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Previous Article | Next Article 
Journal of Bacteriology, January 2000, p. 211-215, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Uptake of 2-Oxoglutarate in
Synechococcus Strains Transformed with the Escherichia
coli kgtP Gene
María Félix
Vázquez-Bermúdez,
Antonia
Herrero, and
Enrique
Flores*
Instituto de Bioquímica Vegetal y
Fotosíntesis, Consejo Superior de Investigaciones
Científicas-Universidad de Sevilla, E-41092 Seville, Spain
Received 7 June 1999/Accepted 14 October 1999
 |
ABSTRACT |
A number of cyanobacteria from different taxonomic groups exhibited
very low levels of uptake of 2-[U-14C]oxoglutarate.
Synechococcus sp. strain PCC 7942 was transformed with DNA
constructs carrying the Escherichia coli kgtP gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The Synechococcus sp. strains bearing the
kgtP gene incorporated 2-oxoglutarate into the cells
through an active transport process. About 75% of the radioactivity
from the 2-[U-14C]oxoglutarate taken up that was
recovered in soluble metabolites was found as glutamate and glutamine.
2-Oxoglutarate was, however, detrimental to the growth of a
Synechococcus sp. strain bearing the kgtP gene.
| |
TEXT |
The dominant mode of growth of
cyanobacteria is photoautotrophy. These organisms show very simple
nutritional requirements and are able to grow in water supplemented
with a few mineral salts. An analysis of the putative transport
proteins encoded in the chromosome of the unicellular cyanobacterium
Synechocystis sp. strain PCC 6803 (11) has shown
a relatively high proportion of transport systems whose predicted
substrates would be ions (metals plus anions), i.e., 47% of all
putative transporters, compared to 16 and 21% in Escherichia
coli and Bacillus subtilis, respectively
(22). On the other hand, only 6% of the putative transporters of strain PCC 6803 are predicted to have carbon compounds (carboxylates plus carbohydrates) as substrates, while carbon compound
transporters represent 28 and 20% in E. coli and B. subtilis, respectively (22). One of the few carbon
compound transporters experimentally identified to date in
Synechocystis sp. strain PCC 6803 is the glucose permease
encoded by the glcP (or gtr) gene (27,
34). This is a monocomponent permease consisting of a
polypeptide of 468 amino acids with 12 putative membrane-spanning segments which is homologous to sugar transporters from several biological sources.
In cyanobacteria, the carbon compound that provides the skeleton for
the assimilation of inorganic nitrogen, via the glutamine synthetase-glutamate synthase pathway, is 2-oxoglutarate (5, 18). In the unicellular cyanobacterium Synechococcus
sp. strain PCC 7942, 2-oxoglutarate also appears to have a key
regulatory role in the integration of carbon and nitrogen metabolisms,
e.g., it determines the phosphorylation level of the PII
protein (glnB gene product) (6, 10), which is
required for the inhibition by ammonium of nitrate uptake to take place
(12). We were interested in studying the fate and roles of
2-oxoglutarate in cyanobacteria, but found that these organisms take up
2-oxoglutarate very poorly when it is supplied to the cells at a low
concentration (see below). We therefore sought the construction of a
strain of Synechococcus sp. able to transport
2-oxoglutarate.
The kgtP gene of E. coli encodes a 432-amino-acid
polypeptide which bears 12 putative membrane-spanning regions and
mediates 2-oxoglutarate transport (28). Because the KgtP
protein is similar (28% overall amino acid sequence identity) to
Synechocystis sp. strain PCC 6803 GlcP, we wondered whether
it could be functional in a cyanobacterium. In this note, we report the
construction of some Synechococcus sp. strain PCC 7942 derivatives transformed with kgtP and show that they can
efficiently transport 2-oxoglutarate.
Methods.
