Download Obligate phototrophy in cyanobacteria: more than a lack of sugar

FEMS Microbiology Letters 161 (1998) 285^292
Obligate phototrophy in cyanobacteria:
more than a lack of sugar transport
Cheng-Cai Zhang a , Robert Jeanjean b , Franc°oise Joset b; *
b
a
ESBS, Boulevard S. Brant, 67400 Illkirch, France
LCB-CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20, France
Received 19 December 1997; revised 17 February 1998; accepted 19 February 1998
Abstract
DNA hybridization using the Synechocystis PCC6803 glucose transporter gene, glcP, revealed a single homologous region in
two facultative photoautotrophic strains out of three tested, and none in three obligate autotrophs. In one of the latter,
Synechococcus PCC7942, integration of glcP into the chromosome resulted in glucose sensitivity. A subclone isolated as
glucose-tolerant had lost glcP. Integration in a replicative vector allowed glucose transport and photoheterotrophic growth,
but could not be maintained. Thus lack of sugar transport could explain cyanobacterial obligate autotrophy. However, at least
in Synechococcus PCC7942, acquisition of such a transport capacity created a metabolic disequilibrium barely compatible with
survival. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Keywords : Cyanobacteria; Glucose transport; Heterotrophy
1. Introduction
Cyanobacteria are naturally photoautotrophic,
through utilization of their plant-like photosynthetic
process. They nevertheless possess a respiratory activity (for a review see [1]) and all the enzymes necessary for sugar catabolism [2,3]. Only approximately half of the strains tested, however, are
capable of using this pathway to perform heterotrophic growth [4]. Usually only one sugar per strain
(most often glucose) is an acceptable substrate. In
most cases only photoheterotrophic capacities have
been observed, i.e. use of the sugar exclusively as
carbon source, ATP being provided through either
* Corresponding author. Tel.: +33 4 91 16 40 96;
Fax: +33 4 91 71 89 14; E-mail: [email protected]
cyclic electron transfer via Photosystem I or respiration. The inability of the other strains to grow heterotrophically has been postulated to result from a
lack of e¤cient uptake of the possible substrates [5].
A de¢ciency of glucose concentrating capacity was
indeed demonstrated in three cases, Nostoc sp.
PCC7118 (renamed Anabaena variabilis) and two
Synechocystis
(former
Aphanocapsa)
strains,
PCC6308 and PCC7008 [5]. The presence of an active glucose uptake system has been described for
four facultative phototrophs, all capable of both
photo- and chemoheterotrophy (the sugar providing
both the carbon and the energy source), Plectonema
boryanum [6], Synechocystis PCC6714 [5,7], Synechocystis PCC6803 [7,8] and Nostoc MAC (PCC8009)
[5]. The transport mechanism of the ¢rst two strains
was described as a proton/glucose symport [6,9].
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 8 4 - 6
FEMSLE 8094 31-3-98
286
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
That of Synechocystis PCC6803 is probably similar
[7,8]. Fructose is toxic for the two latter Synechocystis strains, in which it enters by facilitated di¡usion
through the glucose transporter [7].
The gene encoding the glucose transporter of Synechocystis PCC6803, glcP, has been cloned [10,11].
The deduced protein displays about 30 to 35% amino
acid sequence identity with, and a predictable structure similar to, that of a family of non-phosphorylating sugar carriers largely distributed among prokaryotes and eukaryotes. It thus appeared probable that
the carrier of other facultative phototrophic cyanobacteria using glucose as substrate would be homologous to that of Synechocystis PCC6803. It could be
anticipated that the DNA sequence, at least in some
well chosen cases, would also share enough homology to allow direct detection of the corresponding
genes. In spite of their incapacity to actively take
up sugars, it cannot be excluded that obligate photoautotrophic strains possess equivalent genes, either
non-expressed or encoding functionally de¢cient proteins. DNA hybridization search and interspeci¢c
transformations have been used to challenge such
possibilities in the three facultatively phototrophic
strains mentioned above, and in three obligate phototrophic ones. It could also be expected that synthesis of the Synechocystis PCC6803 glucose transporter in obligate phototrophs could render them
heterotrophic. The consequences of the introduction
of the glcP gene into Synechococcus PCC7942, a
model strain for studies on photosynthesis, are described.
