Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment

Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution).


Introduction
In response to the constant increase in energy consumption and resulting pollution by the growing and developping world population, it is important to develop new energy sources that are plentiful, renewable and environmentally friendly. Thus, the solar driven production of hydrogen (H 2 ) is of special interest for many reasons. The annual solar flux received by Earth In addition to the level of production per se, one potential approach to limit the cost of H 2 production is to couple it with the treatment of wastewaters, which are often polluted with urea originating from mammalian wastes or its use as an agricultural fertilizer [17]. Most organisms that can use urea (CO(NH 2 ) 2 ) as a nitrogen source employ a nickel-dependent metalloenzyme called urease (EC 3.5.1.5, also named urea amidohydrolase) to catalyze the ATP-independent hydrolysis of urea into ammonia (NH 3 ) and carbon dioxide (CO 2 ) [18]. Furthermore, the urease activity consumes no reducing power, unlike the nitrate reductase enzyme. Hence, these spare electrons can be used for H 2 production driven by the HoxEFUYH enzyme. In most bacteria and cyanobacteria, urease is a trimer of three subunits (UreA, UreB and UreC) that requires up to four (accessory) chaperone proteins (UreD, UreE, UreF and UreG) for activation and incorporation of two nickel atoms into its metallocenter active site [18]. In this study, we tested the influence of urea on the production of biomass and hydrogenase activity in wild-type and hydrogenase overproducing strains. We show here that (i) decreasing the activity of urease is beneficial for cell growth on urea as the sole nitrogen source, and (ii) the activity of hydrogenase was similar in cell growing on urea or nitrate. These novel results and strains have important implication for the future production of H 2 from solar energy, CO 2 and urea-polluted waters.

Bacterial strains and growth conditions
Synechocystis PCC6803 (Synechocystis) was grown at 30˚C under continuous white light (2,500 lux; 31.25 μE m -2 s -1 ) on the mineral medium BG11 [19] enriched with 3.78 mM Na 2 CO 3 [20] hereafter designated as MM. For some experiments MM was supplemented with 1-2.5 μM NiSO 4 and/or 17 μM Fe (provided as green ferric ammonium citrate) and/or nitrate was replaced by urea (5 mM) and/or ammonium chloride (1-20 mM) as indicated. In these latter cases nitrate-grown cells were washed twice with sterile water before resuspension in ureaand/or ammonium-containing media).

Gene cloning and manipulation
Synechocystis DNA was PCR amplified with specific primers (S2 Table) to generate the studied genes that were either overexpressed or eliminated. For gene overexpression experiments, the protein coding sequences were cloned downstream of the strong λp R promoter and associated ribosome binding site of our replicative plasmid vector derived from the broad-host-range plasmid RSF1010 [13]. The resulting plasmids were introduced by conjugation in Synechocystis [12]. For gene deletion, the genomic regions encompassing each studied gene were PCR amplified, or synthezised (Eurofins), prior to replacement by an antibiotic resistance marker for selection. The resulting deletion cassettes (S1 Table) were introduced by transformation into Synechocystis [21] where homologous DNA recombination between the deletion cassette and the recipient chromosome integrated the antibiotic resistant marker in place of the studied gene.
All DNA constructions were verified through PCR and DNA sequencing (Big Dye kit, ABI Perkin Elmer, or Eurofins), before and after propagation in Synechocystis. PCR was also used to test whether the segregation between the mutant (antibiotic resistant) and WT copies of the polyploïd chromosome (about 10 per cell, [21] was complete (the studied gene is dispensable to cell growth) or not (the gene is essential to cell viability).

RNA isolation and analysis by quantitative RT-PCR
Exponentially growing cells were rapidly harvested and broken (Eaton press), prior to RNA purification and analysis by quantitative RT-PCR using primers (S2 Table) that generated DNA fragments of similar size (163-234 bp), as previously described [6,14]. Each assay was performed in triplicate to allow the calculation of the mean threshold cycle value (C T ) for each studied gene, which was converted to gene copy number per ng of template cDNA.

Western blot analysis of the HoxF and HoxH proteins
40 μg of Synechocystis proteins were separated, transferred to nitrocellulose membrane (Invitrogen) and immunodetected with rabbit anti-HoxF and anti-HoxH antibodies, and the R800 goat anti-rabbit IgG secondary antibodies, as previously described [14].

Hydrogenase activities
They were measured by the standard amperometric method in an anaerobic glove box equipped with an inverted Clark-type electrode (Hansatech, UK), using Na-dithionite (20 mM, reducing agent) and methylviologen (5 mM, electron donor) as previously described [6,14].