Cyanobacteria were grown in BG11 medium, which
contains nitrate as the nitrogen source (25), at 30°C in
the light, with shaking (80 to 90 rpm) for liquid cultures. When
ammonium replaced nitrate as the nitrogen source, it was added as 2.5 mM NH4Cl, and the medium was buffered with 5 mM TES
[N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]-NaOH (pH 7.5). For plates, the medium was solidified with 1%
separately autoclaved agar (Difco). Synechococcus sp.
strains carrying gene cassette C.K1 or C.K2 (4), both
of which determine resistance to kanamycin (Kmr),
were grown in medium supplemented with 10 µg of kanamycin
· ml
1. Cyanobacterial cell mass was estimated by
measuring the concentration of chlorophyll a (Chl),
determined in methanolic extracts of the cells (15), or the
concentration of protein, determined by a modified Lowry procedure
(16) with bovine serum albumin as a standard. The growth
rate constant, µ (µ = ln 2/td [where
td represents the doubling time]) was
calculated from the increase of protein concentration in the cultures.
Genomic DNA from cyanobacteria was isolated as previously described
(2).
For uptake assays, the cells were harvested by centrifugation at room
temperature, washed with 25 mM Tricine-NaOH buffer (pH 8.1), and
resuspended in the same buffer supplemented with 2.5 mM
NH4Cl and 5 mM TES-NaOH buffer (pH 7.5) to give a cell
density corresponding to 10 to 15 µg of Chl · ml1. After a preincubation at 30°C in the light (100 W · m2 [white light]) for 5 to 30 min, the
assays were started by the addition of
2-[U-14C]oxoglutarate (3.84 to 280 µCi · µmol1 [NEN]) at the concentration indicated in each
experiment. After incubation for different time periods up to 30 min,
0.1- to 1-ml samples were withdrawn, filtered (0.45-µm-pore-size
Millipore HA filters), and washed with 2 to 5 ml of 5 mM Tricine-NaOH
buffer (pH 8.1). The filters carrying the cells were then immersed in scintillation cocktail, and their radioactivity was measured. Retention
of radioactivity by boiled cells was used as a blank. In some
experiments, as indicated, ammonium and 0.1 mM
L-methionine-D,L-sulfoximine (MSX)
were either absent or added to the cell suspension 30 min before the
assay was started. To identify intracellular radioactively labelled
metabolites, filters containing cells that had been used in 30-min
uptake assays were immersed in 2 ml of water and incubated at 100°C
for 5 min. The filters were removed, and the resulting suspensions were
centrifuged. Samples from the supernatants were lyophilized and
dissolved in a small volume of water. Metabolites present in these
samples were resolved by thin-layer chromatography (TLC), with
0.1-mm-thick cellulose plates (20 by 20 cm; Merck). Two-dimensional separation of metabolites was effected by using the
following solvents: first dimension,
n-butanol-acetone-ammonium hydroxide-water
(20:20:10:4 [vol/vol]); second dimension, isopropanol-formic acid-water (20:1:5 [vol/vol]). The resulting radioactive areas were
quantified in an InstantImager scanner for 
particles (Packard) whose efficiency in detection of 14C particles was
1.12%. To calculate intracellular metabolite concentrations, an
intracellular volume of 125 µl · mg of Chl1 (5 µl · mg of protein1) was assumed (9, 24,
30).
E. coli DH5
, used for all plasmid constructions, was
grown in Luria-Bertani medium with, when necessary, 25 µg of
kanamycin · ml1, 50 µg of ampicillin · ml1, 25 µg of streptomycin · ml1,
and 100 µg of spectinomycin · ml1. Molecular
biology manipulations were performed by standard procedures (1). The 2.8-kb AflII-SalI fragment
from plasmid pCW28 (isolated by using polylinker restriction
endonuclease sites as a BamHI-HindIII fragment), which carries the E. coli kgtP gene and its
putative promoter (28), was cloned (after filling in with
Klenow enzyme) together with the C.K2 or C.K1 gene cassette
(4) into an EcoRV site present in the
Synechococcus sp. strain PCC 7942 chromosomal DNA fragment
of plasmid pCSI49b (see below) (13), rendering the
constructs depicted in Fig. 1.
Transformation of Synechococcus sp. strain PCC 7942 (8) and Southern analysis under high-stringency conditions
with Hybond-N+ membranes (Amersham) were performed essentially as
described previously (7).