[12]. The glucose analogues 2-deoxy-glucose (DOG)
and 3-O-methyl-glucose (OMG) were purchased
from Sigma. When appropriate, streptomycin or
spectinomycin were added at 20 Wg ml31 , ¢nal concentration.
2.2. DNA extractions
Extractions of genomic DNA from cyanobacterial
cells were performed as in [10], and plasmid preparations from E. coli as in [12]. Plasmid isolation from
Synechococcus PCC7942 followed [13] except that
elimination of chromosomal DNA was achieved by
incubation for 1 h at 4³C in the presence of NaCl
1 M ¢nal concentration.
2.3. Recombinant DNA methods
2. Materials and methods
The probes used for hybridization covered the entire glucose permease encoding sequence, glcP (1404
bp), of Synechocystis PCC6803, preceded by, respectively, either the 90 bp (plasmid pGT19, 1.5 kbp
EcoRI-KpnI fragment) or the 500 bp (plasmid
pGT12, 2 kbp BamHI fragment) upstream from
the open reading frame (ORF) [10]. The latter fragment, carried on the pUC19 vector, was used for
transformation. The primers for polymerase chain
reaction (PCR) encompassed nucleotides 826^860
and 1257^1240 within the glcP ORF. Southern hybridizations were performed under low stringency
conditions as described in [14]. All other procedures
followed [12]. The nick-translation kit and 32 P-dCTP
were from Amersham, France. The Synechococcus
PCC7942 vector pUC303 was constructed by Kuhlemeier and van Arkel [13].
2.1. Strains and growth conditions
2.4. Cyanobacterial transformation procedures
All wild-type (WT) cyanobacterial strains used
(Table 1) came from the Pasteur Culture Collection,
Paris. The spontaneous single mutant Synechocystis
PCC6803-Frur B5, glucose transport de¢cient (Glc3 )/
fructose resistant (Frur ), was described in [7]. Photoautotrophic (light, 5% CO2 in air) and photoheterotrophic (light, 5 WM DCMU (3-(3,4-dichlorophenyl)1,1-dimethylurea), 1% w/v glucose) culture conditions followed [10]. Escherichia coli XL1-blue and
HB101 were used as hosts for plasmid preparations
Transformation of Synechocystis PCC6803-Frur B5
was performed as in [10]. Selection was made for
recovery of the capacity to grow photoheterotrophically. Transformation of Synechococcus PCC7942
was as described in [13], selection being described
in the text.
2.5. Kinetics of sugar uptake
The procedure for determination of sugar uptake
FEMSLE 8094 31-3-98
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
activity followed [7], using U-14 C-glucose or U-14 COMG (from Amersham). Approximately 5U107
(Synechocystis PCC6803) or 5U108 (Synechococcus
PCC7942 WT or derivatives) cells/ml were incubated
with 0.4 to 13 Gbq/mol labelled glucose or OMG
(¢nal speci¢c activities). The suspension was maintained under standard growth conditions at 34³C.
Uptake was recorded during up to 24 h.
3. Results and discussion
3.1. Search for glcP homologous genes in various
cyanobacteria
The strains tested (Table 1) were chosen upon two
criteria, their (in)capacity of heterotrophic growth on
glucose and when known of glucose uptake, and a
G+C content as close as possible to that of Synechocystis PCC6803 [15,16]. Total DNA from these cells
was digested, in parallel or simultaneously, by EcoRI
or HaeII, and hybridized with the Synechocystis
PCC6803 glcP gene (the 1.5 EcoRI-KpnI fragment)
as probe. A clear positive signal was found with
Synechocystis PCC6803 and PCC6714 DNAs, and
a weak one with Nostoc MAC DNA (Fig. 1). One
band (except for the HaeII digestion of Synechocystis
PCC6714 DNA, which showed two bands) was revealed. These results indicate that the three strains
possess a homologous sugar transporter gene. The
presence of a single copy per genome in Synechocystis PCC6803 was established from previous work [10]
and from its genome sequence [17]. This was also
expected in Synechocystis PCC6714 on the basis of
the high (1036 ) spontaneous frequency of appearance
of glucose transport de¢cient mutants [7], and the
close phylogenic relationship between the two Synechocystis strains [4]. The strong hybridization signal
observed with Synechocystis PCC6714 DNA also
corroborates the similar characteristics of the glucose
transport process in the two strains [7]. The weak
hybridization signal revealed with Nostoc MAC
DNA is consistent with the larger evolutionary distance between the two strains.