Urease activities
They were monitored by the release of ammonia from urea [22]. Cells collected by centrifugation (5,000 rpm, 10 min, 20˚C) were resuspended in 400-1000 μL of buffer (PBS 1X pH 7.5, EDTA 10 mM, Protease Inhibitor Cocktail, Roche) and disrupted in a pre-cooled Eaton press, as described [14]. 10 μg of total proteins (measured with the Bradford protein assay, Biorad) were mixed with 90 μL of PBS EDTA buffer lacking (reaction blank) or containing (samples) 50 mM urea and then incubated for 10 min at 30˚C. Tubes were transferred on ice before the addition of 150 μL of phenol nitroprusside (Sigma-Aldrich), 150 μL of alkaline hypochlorite (Sigma-Aldrich) and 700 μL H 2 O. Chromophore formation was achieved at 37˚C for 10 min before measuring absorbance at 570 nm. After substracting the A 570 values of blank reactions, the ammonia concentration of the samples was determined from a standard curve constructed with varying NH 4 Cl concentrations.

In silico modeling and docking simulations
Each amino acids (AA) sequence was modelled with the intensive search mode of Phyre2 [23]. Poorly modelled AA residues or regions were manually removed with Swiss PDB Viewer. For each structure, a Ramachandran plot was generated using Chimera to verify that less than 5% of residues were modelled with conflicting sidechain orientations. Reliability of models was further evaluated using ModFOLD, ModEval and Qmean servers [24][25][26]. Multimeric complexes were obtained with Chimera by superposition of finihed models to published oligomeric structures.

Construction and analysis of a mutant for high level expression of all hoxEFUYHW genes from the strong lambda-phage p R promoter
In our previous attempt to engineer Synechocystis for high-level photoproduction of H 2 , we increased the expression of the hoxEFUYH operon by replacing its weak natural promoter [6] by the strong λp R (lambda-phage) promoter [27], thereby yielding the CE-hoxEFUYH mutant also called CE1 (CE for constitutive strong expression) [14] and this work (Fig 1). The CE1 mutant over-producing the hoxEFUYH transcripts (at least 100-fold) and HoxEFUYH proteins exhibited only a small increase in hydrogenase activity as compared to wild-type (WT) cells [14] and this work (Fig 2). We also found that the hydrogenase activity of both WT and CE1 cells could be increased by growing the cells in the mineral medium appropriately supplemented with extra iron (17 μM) and nickel (2.5 μM) metals required for the [NiFe] hydrogenase cluster. Furthermore, the gains conferred by the genetic engineering and growth-medium improvement could be cumulative as the hydrogenase activity of the CE1 mutant grow in the improved medium was about 10-fold higher than hydrogenase activity of WT cells grow in the standard medium cells [14] and this work (Fig 2). However, this increase in hydrogenase activity was modest in comparison to the large increase in abundance of the HoxEFUYH transcripts and proteins in the CE1 mutant (see [14] and this work (Fig 2)). Thus, we hypothesized that the natural level of the HoxW protease needed for the proteolytic maturation of the natural quantity of HoxH, might be limiting in CE1 cells that overproduce the HoxEFUYH proteins. This assumption is consistent with the finding that several cyanobacteria (Synechococcus PCC7335, Halothece PCC741 and Leptolyngbya PCC7375) harbor several copies (2-3) of hoxW, while they maintain hoxEFUYH as single copy genes [3]. To further increase the level of hydrogenase activity of our CE1 mutant we cloned the hoxW protein-coding-sequence, downstream of the chromosomal hoxEFUYH operon expressed from the strong [27] λp R promoter. To achieve this, a hoxW-Gm r DNA cassette was constructed by PCR in such a way that it was flanked by appropriate Synechocystis DNA sequences to serve as regions of homology for DNA recombinations promoting the introduction of the hoxW-Gm r cassette downstream of hoxH (S1 Fig). The resulting hoxW-Gm r cassette was verified by PCR (S2 Fig) and nucleotide sequencing, and subsequently transformed [21] to CE1 cells (Km r ), yielding Km r -Gm r clones. This Km r -Gm r mutant grew as well as the WT strain in standard photoautotrophic conditions (Fig 2). This mutant was analyzed by PCR (S2 Fig) in order to verify that the hoxW protein-coding-sequence was successfully integrated downstream of the hoxH gene of the hoxEFUYH operon, generating the hoxEFUYHW operon, as expected. Furthermore, we found that all copies of the Synechocystis polyploid chromosome [21], harbor the hoxEFUYHW operon. This mutant was designated as CE4 (constitutive strong expression of the hoxEFUYHW genes, Fig 1). We also verified by PCR that CE4 cells possessed only CE4-type (hoxEFUYHW-Gm r ) chromosome copies, even after growth in the absence of Gm to allow the possible re-establishment of any remaining CE1 (Gm s ) chromosome copies (S2 Fig). We then verified, using quantitative RT-PCR, that the CE4 strain produced similar high levels of hoxEFUYH transcripts as the CE1 mutant, and more abundant (about 20-fold) hoxW mRNA than WT and CE1 cells (Fig 2). Collectively, these data demonstrate that the overexpression of the hoxEFUYHW genes is not detrimental to cell viability, in agreement with the absence of negative physiological effects of the overexpression of the hoxEFUYH genes, on one hand (CE1 cells [14] and (Fig 2)), and hoxW on the other hand (Fig 2). Indeed, we also cloned hoxW just behind the strong [27] λp R promoter of the plasmid vector for maximal expression (pFC1ΔcI 857 , S1 Table), and found that the resulting cells grew very well (data not shown).
As expected, the hydrogenase activity of CE4 cells was higher than those of CE1 and WT cells, in that order (Fig 2), irrespectively of the use of the standard MM medium or the improved medium supplemented with Fe (17 μM) and Ni (2.5 μM) metals (the Hox hydrogenase uses a [NiFe] redox center). The higher hydrogenase activity of CE4 (overexpression of hoxEFUYHW), as compared to CE1 (overexpression of hoxEFUYH), was consistent with the positive role of HoxW on hydrogenase activity (proteolytic maturation of the HoxH subunit [9]). This finding indicates that the increased expression of hoxW in CE4 was necessary to yield a large quantity of mature HoxH, and by extension the natural (WT) level of HoxW in CE1 cells was insufficient to cleave the large amount of HoxH to maturity. This interpretation was also supported by the western blot analysis of the HoxF and HoxH proteins (Fig 2). In WT cells, the very faint HoxF and HoxH signals were consistent with the low abundance of these proteins in Synechocystis seen with Western blots and quantitative proteomics [14]. Furthermore, in CE1 cells (Fig 2), the more abundant bands on Western blots of HoxF and HoxH (mostly the large HoxH form not yet cleaved by the low natural quantity of HoxW) also confirmed our previous report [14]. Collectively, the present data (Fig 2) show that CE4 cells (overexpression of hoxEFUYHW) produce similar high quantities of HoxF and HoxH as CE1 (overexpression of hoxEFUYH). The thicker HoxH signal observed in CE4, as compared to CE1, is likely due to the increased abundance of the small form of HoxH cleaved by the increased level of HoxW.