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FIG. 1.
Schematic representation of the Kmr-encoding
gene cassette-kgtP gene constructs used to transform
Synechococcus sp. strain PCC 7942 and
2-[U-14C]oxoglutarate uptake activities of the generated
Synechococcus sp. strains. The orientations of the
npt (in the gene cassette) and kgtP genes and the
positions of some restriction endonuclease cutting sites (C,
ClaI; P, PstI; RV, EcoRV) are
indicated. The hatched boxes denote Synechococcus sp. strain
PCC 7942 genomic DNA. Uptake of 20 µM
2-[U-14C]oxoglutarate was determined as described in the
text; data are the mean and standard deviation of three to five
determinations.
|
|
Uptake of 2-[U-14C]oxoglutarate.
The uptake of
20 µM 2-[U-14C]oxoglutarate was tested, in 10-min
assays, in different cyanobacteria with the following results (mean of
two determinations): Anabaena sp. strain PCC 7120, 0.77 nmol · mg of Chl1; Calothrix sp. strain
PCC 7601, 0.46 nmol · mg of Chl1;
Fischerella muscicola UTEX 1829, 0.65 nmol · mg of
Chl1; Pseudanabaena sp. strain PCC 6903, 0.64 nmol · mg of Chl1; Synechocystis sp.
strain PCC 6803, 0.55 nmol · mg of Chl1; and
Synechococcus sp. strain PCC 7942, 0.45 nmol · mg of
Chl1. These can be considered as relatively poor
activities that are, on average, about 100 times lower than the rates
of uptake of several amino acids (supplied at 10 µM) in the same
cyanobacterial strains (20).
The kgtP gene from E. coli was inserted, along
with a Kmr-encoding gene cassette, into a chromosomal DNA
fragment from Synechococcus sp. strain PCC 7942 that has
been observed to tolerate insertions of heterologous DNA
(13). Four different structures were generated with two
different Kmr gene cassettes, C.K1 and C.K2, positioned in
different relative orientations with respect to that of the
kgtP gene (Fig. 1). The four constructs were transferred to
Synechococcus sp. strain PCC 7942 by transformation and
selection for Kmr. The presence of the kgtP gene
and the relative orientation of this gene and the C.K1 or C.K2 gene
cassette were confirmed by Southern analysis for one
Synechococcus sp. clone from each transformation (not
shown). The names of the Synechococcus sp. strain PCC 7942 derivatives carrying the different constructs are indicated in Fig. 1.
Time course assays of 2-[U-14C]oxoglutarate uptake showed
that for the four Synechococcus sp. strains carrying the
kgtP gene, uptake was linear for at least 30 min. Uptake
rate data are summarized in Fig. 1. Strain PCC 7942 clones transformed
with similar constructs bearing only C.K1 or C.K2 showed
2-[U-14C]oxoglutarate uptake activities identical to that
of the wild-type strain (not shown). The maximal level of expression
attained, observed in strain CSF70, which showed an uptake activity
333-fold higher than that of strain PCC 7942, was facilitated by
positioning of kgtP downstream from the C.K1 gene cassette
so that the npt and kgtP genes are situated in
the same orientation (Fig. 1). Because the C.K1 gene cassette does not
bear any transcription terminator downstream from the npt
gene (14, 17), the high uptake activity detected in strain
CSF70 is likely a consequence of a high level of transcription of the
kgtP gene from the npt gene promoter.