The absence of hybridization signal with P. boryanum DNA could appear surprising, since this strain
can e¤ciently take up and use exogenous glucose
[18]. However, apart from the phylogenic distance
287
separating P. boryanum from Synechocystis
PCC6803, the two glucose transport systems display
distinct characteristics. In P. boryanum uptake is inducible by glucose, and the glucose analogue Kmethyl-glucoside shows high competitive e¡ect
rather than OMG in Synechocystis [6^8]. The transport system has no a¤nity for fructose, which is
transported via a distinct system and also used as a
substrate for heterotrophic growth [18].
No hybridization signal was revealed with DNA
from any of the obligate phototrophic strains. The
presence of a poorly or non-homologous gene is unprobable since these strains do not show any uptake
of glucose ([6]; data not shown). In the case of the
Synechocystis strains, this result stresses genomic divergences among strains belonging to the same taxonomic group.
3.2. Search for glucose transport capacity by
complementation of Synechocystis PCC6803Frur B5 by heterologous cyanobacterial DNAs
Glucose uptake pro¢ciency is associated in Synechocystis PCC6803 with sensitivity to fructose [7,8].
Fructose enters, probably through facilitated di¡usion, via the glucose transporter, as attested by competition assays and loss of fructose sensitivity in glucose uptake de¢cient mutants [7,8,10]. One such
mutant, Synechocystis PCC6803-Frur B5, was used
as the recipient for transformation assays with total
DNA isolated from Synechocystis PCC6714, P. boryanum, Nostoc MAC and Synechococcus PCC7942.
Selection for recovery of photoheterotrophic growth
capacity yielded 5U1035 transformants per cell with
Synechocystis PCC6714 DNA. This frequency is only
slightly smaller than that obtained in the homologous transformation (1034 ). All transformants tested
(50 clones) had acquired the wild-type phenotype
(glucose transport capacity and fructose sensitivity).
No transformant ( 6 1038 per cell under saturating
DNA concentration) was obtained with any of the
other three DNAs tested. The negative result observed with Synechococcus PCC7942 DNA was expectable. Restriction incompatibility, lack of su¤cient homology and/or di¡erent chromosomal
organizations of the Nostoc MAC DNA, and at least
absence of homology of the P. boryanum DNA, may
have prevented recombination in these cases.
FEMSLE 8094 31-3-98
288
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
high level of conservation of this class of sugar transporters, and their ubiquiteness, in distant organisms
[10,11].
3.3. Transfer of the glcP gene into Synechococcus
PCC7942. Phenotype of the transgenic clone
R2-1
The demonstration that a glcP transgenic strain
could transport glucose, and possibly perform heterotrophic growth, would support the hypothesis that
the cause for obligate autotrophy resides in an incapacity to actively transport this (any?) sugar. This
hypothesis was challenged in Synechococcus
PCC7942. The 2 kbp long BamHI fragment from
plasmid pGT12 [10], including the whole glcP coding
sequence and 0.5 kbp upstream of its translational
start site, was used as transforming DNA. Since no
prokaryotic consensus promoter could be recognized
upstream of the ORF ([10]; S. Golden, personal
communication), this insured with su¤cient probability the presence of the elements necessary for expression of the gene. The 2 kbp fragment, with glcP
under its own promoter, was inserted into the cat
gene of the E. coli-Synechococcus PCC7942 shuttle
vector pUC303, a low copy number plasmid in the
cyanobacterium [13]. The resulting construction,
pUC303-glcP-Cms -Smr , was checked for its capacity
Fig. 1. Hybridization signals of genomic DNA from several cyanobacterial strains with the glcP glucose transporter gene from
Synechocystis PCC6803. The probe (1.5 kbp) covered the whole
glcP ORF. Total genomic DNAs were digested with HaeII (A,
lanes 1 to 5), EcoRI (A, lanes 7 to 11) and both enzymes (B).