Construction and analysis of a mutant for strong constitutive expression of all hoxEFUYHW and hypABCDEF genes in Synechocystis
In a previous study, we showed that the gain in hydrogenase activity conferred by the overproduction of the hoxEFUYH proteins can be further increased by the simultaneous overproduction of the HypABCDEF proteins directed by the (Sm r /Sp r ) plasmid pCE-hypABCDEF [14]. This pCE-hypABCDEF vector, derived from the promiscuous plasmid RSF1010 that replicates autonomously to the same 10-20 copies per cell as the chromosome [13], strongly express the hypABCDEF gene from the very-active λp R promoter [14]. Consequently, we decided to simultaneously overproduce all HoxEFUYHW proteins and all HypABCDEF proteins. To accomplish this, we introduced the Sm r /Sp r pCE-hypABCDEF plasmid by conjugation [12] into the Km r -Gm r CE-hoxEFUYHW (CE4) mutant (Fig 1). This yielded the CE-hoxEFUYHW-hypABCDEF mutant (Fig 1) designated as CE5 (note that CE5 cells also carry the weakly expressed WT alleles of hoxW and hypABCDEF in their chromosome). CE5 cells grew as well as WT cells (data not shown) and CE4 cells in standard photoautotrophic conditions (Fig 2). As expected, the CE5 strain strongly expressed the hoxEFUYHW and hypABCDEF genes ( Fig  2) and its hydrogenase activity was higher than that of the CE4, CE1 and WT strains, in that order (Fig 2). Altogether, the constitutive overexpression of the hoxEFUYHW and hypABC-DEF genes, and the increased Ni-and Fe-availabilities led to the strongest increase in active hydrogenase (25-fold) as compared to WT cells cultivated in normal medium (Fig 2).