The effect of a wide range of concentrations of 2-oxoglutarate on the
rate of uptake was investigated in wild-type strain PCC 7942 and in
strain CSF70 (Fig. 2). Based on the data
shown in Fig. 2, the following 2-oxoglutarate uptake kinetic parameters were calculated for strain CSF70: Ks, 205 µM;
Vmax, 70 nmol · min1
· mg of Chl1 (about 2.5 nmol · min1 · mg of protein1). In an
independent experiment in which 2-oxoglutarate was supplied at
concentrations from 20 to 500 µM, the following kinetic parameters were determined: Ks, 147 µM;
Vmax, 165 nmol · min1
· mg of Chl1 (about 6 nmol · min1 · mg of protein1). In E. coli MC1061 or in membrane vesicles prepared from it, the
following kinetic parameters have been reported for 2-oxoglutarate uptake: Ks, 13 to 46 µM;
Vmax, 8 to 11 nmol · min1 · mg of protein1
(29). Some retention of radioactivity by cells of
Synechococcus sp. strain PCC 7942 incubated with 1 mM
2-[U-14C]oxoglutarate (see Fig. 2) was repetitively
observed. Rather than taking place via a specific transport system,
this low-affinity uptake might be the result of diffusion or might be
mediated by another permease, e.g., the acidic amino acid permease
present in Synechococcus sp. strain PCC 7942 (20).
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FIG. 2.
Effect of the concentration of 2-oxoglutarate on the
rate of 2-oxoglutarate uptake in Synechococcus sp. strains
PCC 7942 (circles) and CSF70 (triangles). The cells were supplemented
with the indicated 2-[U-14C]oxoglutarate concentrations,
and uptake was determined as described in the text.
|
|
Fate of 2-[U-14C]oxoglutarate.
The presence of
labelled metabolites was studied with cells of strain CSF70 incubated
with 2-[U-14C]oxoglutarate for 30 min under the standard
conditions used in this work (Fig. 3).
Most label was associated with glutamate (53.9% of the radioactivity
in soluble metabolites) and glutamine (18.3%), while five other
metabolites together accounted for 15.0%, and a spot that could be
identified as 2-oxoglutarate represented 12.8%. Although ammonium was
routinely included in our standard assays to ensure metabolism of
2-oxoglutarate, this did not have a major effect on 2-oxoglutarate
uptake, at least when a low concentration of 2-oxoglutarate was used
(Table 1). MSX, an inhibitor of glutamine synthetase, on the other hand, blocked production of glutamine and
impaired 2-oxoglutarate uptake (Table 1). This indicates that the rate
of uptake of 2-oxoglutarate was dependent on its metabolism via
glutamate synthase, which catalyzes the synthesis of two glutamate
molecules from 2-oxoglutarate and glutamine. Synthesis of labelled
glutamate from 2-[U-14C]oxoglutarate in MSX-treated
cells will depend on the intracellular pool of unlabelled glutamine
present in those cells before the addition of MSX.
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FIG. 3.
Fate of 2-[U-14C]oxoglutarate in
Synechococcus sp. strain CSF70. The cells were supplemented
with 30 µM 2-[U-14C]oxoglutarate and incubated for 30 min under standard conditions (i.e., in the presence of ammonium).
Metabolites were then extracted and analyzed by TLC as described in the
text. The amount of extract loaded onto the TLC plate corresponded to
an amount of cells containing 0.52 µg of Chl. The arrow points to the
origin of the chromatogram, where some radioactive material accumulated
(Table 1). Glu, glutamate; Gln, glutamine; 2-OG, 2-oxoglutarate. The
glutamate and glutamine spots were identified by cochromatography with
unlabelled amino acids that were revealed by the standard ninhydrin
reaction. The 2-oxoglutarate spot was identified by cochromatography
with labelled 2-oxoglutarate. Controls run with authentic
2-[U-14C]oxoglutarate indicated that some decomposition
of 2-oxoglutarate took place during the development of the
chromatography, so that the radioactivity in the spot identified as
2-oxoglutarate accounted for 24.7% of the total radioactivity of the
2-[U-14C]oxoglutarate applied to the TLC.
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TABLE 1.
Effect of ammonium and MSX on the uptake and fate of
2-[U-14C]oxoglutarate in Synechococcus sp.
strain CSF70a
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|
The highest levels of 2-[U-14C]oxoglutarate accumulated
(Table 1) corresponded to intracellular concentrations of
about 50 to 60 µM. Considering a membrane potential of 
120 mV
(21, 26), the electrochemical potential of 2-oxoglutarate
(intracellular concentration of labelled 2-oxoglutarate, 60 µM;
extracellular concentration at the time of sampling, 27 µM) would
correspond to +260 mV, implying that 2-oxoglutarate is actively
transported into the cell. Moreover, this figure was derived without
taking into account the intracellular pool of unlabelled
2-oxoglutarate, which can be in the order of 16 to 340 µM (calculated
from data in references 3 and
19). The KgtP permease is a proton symporter (29), and photosynthetically active cyanobacterial cells
maintain, under incubation conditions similar to those used in this
work, an electrochemical proton potential of about 100 mV
(26). The active transport of 2-oxoglutarate in
Synechococcus sp. can therefore proceed, as is the case in
E. coli (29), through a proton symport mechanism.
Growth rates.