Hybridizations were performed under low stringency. A: Lanes 1
and 7, Synechocystis PCC6714 ; lanes 2 and 8, Synechocystis
PCC6803 ; lanes 3 and 9, Synechococcus PCC7942 ; lanes 4 and
10, Nostoc MAC ; lanes 5 and 11, Plectonema boryanum. B :
Lane 1, Synechocystis PCC6714; lane 2, Synechocystis PCC6902 ;
lane 3, Synechocystis PCC7008; lane 4, pGT19; lane 5, Synechocystis PCC6803.
The presence and/or conservation of a glcP homologous gene appears restricted among cyanobacteria. This observation is surprising considering the
Table 1
Test for the presence of a glcP homologous gene in cyanobacterial strains
Strain
Taxonomic
group [4]
a
Synechocystis PCC6803
Synechocystis PCC6714a
Synechocystis PCC6902
Synechocystis PCC7008
Synechococcus PCC7942
Plectonema boryanum
PCC73110
Nostoc MAC PCC8009
G+C content
[ref.]
Growth
capacitiesb;c
PA
PH
CH
I
I
I
I
I
III
47 [15]
47 [15]
42.1 [15]
44.9 [15]
55 [16]
47 [15]
+
+
+
+
+
+
+
+
3
3
3
+
+
+
3
3
3
+
IV
42^45e [15]
+
+
+
Growth
substrates
Glucose
transport
glcP
homologuec
glucosed
glucosed
/
/
/
glucose,
fructose
glucose
fructose
constitutive [7]
constitutive [7]
none
none [9]
none
inducible [6]
+
+
3
3
3
3
constitutive [5]
+
(weak signal)
Hybridizations of total DNA from the strains listed were run at low stringencies, using a probe encompassing the whole glcP gene from
Synechocystis PCC6803. See Section 2 for details.
a
Formerly referred to as Aphanocapsa.
b
PA, photoautotrophy; PH, photoheterotrophy; CH, chemoheterotrophy ; data from [4].
c
+ or 3 indicate presence or absence of the property tested.
d
Fructose is toxic, toxicity being reversed by glucose [7,8].
e
Estimated from the values of other Nostoc strains.
FEMSLE 8094 31-3-98
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
289
Fig. 2. Growth (A) and glucose uptake (B) of Synechococcus PCC7942 WT and R2-1 cells incubated under the indicated trophic conditions. See Section 2 for details. All cultures of R2-1 contained streptomycin. A: WT, photoautotrophy (R) ; R2-1, photoautotrophy (E),
transferred from photoautotrophy to photoheterotrophy (F, lower line), maintained under photoheterotrophy (F, upper line). B: WT
(R); R2-1 pre-grown under photoautotrophy (E), adapted to photoheterotrophy (F); WT Synechocystis PCC6803 grown under photoautotrophy (+). The growth curves shown represent one typical set of 3^4 repeats.
to transform Synechocystis PCC6803-Frur B5 (via
homologous recombination) for glucose transport
pro¢ciency. It was transferred in parallel into WT
cells of Synechococcus PCC7942. Direct selection
for acquisition of a photoheterotrophic capacity
proved negative, whether streptomycin was present
or not, whilst Smr transformants were recovered at a
frequency of 5U1035 per treated cell. Five of these
transformants were tested and harbored an intact
pUC303-glcP plasmid after sub-culturing under photoautotrophic conditions in the presence of streptomycin. The physiology of one of these clones, R2-1,
was studied.
