading frame, rbcX, is present in the intergenic space between
the large (rbcL) and small (rbcS) subunit genes for Rubisco
(Larimer and Soper 1993). It was first reported that the rbcX
gene of the filamentous cyanobacterium Anabaena sp. strain
PCC7120 does not influence the expression levels of recombinant Rubisco in E. coli under conditions favoring maximum
E. coli GroEL and GroES synthesis (Larimer and Soper 1993).
More recently, the rbcX gene of Anabaena sp. strain CA was
found to affect the levels of recombinant Rubisco activity in E.
coli strongly, when the chaperonin synthesis was not maximized (Li and Tabita 1997). This suggests chaperone-like functions of the rbcX gene product. We recently found that in other
unicellular cyanobacteria, including Synechocystis sp. strain
6803 and Synechococcus sp. PCC7002 (Agmenellum quadruplicatum PR-6), the rbcX gene is juxtaposed with the rbcL and
rbcS genes and is likely to be cotranscribed with the rbcL and
rbcS genes (Onizuka et al. 2002). This suggests that RbcX is
involved in the synthesis and the subsequent assembly of RbcL
and RbcS in this organism.
The aim of this study with genes from Synechococcus sp.
PCC7002 is to gain insight into the mechanism of the involvement of RbcX in the synthesis and assembly of the Rubisco
subunits.
Results
Effect of mutation in rbcX gene on RbcLS production
We first examined the influence of the rbcX gene on production of RbcLS in Synechococcus sp. PCC7002. A 2.6-kb
DNA fragment containing the rbc operon with a frameshift
Corresponding author: E-mail, [email protected]; Fax, +81-467-320414.
1390
RbcX promotes production and assembly of Rubisco
1391
Fig. 1 Effect of partial inactivation of
the rbcX gene on RbcLS production in
Synechococcus sp. PCC7002. (A) Scheme
for the frameshifted inactivation of the
rbcX gene. A 1.3-kb kanamycin resistance cassette was inserted at a SalI site
created between the rbcL and rbcX genes
for selection. Wild-type cells were transformed with this construct to generate the
strain containing the partially inactivated
rbcX gene. (B) Amino acid sequences of
the wild-type RbcX (RbcX) and the
mutated RbcX (*RbcX). Translation
frameshift was introduced from the SacI
site located at codon 66 of the rbcX gene.
Identical amino acids between RbcX and
*RbcX are indicated in white on a black
background. (C) SDS-PAGE of cell
lysates, revealed by immunodetection with
the antiserum raised against RbcL. Wildtype cells (lane 1) or cells containing the
partially inactivated rbcX gene (lane 2)
were grown to mid-log phase. Cells were
harvested, disrupted and separated into
soluble fractions. Soluble proteins were
analyzed by Western blot analyses as
described in Materials and Methods. (D)
SDS-PAGE of cell lysates revealed by
immunodetection with an antiserum raised
against RbcS. The experiment was carried out as described in (C). (E) Rubisco
activities of cell lysates from wild-type
cells (1) or cells containing the mutated
rbcX gene (2). Data represent mean ± SD
obtained from three independent measurements. *Translation frameshift.
mutation in the rbcX gene was constructed (Fig. 1A). Fig. 1B
shows the resulting amino acid sequences of the mutated
RbcX. We transformed Synechococcus sp. PCC7002 wild-type
cells using the construct and selected the mutant strain containing the partially inactivated rbcX gene. As shown in Fig. 1C, D,
Western blotting revealed that the amount of soluble Rubisco
proteins from the mutant strain was decreased compared with
that from the wild-type cells. In the same manner, mutation of
the rbcX gene lowered the Rubisco activity (Fig. 1E). Because
a complete loss of the rbcX gene hampers the viability of
cyanobacterial cells, we examined the effects of the rbcX gene
on production of RbcLS in E. coli using the rbc expression
plasmids. Two rbc expression plasmids, in which the rbc genes
were placed under the control of the rbc promoter, were constructed. The one that could express rbcLXS completely was
designated pRbcLXS, and the other in which the rbcX gene
was inactivated by introducing a translational frameshift was
named pRbcLS (Fig. 2). When the rbc genes were expressed in
the strain JM109, a large part of the recombinant Rubisco was
insoluble. The soluble Rubisco was hardly detected by SDSPAGE, because its level was significantly lower than the insoluble one. However, subsequent immunoblotting revealed that the
recombinant Rubisco was also produced in soluble proteins in
the E. coli cells containing the plasmid pRbcLXS (Fig. 3A, B,
lanes 1). When the rbcX frameshift plasmid (pRbcLS) was
transformed into cells, RbcL and RbcS could hardly be
detected in soluble proteins (Fig. 3A, B, lanes 2). Similarly, the
disruption of the rbcX gene eliminated the Rubisco activity
(Fig. 3C). Moreover, SDS-PAGE of corresponding insoluble
proteins revealed that cells containing pRbcLS produced significantly less RbcLS than cells containing pRbcLXS (Fig. 3D).
