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Journal of Bacteriology, December 1998, p. 6776-6779, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sucrose-Phosphate Synthase from Synechocystis sp.
Strain PCC 6803: Identification of the spsA Gene and
Characterization of the Enzyme Expressed in Escherichia
coli
Leonardo
Curatti,1
Eduardo
Folco,1
Paula
Desplats,1
Gustavo
Abratti,1
Verónica
Limones,2
Luis
Herrera-Estrella,2 and
Graciela
Salerno1,*
Centro de Investigaciones Biológicas,
Fundación para Investigaciones Biológicas Aplicadas (FIBA)
and PROBIOP-CONICET, 7600 Mar del Plata,
Argentina,1 and
Departamento de
Ingeniería Genética de Plantas, Centro de
Investigación y Estudios Avanzados del IPN, Unidad Irapuato,
Irapuato, Gto., México2
Received 29 July 1998/Accepted 8 October 1998
 |
ABSTRACT |
The first identification and characterization of a prokaryotic gene
(spsA) encoding sucrose-phosphate synthase (SPS) is
reported for Synechocystis sp. strain PCC 6803, a
unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the
deduced amino acid sequence and some relevant biochemical properties of
the enzyme with those of plant SPSs revealed important differences in
the N-terminal and UDP-glucose binding site regions, substrate
specificities, molecular masses, subunit compositions, and regulatory properties.
| |
TEXT |
Sucrose is the major product of
photosynthesis in most plants and is exported from leaves to all
heterotrophic tissues. Sucrose-phosphate synthase (SPS) (UDP-glucose:
D-fructose-6-phosphate
2-
-D-glucosyltransferase) (EC 2.4.1.14), one of the key
enzymes in the control of sucrose synthesis, has been studied
extensively in various plant species. Importantly, SPS activity is
controlled by allosteric effectors (glucose-6-phosphate and
orthophosphate) and by reversible covalent modification (6).
On the other hand, the knowledge of sucrose metabolism in unicellular
organisms is very limited. Biochemical properties of SPSs isolated from
several species of green algae (21) were similar to those of
plant enzymes. The first evidence of the biosynthesis of sucrose
through the action of SPS and sucrose-phosphate phosphatase in
prokaryotes was shown by Porchia and Salerno (15), who
described the isolation and characterization of two forms of SPS from
Anabaena sp. strain PCC 7119, a filamentous heterocystous
cyanobacterium. Biochemical properties of these enzymes were strikingly
different from those of plant SPSs. We report here that SPS is also
present in a unicellular non-nitrogen-fixing cyanobacterium and prove that the Synechocystis sp. strain PCC 6803 open reading
frame (ORF) sll0045 (19) encodes a protein with SPS
activity. This enzyme has distinct biochemical regulatory properties
and molecular structure in comparison with those of plant SPSs.
(Part of this research was conducted by L. Curatti and P. Desplats in
partial fulfillment of the requirements for Ph.D. degrees from
Universidad Nacional de Mar del Plata, Mar del Plata, Argentina.)
Detection of SPS activity in Synechocystis sp. strain
PCC 6803 extracts.
The presence of SPS in unicellular
cyanobacteria has not been demonstrated until now. Therefore, we
decided to examine the occurrence of SPS activity in
Synechocystis, a unicellular non-nitrogen-fixing prokaryote.
When Synechocystis cell extracts were subjected to ion-exchange chromatography on DEAE-Sephacel, a single peak of SPS
activity eluted at a NaCl concentration similar to that of SPS-I from
Anabaena sp. strain PCC 7119 (15). The reaction
products were identified as UDP (27) and sucrose-6-phosphate
(15). The apparent molecular mass (MM) of the native enzyme
was about 85 kDa, as estimated by gel filtration through a Sephadex
G-100 column (15; also data not shown).
Expression of the Synechocystis sp. strain PCC 6803 ORF
sll0045 in Escherichia coli.
Recently, Kaneko and coworkers
determined the complete sequence of the Synechocystis sp.
strain PCC 6803 genome (9). Sequence comparison analysis
revealed the presence of an ORF (sll0045) which shares about 30%
identity with plant SPSs. To ascertain if this ORF encodes a protein
with SPS activity, we constructed the plasmid pSySPS containing the
putative SPS ORF flanked by 500 bp of upstream sequence and 1,200 bp of
downstream sequence by standard protocols (25). Extracts
from E. coli harboring pSySPS harvested in late exponential
phase showed an SPS activity of about 0.25 µmol · min
1 · mg of protein1 (14, 15,
19), indicating that the ORF sll0045 is contained in an SPS gene
(spsA) and codes for an active SPS protein. Under similar
conditions, E. coli harboring pBluescript II SK(+) had no
SPS activity.
spsA gene disruption.