The photoautotrophic growth rate of R2-1 was
Table 2
Toxicity of fructose and DOG on Synechococcus PCC7942 WT and the glcP-derived transgenic strains
Strain
Synechococcus PCC7942 WT
Synechococcus PCC7942 R2-1
Synechococcus PCC7942 AMC262
Synechocystis PCC6803 WT
Condition
PA
PA
PA
PA
PA
PH
PA
PA
PA
PA
PA
Control
Fructose
DOG
Growth
capacity
Addition
(mM)
Growth capacity
or survival (S)
Addition
(mM)
Growth
capacity
30 ( s 5)
10
50
30 (3)
19 (3)
(3)
(3)
(3)
(3)
1
1
1
10
50
S = 2U1032
S = 4U1034
nd
25 (3)
15 (3)
20
14
4
1
20 (4)
7.5 (4)
26 ( s 5)
(3)
(3)
(3)
(3)
1
S = 1U1034
15
11
3
1
30 ( s 5)
1
2
5
10
nd
nd
1
2
5
10
nd
Cultures growing exponentially under photoautotrophic (PA) or photoheterotrophic (PH) conditions were washed, diluted into mineral
medium containing fructose or DOG at the indicated ¢nal concentrations, and incubated under standard conditions. Growth capacity is
indicated via the factor of increase in OD580 after 4 days. Figures in parentheses indicate number of repeats of assays. Actual survival (S, ratio
of survivors after 3 days of treatment to total viable cells at time 0) is given for 1 mM fructose. Survival of WT Synechocystis PCC6803 after
24 h of a similar treatment is shown for comparison. nd, not determined.
FEMSLE 8094 31-3-98
290
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
identical to or slightly lower than that of the WT
(Fig. 2A). When transferred to photoheterotrophic
conditions, the R2-1 clone showed an adaptation
period of approximately 5 days followed by an exponential growth (Fig. 2A). No such adaptation was
needed for the onset of growth under the reverse
conditions. A maximal photoheterotrophic growth
rate (doubling time 20^30 h) was stably reproduced
if heterotrophic conditions were maintained, whether
streptomycin was present or not. The reproducibility
of the adaptation pattern after several cycles of reverse transfers between photoauto- and photoheterotrophic conditions indicated that the lag did not
correspond to a stabilization of the transforming
plasmid, but must be interpreted as a genuine adaptive process. The possible role of glucose as inducer
was tested by growing a suspension for up to 10
generations in the presence of the sugar and streptomycin before application of the photoheterotrophic
conditions (addition of DCMU). A lag identical to
that of a control culture was always observed before
growth started. The actual inducing trigger is thus
not glucose, but possibly related to the inhibition
by DCMU of electron transfer through Photosystem
II.
The rate of glucose uptake of R2-1 cells grown in
the absence of glucose was above the background
level observed for WT cells, and had increased by
a factor of approximately 1.4 (mean of three determinations) in cells fully adapted to photoheterotrophy (Fig. 2B). The latter cells took up glucose at a
rate about 70% that of Synechocystis PCC6803. The
apparent Km s for glucose uptake of the photoautotrophic versus photoheterotrophic Synechococcus
R2-1 suspensions were slightly higher (0.83 and 2.5
mM, respectively) than their counterparts in Synechocystis PCC6803 (0.58 and 1.66 mM). The Vmax
values in photoheterotrophic R2-1 and in Synechocystis PCC6803 cells (33 and 50 nmoles mg31 protein
min31 , respectively), were close. These ¢gures suggest an e¤cient insertion of the protein in the plasma
membrane of Synechococcus PCC7942. Glucose did
not stimulate uptake in samples of photoautotrophically grown cells. Uptake of OMG, the glucose analogue with the highest a¤nity in Synechocystis
PCC6803 [7], followed kinetics close to those for
glucose, re£ecting the situation in Synechocystis
PCC6803. Fructose showed a lethal e¡ect in Syne-
chococcus R2-1 (Table 2). Its killing e¤ciency was
directly related to the rate of glucose (thus presumably of fructose) transport activity present in the
cells at the time of addition of the sugar. As in the
Synechocystis strains PCC6714 and PCC6803, glucose prevented fructose toxicity, probably through
competition for entry.
The pUC303-glcP plasmid proved unstable in R21. Several months of sub-culturing in the presence of
streptomycin but under photoautotrophic conditions
led to loss of the capacity to grow photoheterotrophically. A similar result was observed when R2-1 cells
were transferred from a photoheterotrophic preculture, thus obligately using glucose as sole carbon
source, and in the presence of streptomycin, to totally non-selective conditions, i.e. photoautotrophy
with no added antibiotic. After maintenance in exponential growth for approximately 10 generations,
by appropriate sub-culturings, the proportion of
streptomycin resistant cells had dropped to 2%. No
modi¢cation of the restriction pattern of the plasmid
present in these cells was observed (data not shown).