These results suggest that the rbcX gene is involved in a process of RbcLS production and/or subsequent assembly.
Fig. 2 Structure of rbc expression plasmids used in this study. pRbcLXS contains the rbc promoter followed by Synechococcus rbcL,
rbcX and rbcS genes cloned in pAQJ4-MCS (Akiyama et al. 1998,
Akiyama et al. 1999). pRbcLS contains a frameshifted rbcX gene
between rbcL and rbcS genes under the control of the rbc promoter
cloned in pAQJ4-MCS. *Translation frameshift.
1392
RbcX promotes production and assembly of Rubisco
Fig. 3 Expression of rbc genes in E.
coli cells containing rbc expression plasmid pRbcLXS (lanes 1) or pRbcLS
(lanes 2). (A) SDS-PAGE of cell lysates
revealed by immunodetection with an
antiserum raised against RbcL. Cells
were grown in LB medium under selective conditions. Cultures were grown to
mid-log phase. (B) SDS-PAGE of cell
lysates revealed by immunodetection
with the antiserum raised against RbcS.
(C) Rubisco activities of cell lysates.
Data represent mean ± SD obtained from
three independent measurements. (D)
SDS-PAGE of insoluble fractions. The
gel was stained with Coomassie brilliant
blue. (E) Expression of the rbc mRNA
revealed by RT-PCR using rbc primers.
We also examined the effect of the rbcX gene on mRNA
levels of rbc genes in E. coli harboring the above plasmids. As
shown in Fig. 3E, the mRNA levels of rbc genes were almost
the same in cells containing pRbcLXS and pRbcLS, indicating
that the rbcX gene had no effect on the transcription of rbc
genes.
Effect of RbcX protein on RbcLS production
We constructed an rbcX-expression plasmid in which the
gene was placed under the control of the rbc promoter and designated it pACYC-rbcX (Fig. 4A). This plasmid was a derivative of pACYC184 and carried the tetracycline resistance gene
for selection. The pACYC-rbcX contained a p15A replicon and
was compatible with pRbcLS. E. coli cells harboring the plasmid pACYC-rbcX synthesized the soluble RbcX detected by
immunoblotting (Fig. 4B), indicating that the plasmid pACYCrbcX could facilitate studies on the effect of the synthesized
RbcX on RbcLS production.
To examine the effects of the synthesized RbcX on the
production of RbcLS, we used E. coli cells harboring a pair of
compatible plasmids for expression of rbcX, pACYC-rbcX, and
for expression of rbcLS, pRbcLS, in which the rbcX gene was
inactivated by introducing a translational frameshift. As shown
in Fig. 5, the cells containing the plasmid encoding the Synechococcus rbcLXS genes (pRbcLXS) produced a large amount
of RbcLS (Fig. 5A, B, lanes 1), while the cells containing the
pRbcLS lacking the active rbcX gene together with the control
plasmid pACYC184 did not (Fig. 5A, B, lanes 2). However,
Fig. 4 Synthesis of RbcX in E. coli cells containing the rbcX expression plasmid pACYC-rbcX. (A) Structure of the rbcX expression plasmid; p15A, p15A replicon; tetR, tetR repressor gene; rbcP, rbc
promoter; rbcX, rbcX gene. (B) Synthesis of RbcX in E. coli cells containing pACYC-rbcX. Cells were grown in LB medium containing
25 µg ml–1 of tetracycline at 37°C. Cultures were grown to mid-log
phase and soluble proteins were analyzed by SDS-PAGE, revealed by
immunodetection with an antiserum raised against recombinant RbcX.