Two insertion mutants were
constructed to further confirm the identity of the ORF sll0045. Several
complete segregant clones were obtained after transformation of
Synechocystis sp. strain PCC 6803 with pLC20 or pLC21 to
generate strain LC20 or LC21 (16), respectively (Fig.
1A). The segregation was confirmed by PCR
analysis (Fig. 1B). No SPS activity could be detected in the mutant
cells, providing additional evidence that the ORF sll0045 encodes an SPS protein. The growth curves of Synechocystis sp. strain
PCC 6803, LC20, and LC21 under standard conditions (BG-11 liquid
medium, 28°C, continuous illumination) did not show significant
differences, suggesting that SPS activity is not essential for their
growth.
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FIG. 1.
Insertion inactivation of Synechocystis sp.
strain PCC 6803 ORF sll0045. (A) Integration sites of the
cat cartridge used to obtain the insertion mutants LC20 and
LC21. The arrows indicate the direction of transcription. (B) PCR
analysis done with primers ol22 and ol23 (shown in panel A) and genomic
DNA from Synechocystis sp. strain PCC 6803 (lane 2), LC20
(lanes 3 to 5), and LC21 (lanes 6 to 8). Lane 1, 1-kb ladder of
molecular size markers (Gibco BRL).
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|
Biochemical characterization of Synechocystis sp.
strain PCC 6803 SPS expressed in E. coli.
To determine
the biochemical properties of Synechocystis SPS, the enzyme
was partially purified from extracts of E. coli harboring pSySPS. When extracts were chromatographed onto a DEAE-Sephacel column
(15), a single peak of SPS activity was detected at an elution position similar to that of the enzyme isolated from
Synechocystis cells. The concentrated SPS fraction at this
stage (purified ca. 25-fold; free of phosphoglucose isomerase
[13] and inorganic phosphatases [15])
was used to analyze some biochemical properties of the enzyme.
SPS activity showed a broad pH dependence, with a maximum around pH 7.5 to 8.5. The addition of divalent cations to the incubation
mixture (10 mM MgCl
2 or MnCl
2) increased enzyme activity
about
80 to 90%.
Specificity for the glucosyl donor was then investigated, and the
kinetic constant values from Wolf's plots were determined.
As shown in
Table
1, the enzyme was not specific for
UDP-glucose:
it could also use ADP-glucose and, to a minor extent,
GDP-glucose
as substrates. The apparent
Km for
fructose-6-phosphate was 1.5
mM. The enzymic preparation was not able
to yield sucrose from
fructose and UDP-glucose (or ADP-glucose).
The actions of the two allosteric effectors of plant SPS
(glucose-6-phosphate [activator] and orthophosphate [inhibitor]
[
6])
on enzyme activity were assayed.
Glucose-6-phosphate did not activate
the enzyme, and orthophosphate
caused only a 17% reduction of
enzyme activity at concentrations as
high as 20 mM. A similar
result was obtained for
Anabaena
SPS (
14). By comparison, 2
to 5 mM glucose-6-phosphate
increases activity about fivefold,
whereas 10 mM orthophosphate
inhibits SPS activity from rice leaves
by 80 to 90%, (
20),
indicating that the regulation of plant
and cyanobacterium SPSs may be
different.
Sequence analysis of the Synechocystis SPS
protein.
The Synechocystis ORF sll0045 encodes a
protein with a predicted MM of 81,421 Da. This value is similar to the
apparent MM calculated for the native SPS isolated from
Synechocystis extracts (see above). These results indicate
that Synechocystis SPS protein is composed of a single
polypeptide. Our recent studies showed that Anabaena SPS is
also a monomer, with an apparent MM of 45 to 47 kDa (15).
The different MMs of both cyanobacterial enzymes could be due either to
species differences or to proteolysis of Anabaena SPS during
the isolation procedure. By contrast, plant SPSs are either dimers or
tetramers of 116- to 138-kDa subunits, depending on the experimental
procedure used to determine the relative MM of the holoenzyme (1,
20, 22).
A comparison of the deduced amino acid sequences of
Synechocystis and plant SPSs (Fig.