However, these plasmids were no longer capable of
transforming the Synechocystis PCC6803-Frur B5
mutant to heterotrophy, or the WT Synechococcus
PCC7942 to streptomycin resistance. The streptomycin resistant phenotype could, in contrast, be acquired by the latter after transformation by total
DNA extracted from these R2-1 subclones, indicating that the resistance was due to a newly acquired
mutation in a chromosomal gene. Cell samples frozen (in 5% DMSO) from suspensions grown either
photoauto- or photoheterotrophically, in the presence of streptomycin, were also unable to resume
heterotrophic growth, though their viability under
autotrophic conditions was unaltered. The plasmid
content of these cells proved variable in number,
size and restriction pattern, suggesting that various
rearrangements had taken place (data not shown).
3.4. Attempts to stabilize glcP in the Synechococcus
PCC7942 genome
Another strategy was applied in order to insure a
better stability of the transgenic glcP gene in the
transformed cells. The 2 kbp fragment was inserted,
next to a Smr /Spr 6 integron, into a `neutral site' of
the chromosome of Synechococcus PCC7942 incor-
FEMSLE 8094 31-3-98
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
porated in plasmid pAMC128 [19]. Stable Spr transformants were recovered and Southern hybridization
with the glcP probe indicated the expected chromosomal insertion inside the neutral site. Glucose exerted a toxic e¡ect on these transformants, suggesting that it was transported. When transferred to a
glucose DCMU containing medium, a subclone,
AMC262, capable to persist, i.e. to remain green
but not to grow, in these conditions could be isolated. This subclone was capable to grow in the presence of glucose in the light, but the sugar did not
improve the growth parameters, and no glucose
transport activity could be detected. The AMC262
transformant and the WT showed similar insensitivities to both fructose and DOG (Table 2). PCR ampli¢cation, using primers encompassing the 860 to
1260 nucleotide region of the ORF, critical for the
activity of the transporter, did not produce any ampli¢ed fragment, in conditions which allowed clear
ampli¢cation from the Synechocystis PCC6803 genomic DNA.
Concomitant acquisition of the glcP gene and of a
photoheterotrophic capacity in the Synechococcus
PCC7942 transformants supports the hypothesis
that the presence of an e¤cient sugar transport is
su¤cient to allow heterotrophy [5], indicating that
(a) complete set(s) of glucose catabolism enzymes
can be synthesized, at least in this strain. The possibility to promote glucose transport by expression of
the glcP gene con¢rms the previous conclusion that
the transporter consists of a single protein [10]. It
appears, however, that this acquired capacity is
hardly compatible with a long-term survival of the
transgenic cells. The genomic location of glcP (on
the chromosome or on a low copy number plasmid)
did not seem critical, since in both cases major modi¢cations of the inserted construction took place in
the surviving clones. Lethality could emerge from a
toxic e¡ect of glucose similar to that of fructose in
Synechocystis PCC6803 and PCC6714. Natural toxicity of fructose is speci¢c to the two Synechocystis
strains, since some heterotrophic cyanobacteria can
actually use this sugar as a carbon source [4]. It may
be linked to the intracellular accumulation of (a derivative of) the sugar to a higher than normal concentration [7,8]. These molecules could act as poisons, disequilibrating normal regulatory processes
(e.g. via feedback inhibition) of metabolic activities.
291
Cyanobacteria have means to control their carbonassimilating capacities when using the usual inorganic carbon source CO2 . Similar regulatory patterns
when an exogenous reduced organic molecule plays
the same role may not be available in obligate photoautotrophic strains. Loss of the sugar transport system(s) might have been selected during evolution as
a means of protection against chance encounter with
such molecules. A non-exclusive hypothesis is that
lethality of GlcP in Synechococcus PCC7942 results
from disorganization of the (cytoplasmic) membrane
due to the presence of this protein.
Acknowledgments
We thank R. Rippka for sending us the WT cyanobacterial strains, and N. Tandeau de Marsac for
providing the pUC303 vector. Construction of the
AMC262 transformant was performed by Mark S.
Nalty and S. Golden (Texas A and M University),
who kindly provided the strain. Sequencing of the
upstream region of glcP was performed at the Texas
A and M Gene Technologies Laboratory.