Influence of urea and urease activity on cell growth and hydrogenase activity
As a step towards the future development of an economic cyanobacterial process for the photoproduction of H 2 coupled with wastewater treatment, we have tested the influence of urea (a frequent water pollutant) on the biomass production and hydrogenase activity of our strains. Therefore, Synechocystis WT was incubated for increasing periods of time in liquid mineral medium containing urea as the sole nitrogen source, and nickel (both urease and hydrogenase require Ni). WT cells grew well on urea up to 5 mM, whereas higher urea concentrations reduced the duration of healthy growth and production of biomass (Fig 3). We then tested the influence of 5 mM urea on the growth and hydrogenase activity of the WT and CE1-CE5 strains, and the previously constructed delta hoxEFUYH mutant [14]. All these strains grew well and displayed the normal blue green color for more than 7 days (Fig 3). During this time, the hydrogenase activity of the WT and CE5 strains did not vary (data not shown).
After 14 days of cultivation on urea (5 mM, sole N source) and Ni (2.5 μM) cells reaching the stationary phase of growth (OD 750 > 5.0) became invariably yellowish (Fig 3). Once the chlorosis process became visibly detectable, a large decrease in cell viability was observed by plating assay on solid standard growth medium (it contains nitrate, not urea).
A similar urea-induced chlorosis was observed in the cyanobacteria Anabaena cylindrica and Synechococcus PCC7002, distantly related to Synechocystis PCC6803, when they were cultivated on urea as the sole nitrogen source [28,29]. The urea-mediated chlorosis and cell death were found not to occur in a urease defective mutant harboring an inactivated allele of ureC, the gene normally encoding the large urease subunit. To test whether the same is true in Synechocystis, we inactivated its ureC gene by replacing its first 971 bp by the Km r marker gene (S3 Fig), yielding the λureC::Km r DNA cassette. Following transformation to Synechocystis [21], Km r clones growing in standard medium (i.e. on nitrate as the sole nitrogen source) supplemented with Km were analyzed by PCR to verify that all chromosome copies harbor the ΔureC::Km r cassette in place of ureC (S3 Fig). The complete absence of WT (ureC + ) chromosome copies in the ΔureC::Km r mutant was confirmed by analyzing cells that were subsequently grown in absence of Km to allow a possible re-invasion of ureC + chromosome copies if any remained. We also verified that the ΔureC::Km r mutant had lost the urease activity and the capability to grow on urea as the sole nitrogen source (data not shown). These results demonstrate that the Synechocystis ureC gene is essential for urease activity and cell growth on urea as the sole nitrogen source, as observed in the distantly related cyanobacteria Synechococcus PCC7002 [29] and Synechococcus PCCWH7805 [30].
During our study, we found one mutant clone of each hydrogenase overproducing strains CE4 and CE5 that grew stably on urea as the sole N source and retained the normal blue-green color. These clones escaping the urea-promoted chlorosis (Fig 3) were named CE4u and CE5u (u for withstanding urea as the sole nitrogen source). Interestingly, the CE4u and CE5u strains retained their high hydrogenase activity (Fig 2). Knowing that the urea-mediated chlorosis depends on an active urease (see above), we confirmed that the urease in the CE4u and CE5u strains were less effective than the enzyme in the WT, CE4 and CE5 strains (Fig 2). To identify the mutation in CE4u and CE5u responsible for their low urease activity, we used specific oligonucleotide primers for PCR amplification and nucleotide sequencing of all seven urease genes (ureABCDEFG) including their upstream and downstream regions (about 200 bp in each case). We also used qRT-PCR to monitor and compare the expression of the ureABCDEFG genes in CE4u and CE5u (urea-tolerant) and WT (urea-sensitive) strains. The results showed that the expression of ureABCDEFG was not affected in CE4u and CE5u (S4 Fig and data not shown). Interestingly, both CE4u and CE5u carried the same (single) mutation, a C to T transition at position 254 of the ureG coding sequence, which substituted the alanine amino-acid residue at position 85 by a valine residue (mutation A85V; S4 Fig). To confirm that the low urease activity of CE4u is due to the A85V mutation in ureG, we performed a plasmid complementation test. We cloned the WT ureG protein-coding-sequence in the replicative plasmid vector for strong constitutive gene expression (S5 Fig). The resulting plasmid pCE-ureG was introduced by conjugation in the CE4u strain, which harbors the ureG A85V gene in its chromosome, and in the WT, CE1 and CE4 control strains, which have ureG in their chromosome. As expected, in the CE4u/pCE-ureG test strain, the production of the wild-type UreG protein directed by the pCE-ureG plasmid increased the urease activity (3-fold) well above the level observed in the CE4u strain producing only the UreG A85V mutant protein (S4 Fig). In contrast, the control strains WT/pCE-ureG, CE1/pCE-ureG and CE4/pCE-ureG, which produced UreG from both their chromosome and their pCE-ureG plasmid, exhibited no increase in urease activity when compared to their plasmid-free parental strains WT, CE1 and CE4, which produced UreG only from their chromosome. It appeared that the stronger level of ureG expression driven by both the plasmid and the chromosome slightly decreased the overall urease activity for an unknown reason (S4 Fig; compare WT/pCE-ureG with WT).
The alanine 85 of UreG belongs to an α-helix situated near the Ni-binding site of UreG, which physically interacts with the groove formed by the UreF dimer (S4 Fig). It is thus possible that the steric hindrance generated by the A85V mutation in ureG somehow impaired the UreG-UreF physical interaction, thereby decreasing the incorporation of Ni atoms in the urease active site. Collectively, these findings showed that UreG plays a crucial role on urease activity of Synechocystis as observed in the evolutionary distant cyanobacterium Anabaena PCC7120 [Valladares, 2002 #108]. Interestingly, a similar alanine to valine mutation (A142V) near the Ni-binding of the soybean UreG (A142 corresponds to G64 in the Synechocystis protein) was also shown to decrease urease activity [31].
Finally, we found that hydrogenase activity is not affected by the decline in urease activity (Fig 2) that allows cell to grow stably on media containing urea as the sole nitrogen source.