2) revealed two important differences:
(i)
Synechocystis SPS lacks the N-terminal
178-amino-acid-residue
sequence of plant SPSs, which contains the
phosphorylation site
reported to be critical for the regulation of the
enzyme activity
(Ser-158 in spinach [
12]; Ser-162 in
maize [
7]), and (ii)
the UDP-glucose binding site
determined in spinach SPS by photoaffinity
labeling (
23) and
highly conserved in plant species is remarkably
divergent in
Synechocystis SPS (amino acid residues 59 to 72).
We
speculate that this divergence may be responsible for the observed
differences in substrate specificity: plant SPSs are specific
for
UDP-glucose, whereas
Synechocystis (Table
1) and
Anabaena (
15) SPSs are not. The putative
fructose-6-phosphate (the other
SPS substrate) binding site (residues
199 to 206 in maize SPS
[
24]) is highly conserved in
the
Synechocystis SPS sequence
(Fig.
2).
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FIG. 2.
Alignment of the N-terminal regions of the predicted SPS
amino acid sequences from Synechocystis sp. strain PCC 6803 (residues 1 to 83) (9), Zea mays (residues 1 to
256) (30), and Spinacia oleracea (residues 1 to
256) (10). This alignment was generated with the program
Megalign of the DNAStar package, using the Clustal method with PAM 250 residue weight table. Positions that are identical in different
proteins are indicated in bold type. Stippled boxes indicate the
UDP-glucose (box 2) and putative fructose-6-phosphate (box 1) binding
domains. The sites of phosphorylation in plant SPSs (Ser-162 for maize
and Ser-158 for spinach) (6) are underlined and indicated by
arrowheads. Gaps introduced to maximize alignment are indicated by
dashes.
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|
A dendogram generated using the deduced amino acid sequences reported
for various SPSs shows that the
Synechocystis enzyme
clearly
diverges from plant SPSs (Fig.
3).
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FIG. 3.
Phylogenetic tree of SPSs. SPSs from Craterostigma
plantagineum (8), Solanum tuberosum
(2), Vicia faba (4), Beta
vulgaris (5), Spinacia oleracea
(10), Zea mays (30), Saccharum
officinarum (GenBank accession no. AB001338), Oryza
sativa (28), Citrus unshiu (11),
Musa acuminata (GenBank accession no. U59489), and
Synechocystis sp. strain PCC 6803 (9) were used
to construct this tree. The tree was generated with the program
Megalign of the DNAStar package, using the Clustal method with PAM 250 residue weight table.
|
|
By using Southern blot analysis, we failed to detect
spsA
homologous sequences in other cyanobacteria corresponding to different
taxonomical groups (
18):
Synechoccocus sp. strain
PCC 7942 (group
I);
Anabaena sp. strain PCC 7119,
Anabaena sp. strain PCC 7120,
and
Anabaena
variabilis (group IV); and
Calothrix sp. strain PCC
7601 (group
V).
Conclusions.
We demonstrate by expression in E. coli and insertion inactivation that the Synechocystis
sp. strain PCC 6803 ORF sll0045 encodes a protein with SPS activity.
This is the first identification of a prokaryotic SPS gene
(spsA). However, the role of sucrose in cyanobacteria is
still a point of discussion. Its presence in these organisms has
usually been associated with responses to different environmental
stresses (3, 17), and it was hypothesized that it could be
the carbon carrier substance from vegetative cells to heterocysts in
filamentous cyanobacteria (26, 29). However, the fact that
SPS is present in a unicellular non-nitrogen-fixing cyanobacterium such
as Synechocystis even under standard growth conditions
suggests that sucrose may not be exclusively associated with carbon
transport or stress tolerance.
| |
ACKNOWLEDGMENTS |
We thank S. Tabata (Kazusa DNA Research Institute, Chiba, Japan)
for kindly providing the cosmid CSO415, H. G. Pontis for helpful
discussions, and C. Fernández and C. Rodríguez for
technical assistance.
G.S. is a Career Investigator of the Consejo Nacional de
Investigaciones Cientificas y Técnicas (CONICET). This work was supported partly by grants from the CONICET, the Agencia Nacional de
Promoción Cientifica y Tecnológica from Argentina, and the Rockefeller Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Investigaciones Biológicas (FIBA), Casilla de Correo 1348, 7600 Mar del Plata, Argentina. Phone: (54) 23 748257. FAX: (54) 23 757120. E-mail: fiba{at}mdq.com.ar.
| |
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Journal of Bacteriology, December 1998, p. 6776-6779, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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