This work was supported by CNRS, the Universiteè de la Meèditerraneèe in Marseille and the ESBS of
Strasbourg University.
References
[1] Peschek, G.A. (1987) Respiratory electron transport. In: Cyanobacteria (Fay, P. and Van Baalen, C., Eds.), pp. 119^162.
Elsevier Science Publishers, Amsterdam.
[2] Joset-Espardellier, F., Astier, C., Evans, E.H. and Carr, N.G.
(1978) Cyanobacteria grown under photoautotrophic, photoheterotrophic, and heterotrophic regimes : sugar metabolism
and carbon dioxide ¢xation. FEMS Microbiol. Lett. 4, 261^
264.
[3] Smith, A.J. (1982) Modes of cyanobacterial carbon metabolism. In: The Biology of Cyanobacteria (Carr, N.G. and
Whitton, B.A., Eds.), pp. 47^86. Blackwell Scienti¢c Publications, Oxford.
[4] Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M. and
Stanier, R.Y. (1979) Generic assignments, strain histories and
properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1^61.
[5] Beauclerk, A.A.D. and Smith, A.J. (1978) Transport of Dglucose and 3-O-methyl-D-glucose in the cyanobacteria Aphanocapsa 6714 and Nostoc strain MAC. Eur. J. Biochem. 82,
187^197.
FEMSLE 8094 31-3-98
292
C.-C. Zhang et al. / FEMS Microbiology Letters 161 (1998) 285^292
[6] Raboy, B. and Padan, E. (1978) Active transport of glucose
and K-methylglucoside in the cyanobacterium Plectonema
boryanum. J. Biol. Chem. 253, 3287^3291.
[7] Joset, F., Buchou, T., Zhang, C.C. and Jeanjean, R. (1988)
Physiological and genetic analysis of the glucose-fructose permeation system in two Synechocystis species. Arch. Microbiol.
149, 417^421.
[8] Flores, E. and Schmetterer, G. (1986) Interaction of fructose
with the glucose permease of the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 166, 693^696.
[9] More, J.E., Williams, M.M. and Smith, A.J. (1979) Features
of organic growth substrate utilization by cyanobacteria. In:
IIIrd International Symposium on Photosynthetic Prokaryotes, Dundee, p. 49.
[10] Zhang, C.C., Durand, M.C., Jeanjean, R. and Joset, F. (1989)
Molecular and genetic analysis of the fructose-glucose transport system in the cyanobacterium Synechocystis PCC6803.
Mol. Microbiol. 3, 1221^1229.
[11] Schmetterer, G. (1990) Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis
PCC6803 and mammalian glucose transporters. Plant Mol.
Biol. 4, 697^706.
[12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
[13] Kuhlemeier, C.J. and Van Arkel, G.A. (1987) Host-vector
systems for gene cloning in cyanobacteria. Methods Enzymol.
153, 199^215.
[14] Kallas, T. and Malkin, R. (1988) Isolation and characterization of genes for cytochrome b6/f complex. Methods Enzymol.
167, 779^794.
[15] Herdman, M., Janvier, M., Rippka, R. and Stanier, R.Y.
(1979) Genome size of cyanobacteria. J. Gen. Microbiol.
111, 73^85.
[16] Wilmotte, A.M.R. and Stam, W.T. (1984) Genetic relationship among cyanobacterial strains originally designated as
Anacystis nidulans and some Synechococcus strains. J. Gen.
Microbiol. 130, 2737^2740.
[17] Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E.,
Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M.,
Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S.,
Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda,
M. and Tabata, S. (1996) Sequence analysis of the genome of
the unicellular cyanobacterium Synechocystis sp. strain
PCC6803. II. Sequence determination of the entire genome
and assignment of potential protein-coding regions. DNA
Res. 3, 109^136.
[18] Raboy, B., Padan, E. and Shilo, M. (1976) Heterotrophic
capacities of Plectonema boryanum. Arch. Microbiol. 110,
77^85.
[19] Bustos, S.A. and Golden, S.S. (1991) Expression of the psbDII
gene in Synechococcus PCC7942 requires sequences downstream of the transcription start site. J. Bacteriol. 173, 7525^
7533.
FEMSLE 8094 31-3-98