RbcX promotes production and assembly of Rubisco
1393
Fig. 5 Effect of synthesized RbcX on RbcLS production. Strain
JM109 harboring the rbc expression plasmid pRbcLXS (lanes 1), rbc
expression plasmid pRbcLS together with pACYC184 (lanes 2), or rbc
expression plasmid pRbcLS together with pACYC-rbcX (lanes 3)
were grown in LB medium under selective conditions. Cultures were
grown to mid-log phase and harvested. Whole-cell proteins were subjected to SDS-PAGE and detected with an antiserum raised against
RbcL (A) or RbcS (B).
when the rbcLS genes were coexpressed with the rbcX gene,
RbcLS production was recovered, suggesting that the RbcX
protein could effectively promote production of the RbcLS proteins (Fig. 5A, B, lanes 3). Similar results were obtained when
the E. coli cells carrying pRbcLS were cotransformed with the
expression plasmid for groE genes (pACYC-T7-GroE) instead
of pACYC-rbcX (data not shown). Thus, the RbcX protein and
the GroE chaperonins have similar effects on the production of
the RbcLS proteins.
Similar coexpression experiments were carried out by
using E. coli cells harboring a pair of compatible plasmids
pACYC-rbcX and pT7LS carrying the inactive rbcX gene
whose expression is controlled by the T7 promoter. The effect
of the synthesized RbcX is similar to that shown in Fig. 5,
although there was a decrease in the yield (data not shown).
These results indicate that the RbcX protein could effectively
promote production of RbcLS proteins independently of the
promoter controlling rbcLS expression.
Effect of the RbcX protein on Rubisco assembly
To investigate whether the synthesized RbcX promotes the
assembly of RbcL and RbcS into a L8S8 form, coexpression
experiments were carried out by using E. coli cells harboring a
pair of compatible plasmids pACYC-rbcX and pRbcLS. By
expressing the rbcX gene with the plasmid pACYC-rbcX, the
formation of L8S8 was achieved as revealed by non-denaturing
PAGE and immunoblotting (Fig. 6, lane 2). The recombinant
L8S8 unit appeared to be identical to the control Rubisco
holoenzyme (Fig. 6, lanes 1, 2). On the other hand, the E. coli
cells harboring a combination of plasmids pACYC184 (control) and pRbcLS revealed incomplete assembly of the Rubisco
holoenzyme (Fig. 6, lane 3). These results indicate that the
RbcX protein is involved in the post-translational assembly of
RbcLS into a L8S8 holoenzyme.
Fig. 6 Effect of synthesized RbcX on Rubisco assembly. Strain
JM109 co-transformed with plasmids pRbcLS and pACYC-rbcX (lane
2), or plasmids pRbcLS and pACYC184 (lane 3) were grown in LB
medium under selective conditions. Cultures were grown to mid-log
phase. Cells were harvested, disrupted and separated into soluble fractions. Soluble proteins were analyzed by non-denaturing PAGE with
the endogenous Rubisco protein from Synechococcus sp. PCC7002
(lane 1) and Rubisco detection was performed by Western blot analysis
as described in Materials and Methods.
Discussion
As a prerequisite to the study of the function of cyanobacterial RbcX, we constructed the coexpression system for the
rbc genes from Synechococcus sp. PCC7002 in E. coli. When
the recombinant RbcX was synthesized in the E. coli strain, the
protein was soluble enough to examine the role of RbcX in production and assembly of the Synechococcus Rubisco proteins.
In the present study with the rbcX gene from Synechococcus sp. PCC7002, we found that the introduction of a translational frameshift into the rbcX gene resulted in a significant
decrease in the production of large (RbcL) and small (RbcS)
subunits of the Rubisco protein in Synechococcus sp. PCC7002
and in E. coli (Fig. 1, 3). mRNA levels of rbcL and rbcS were
not affected by the inactivation of the rbcX gene (Fig. 3). Moreover, when the rbcX gene was coexpressed with the rbcLS
genes in E. coli, the synthesized RbcX increased the amounts
of RbcL and RbcS (Fig. 5), and the L8S8 structure of Rubisco
protein was apparently formed (Fig. 6). These findings suggest
that the product of the rbcX gene is not a transcription activator,
but promotes the RbcLS protein production without regulating
the mRNA levels of Rubisco genes, and that the RbcX protein
is involved in the synthesis and/or the post-translational assembly of the Synechococcus Rubisco proteins.