To test any possible effects of the presence of
the KgtP permease on the growth of Synechococcus sp., the
growth rate of strain CSF70 was determined under the conditions
summarized in Table 2. As a control, a
strain, PCC 7942::C.K1, carrying the C.K1 gene cassette in
the same location and orientation as in CSF70, was used. The presence
of the permease itself was not detrimental to the growth of
Synechococcus sp. Addition of 1 mM 2-oxoglutarate, however,
slowed down the growth of the kgtP-bearing strain, but not
that of the control strain. This effect was more pronounced when
ammonium was used as the nitrogen source (Table 2). Nonetheless, the
cells were not killed by 2-oxoglutarate and kept growing for a long
period of time. Whether the observed negative effect is due to an
imbalance of metabolites within the cells, to a bioenergetic shortage
caused by 2-oxoglutarate transport and affecting other membrane
transport functions, or to a general effect on the physiology of the
cell such as a change of intracellular pH caused by the influx of a
dicarboxylate, is currently unknown.
Conclusions.
The results presented above show the feasibility
of expressing a secondary active transporter from a heterologous
biological source in a cyanobacterium. Expression of heterologous genes
in cyanobacteria is well documented: e.g., the antibiotic resistance genes widely used in molecular genetics work (32, 33), the gene encoding ribulose bisphosphate carboxylase from
Rhodospirillum rubrum that has been expressed in
Synechocystis sp. strain PCC 6803 (23), or the
sacB gene encoding a levansucrase that has been expressed
in Anabaena sp. strain PCC 7120 (2). A
derivative of Synechococcus sp. strain PCC 7942 transformed
with the glcP gene from Synechocystis sp.
strain PCC 6803 has been shown to take up glucose (35). We
are not aware, however, of any previous expression in a
cyanobacterium of a permease from a noncyanobacterial source.
Cyanobacteria lack 2-oxoglutarate dehydrogenase and bear an incomplete
citric acid cycle that produces 2-oxoglutarate, which is not futher
catabolized and is used for biosynthetic purposes (31).
Consistently, the fate of 2-[U-14C]oxoglutarate observed
in this work mainly consisted of its incorporation into glutamate
through the reaction catalyzed by glutamate synthase. This limited use
of 2-oxoglutarate may be a rationale to explain why the cyanobacteria,
which when provided with an appropriate permease can transport
2-oxoglutarate, appear not to have acquired transporters specific for
this compound. Additionally, the metabolism and/or transport
capabilities of Synechococcus sp. strain PCC 7942 appear to
have been tuned in a way that makes this organism not tolerate well
exposure to 2-oxoglutarate when it has also been provided with a
2-oxoglutarate permease.
| |
ACKNOWLEDGMENTS |
We thank W. Seol and A. J. Shatkin for kindly providing us
with E. coli kgtP clones and M. M. Allen for
useful discussion.
This work was supported by grant PB95-1267 from the Dirección
General de Enseñanza Superior (Spain). M.F.V.-B. was the
recipient of a fellowship from the Plan de Formación de Personal
Investigador, Ministerio de Educación y Cultura (Spain).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Bioquímica Vegetal y Fotosíntesis, Centro de
Investigaciones Científicas Isla de la Cartuja, Avda.
Américo Vespucio s/n, E-41092 Seville, Spain. Phone: 34 95 448 9523. Fax: 34 95 446 0065. E-mail: flores{at}cica.es.
| |
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Journal of Bacteriology, January 2000, p. 211-215, Vol. 182, No. 1
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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