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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