The difficulty in overexpressing cyanobacterial Rubisco to
yield the active enzyme in E. coli cells was overcome by simultaneous oversynthesis of E. coli chaperonin proteins (GroEL
1394
RbcX promotes production and assembly of Rubisco
and GroES) and Rubisco (Gurevitz et al. 1985, Goloubinoff et
al. 1989, Larimer and Soper 1993). In the presence of an excess
amount of chaperonin proteins, Anabaena rbcX (Anabaena sp.
strain PCC7120) had little effect on the levels of Rubisco activity (Larimer and Soper 1993). On the other hand, the intact
rbcX gene of Anabaena sp. strain CA retained the activity of
recombinant Rubisco in E. coli limiting the level of chaperonins (Li and Tabita 1997). In the present study, the recombinant
RbcX protein promoted the production of RbcLS and the formation of the L8S8 structure of the Rubisco protein, indicating
that the low activity of Rubisco observed in other laboratories
was due to a failure in the formation of the L8S8-form enzyme
of Rubisco in the absence of RbcX. Only the expression of
rbcLS was insufficient for the production of RbcLS and assembly into complete oligomeric form in E. coli expressing endogenous chaperonins only. The product of the rbcX gene was
required for the production of the Rubisco protein and formation of the L8S8 structure. In the E. coli cellular environment,
the RbcX protein had effects apparently similar to those of
GroEL and GroES chaperonins in promoting the production of
RbcLS protein and assembly of RbcLS into the L8S8 structure, in agreement with the conclusion drawn first by
Goloubinoff et al. (1989) using Anacystis Rubisco. Since inactivation of the rbcX gene significantly abolished the production
of RbcLS proteins both in supernatant and in pellet fractions in
our case, RbcX may have functions in translation of transcripts
of rbcLS and/or folding of nascent peptides, preventing the
Rubisco proteins from degradation rather than from aggregation. The proposed maturation pathway of Rubisco consists of
polypeptide translation, folding, L2 dimerization, tetramerization of L2 dimers into L8 octamers and association of small
subunits with the L8 octomers into the hexadecameric L8S8
holoenzyme (Goloubinoff et al. 1989, Fitchen et al. 1990, Lee
and Tabita 1990, Paul et al. 1991). Experimentally, we have
shown that RbcX is required for assembly of the Rubisco protein in E. coli and possibly in the cyanobacterium, but we have
not specified the steps in which the RbcX protein is involved.
However, we have not yet determined the chaperonin dependence and efficacy of the RbcX protein for assembly of the L8S8
structure of Rubisco in Synechococcus. Much more work is
required to determine the role of RbcX in the maturation of
Rubisco in E. coli and also in Synechococcus itself.
The rbcX coexpression system described here will provide a useful way to study the translation, folding and assembly of Rubisco and prompts us to investigate whether this rbcX
coexpression system is able to improve production of various
foreign proteins, including plant Rubisco in E. coli cells.
Materials and Methods
Materials and strains
Restriction enzymes were purchased from Takara Bio (Kyoto,
Japan). The DNA sequencing kits were from Applied Biosystems
(Norwalk, CT, U.S.A.). All chemicals were purchased from Nacalai
Tesque (Kyoto, Japan). The E. coli strain JM109 purchased from
Takara Bio was used throughout this study and cultured at 37°C in LB
medium (Sambrook et al. 1989). A groE expression vector pACYCT7-GroE, which harbored the chloramphenicol resistance cassette, was
a kind gift from Dr. Hiroshi Yamamoto of the Research Institute of
Innovative Technology for the Earth (RITE), Kyoto, Japan. The cyanobacterial strain Synechococcus sp. PCC7002 (A. quadruplicatum PR-6,
ATCC 27264) (Buzby et al. 1983, Buzby et al. 1985), obtained from
the American Type Culture Collection, was cultured at 30°C in
medium A (Tabita et al. 1974) under aeration with 1% CO2. Continuous illumination was provided at 50 µmol photons m–2 s–1 by three
FL40SS (37W) fluorescent lamps. Agar plates were prepared using
medium A solidified with 1.5% agar and the cells were cultured at
30°C.
Plasmid constructions
The rbc expression vector, pRbcLXS was made by cloning the
rbc operon into pAQJ4-MCS (Akiyama et al. 1998, Akiyama et al.
1999) using the PCR technique. A 2.6-kb fragment in the rbc operon
was amplified with Pfx DNA polymerase (Invitrogen, Carlsbad, CA,
U.S.A.) for 25 cycles of 94°C denaturation for 15 s, 55°C annealing
for 30 s and 68°C extension for 3 min with a final extension time of
3 min. A PCR product including the rbc promoter and rbcLXS was
digested with BamHI and XbaI, and cloned into the BamHI and XbaI
sites on pAQJ4-MCS. Plasmid pRbcLS was derived from a partial SacI
digestion of pRbcLXS, treated with T4 DNA polymerase. The resulting repair of the SacI site located at codon 66 of rbcX produced a
frameshift mutation. A 2.3-kb PCR product without the rbc promoter
was amplified from pRbcLS as a template, digested with NdeI and
XhoI and inserted into the NdeI and XhoI sites on pIVEX2.4b-Nde
(Roche Diagnostics GmbH, Penzberg, Germany), constructing plasmid pT7LS. To generate the construct for inactivation of the rbcX gene
in Synechococcus sp. PCC7002, a 2.6-kb DNA fragment containing
the rbc operon was amplified by PCR from pRbcLS with a frameshift
mutation in the rbcX gene. The PCR-amplified DNA fragment was
cloned into plasmid pUC18 and a 1.3-kb kanamycin resistance cassette was inserted into a SalI site created between the rbcL and rbcX
genes to select the mutant strain. The resulting plasmid was linearized
by digestion with BamHI and XbaI, and was used to transform Synechococcus sp. PCC7002 wild-type cells. Plasmid pACYC-rbcX was
derived from plasmid pACYC184 (Nippon Gene, Tokyo, Japan) containing a p15A replicon and tetR repressor gene. A 250-bp NcoI–
EcoRI fragment containing the rbc promoter and a 400-bp NcoI fragment containing the rbcX gene, amplified by PCR, were inserted into
pACYC184 cut with NcoI/EcoRI. All the resulting vectors were confirmed by DNA sequence analysis using a 3100 DNA sequencer
(Applied Biosystems).
Protein extraction and Rubisco assay
The E. coli strains harboring a pair of compatible expression
plasmids were grown in LB medium containing 100 µg ml–1 of ampicillin and 25 µg ml–1 of tetracycline or chloramphenicol at 37°C with
constant aeration for 6–8 h. The cells were harvested, and whole-cell
proteins or insoluble proteins from the same cell number were analyzed by SDS-PAGE and Western blotting. Cells were resuspended in
phosphate-buffered saline (PBS) to examine the soluble proteins. Protein extracts were prepared using a FastPrep system (Qbiogene,
Carlsbad, CA, U.S.A.) as described by the manufacturer, and centrifuged at 10,000×g for 15 min at 4°C. The activity of Rubisco in an
aliquot of the supernatant was determined using the spectrophotometric, enzyme-coupled assay developed by Racker (1962). We defined
the specific activity of Rubisco as the amount of enzyme that catalyzes the carboxylation of 1.0 nmol of ribulose bisphosphate per min
per mg of total proteins. Protein concentrations were determined by
the method of Bradford (1976) with bovine serum albumin as the
standard.
RbcX promotes production and assembly of Rubisco
Western blot analysis
An antiserum raised against Synechococcus RbcX was prepared
as follows. The PCR-generated DNA fragment, containing the rbcXcoding region, was cloned into the NcoI site of pIVEX2.4b-Nde, which
is the N-terminal hexa-His-containing vector. The sequence of the
resulting plasmid was verified by DNA sequencing. The expression
experiment of the rbcX gene was performed using the RTS 500 instrument and the RTS 500 E. coli HY Kit (Roche Diagnostics GmbH)
according to the supplier’s instructions (Martin et al. 2001). The reaction was run with a stirring rate of 120 rpm, at 30°C for 20 h. The content of the reaction mixture (1 ml) was diluted to 10 ml with PBS and
purified using B-PER 6 × His spin purification kit (Pierce, Rockfold,
IL, U.S.A.). The purity and concentration of recombinant RbcX were
estimated using SDS-PAGE, and an antiserum against the purified
recombinant RbcX was raised in rabbits. Whole-cell proteins or soluble proteins (1.3 µg) were separated on a 4–20% gradient SDS-polyacrylamide gel. In the case of non-denaturing PAGE, soluble proteins
(3.3 µg) were applied on a 4–12% gradient polyacrylamide gel. Separated proteins were transferred onto a nitrocellulose membrane. AntiRbcL, anti-RbcS (Onizuka et al. 2003) or anti-RbcX antibodies (1 :
10,000 dilution) and anti-rabbit IgG antibody conjugated with horseradish peroxidase (1 : 10,0000 dilution, Santa Cruz Biotechnology,
Santa Cruz, CA, U.S.A.) were used as primary and secondary antibodies, respectively. Immunodetection was performed by an enhanced
chemiluminescence method as recommended by the manufacturer
(Amersham Biosciences, Piscataway, NJ, U.S.A.).
RT-PCR
Total RNA was extracted from E. coli strains harboring the indicated plasmids using ISOGEN (Nippon Gene). Contaminating DNA
was removed completely using a DNA-free kit (Ambion, Austin, TX,
U.S.A.). The quality and quantity of RNA were examined by conventional 1% agarose gel electrophoresis and spectrophotometric measurement. Aliquots of total RNA (1.0 µg) were amplified using 0.4 µM
of a set of oligonucleotide primers, 1 mM of dNTP-analog mixture
(Takara Bio), 0.8 U µl–1 of RNase inhibitor (Takara Bio), 0.1 U µl–1 of
AMV Rtase XL (Takara Bio) and 0.1 U µl–1 of AMV-optimized Taq
polymerase (Takara Bio) in 50 µl of reaction mixture. A set of oligonucleotide primers for Synechococcus PCC7002 rbc genes were synthesized based on the following nucleotide sequences: rbc sense
primer 5′-CCC TCA GCG ACC AGC AAA TC-3′: rbc antisense
primer 5′-ACG GGT TTG GTT GGG CTT GT-3′. The size of the fragment amplified was 278 bp. Reactions were aliquoted from a master
mix to minimize tube to tube variation. After cDNA synthesis at 50°C
for 30 min, PCR amplification was conducted by the protocol of 85°C
denaturation for 40 s, 55°C annealing for 40 s and 72°C extension for
1 min. The mRNA levels were compared at a logarithmic amplification
of 25 cycles. Amplification products were subjected to electrophoresis
with 2.0% agarose gels and stained with ethidium bromide.
Acknowledgments
We are grateful to Mrs. Mina Usui-Takeshige for DNA sequencing analysis.
References
Akiyama, H., Kanai, S., Hirano, M. and Miyasaka, M. (1998) Nucleotide
sequence of plasmid pAQ1 of marine cyanobacterium Synechococcus sp.
PCC7002. DNA Res. 5: 127–129.
Akiyama, H., Kanai, S., Hirano, M. and Miyasaka, M. (1999) A novel plasmid
recombination mechanism in the marine cyanobacterium Synechococcus sp.
PCC7002. DNA Res. 5: 327–334.
1395
Barraclough, R. and Ellis, R.J. (1980) Protein synthesis in chloroplasts. IX.
Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated pea chloroplasts. Biochim. Biophys. Acta 608: 19–31.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Buzby, J.S., Porter, R.D. and Stevens, S.E. Jr. (1983) Plasmid transformation in
Agmenellum quadruplicatum PR-6: Construction of biphasic plasmids and
characterization of their transformation properties. J. Bacteriol. 154: 1446–
1450.
Buzby, J.S., Porter, R.D. and Stevens, S.E. Jr. (1985) Expression of the
Escherichia coli lacZ gene on a plasmid vector in a cyanobacterium. Science
230: 805–807.
Ellis, R.J. and van der Vies, S.M. (1988) The Rubisco subunit binding protein.
Photosynth. Res. 16: 101–115.
Fitchen, J.H., Knight, S., Andersson, I., Branden, C.I. and Mcintosh, L. (1990)
Residues in three conserved regions of the small subunit of ribulose-1,5bisphosphate carboxylase/oxygenase are required for quaternary structure.
Proc. Natl Acad. Sci. USA 87: 5768–5772.
Gatenby, A.A. and Ellis, R.J. (1990) Chaperone function: the assembly of ribulose bisphosphate carboxylase-oxygenase. Annu. Rev. Cell. Biol. 6: 125–149.
Goloubinoff, P., Gatenby, A.A. and Lorimer, G.H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337: 44–47.
Gurevitz, M., Somerville, C.R. and Mcintosh, L. (1985) Pathway of assembly of
ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed
in Escherichia coli. Proc. Natl Acad. Sci. USA 82: 6546–6550.
Gutteridge, S. and Gatenby, A.A. (1995) Rubisco synthesis, assembly, mechanism, and regulation. Plant Cell 7: 809–819.
Hemmingsen, S.M., Woolford, C., van der Vies, S.M., Tilly, K., Dennis, D.T.,
Georgopoulos, C.P., Hendrix, R.W. and Ellis, R.J. (1988) Homologous plant
and bacterial proteins chaperone oligomeric protein assembly. Nature 333:
330–334.
Larimer, F.W. and Soper, T.S. (1993) Overproduction of Anabaena 7120 ribulose-bisphosphate carboxylase-oxygenase in Escherichia coli. Gene 126: 85–
92.
Lee, B. and Tabita, F.R. (1990) Purification of recombinant ribulose-1,5bisphosphate carboxylase/oxygenase large subunits suitable for reconstitution and assembly of active L8S8 enzyme. Biochemistry 29: 9352–9357.
Li, L.A. and Tabita, F.R. (1997) Maximum activity of recombinant ribulose 1,5bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the
product of the rbcX gene. J. Bacteriol. 179: 3793–3796.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
Martin, G.A., Kawaguchi, R., Lam, Y., DeGiovanni, A., Fukushima, M. and
Mutter, W. (2001) High-yield, in vitro protein expression using a continuousexchange, coupled transcription/translation system. Biotechniques 31: 948–
953.
Miziorko, H.M. and Lorimer, G.H. (1983) Ribulose-1,5-bisphosphate carboxylase-oxygenase. Annu. Rev. Biochem. 52: 507–535.
Onizuka, T., Akiyama, H., Endo, S., Kanai, S., Hirano, M., Tanaka, S. and
Miyasaka, M. (2002) CO2 response element and corresponding trans-acting
factor of the promoter for ribulose-1,5-bisphosphate carboxylase/oxygenase
genes in Synechococcus sp. PCC7002 found by an improved electrophoretic
mobility shift assay. Plant cell Physiol. 43: 660–667.
Onizuka, T., Akiyama, H., Endo, S., Kanai, S., Hirano, M., Tanaka, S. and
Miyasaka, M. (2003) CO2 response for expression of ribulose-1,5-bisphosphate carboxylase/oxygenase genes is inhibited by AT-rich decoy in the
cyanobacterium. FEBS Lett. 542: 42–46.
Paul, K., Morell, M.K. and Andrews, T.J. (1991) Mutations in the small subunit
of ribulosebisphosphate carboxylase affect subunit binding and catalysis. Biochemistry 30: 10019–10026.
Pelham, H. (1988) Coming in from the cold. Nature 332: 776–777.
Racker, E. (1962) Methods in Enzymology 5: p. 266. Academic Press, New
York.
Tabita, E.R., Stevens, S.E. Jr. and Quiano, R. (1974) D-Riburose 1, 5-diphosphate carboxylase from blue-green algae. Biochem. Biophys. Res. Commun.
61: 45–52.
(Received May 3, 2004; Accepted July 15, 2004)