Discussion
We showed here that the simultaneous overproduction of all HoxEFUYHW and HypABCDEF proteins involved in the synthesis, maturation and assembly of the [NiFe] hydrogenase complex, combined with media improvement, led to a strong (about 25-fold) increase in the level of active hydrogenase. We also showed the counterintuitive notion that lowering the activity of the Synechocystis urease can improve the photoproduction of biomass, and in turn H 2 , from urea-polluted waters. The sophisticated strains constructed during this work displayed a higher increase in expression of the hoxEFUYH and hypABCDEF genes than that of hydrogenase activity. This finding indicates that limiting post-transcriptional factors need to be dealt with in order to engineer a powerful H 2 producer of industrial importance. We think that the presently described strain with a healthy growth and an increased abundance of active hydrogenase is a suitable starting point for this important objective. Indeed, this hydrogenase overproducing strain should facilitate the purification and structural analysis of hydrogenase (it structure is as yet unknown), which, in turn, should facilitate the design of meaningfull strategies to increase its (low) tolerance to O 2 that is massively produced by photosynthesis. Furthermore, recent in vitro data suggested that the hydrogenase enzyme could receive electrons not only from NAD(P)H but also the well conserved electron transfer proteins [32] ferredoxins [8]. Thus, it will be interesting to try to further increase the hydrogenase activity of our highhydrogenase level strain by overproducing each of the nine Synechocystis ferredoxins [32] along with its HoxEFUYHW and HypABCDEF proteins. It will also be important to examine the redox state of the cysteine amino-acids of the HoxH and HoxF subunits, since it has been shown that they can be oxidized [33,34], a finding that should stimulate the analysis of crosstalks between hydrogen production and redox (oxidative) stress [1].
Towards a future objective of reducing the cost of H 2 photoproduction by combining it with the treatment of water, we chose to test the influence of urea, a frequent pollutant [17], on Synechocystis growth (production of biomass) and hydrogenase activity. We found that Synechocystis coud grow for several days on urea (5 mM) as the sole nitrogen source, but invariably cells turned yellow and died upon longer incubation times (for example in 14 days, Fig 3). Furthermore, cells were killed faster by higher urea concentration (7.5-20 mM; the higher the concentration the faster was cell death; Fig 3). Similar findings were observed in the cyanobacteria Anabaena cylindrica and Synechococcus PCC7002 [28,29] phylogenetically-distant from Synechocystis. Urea-stressed Synechococcus PCC7002 cells were shown to contain high levels of lipid peroxides. Furthermore, exogenously added polyunsaturated fatty acids triggered a similar death response, while vitamin E suppressed the formation of peroxides and delayed the onset of chlorosis and cell death. These results suggest that cyanobacterial cells grown on urea for several days undergo a metabolic imbalance that ultimately leads to oxidative stress and lipid peroxidation. As observed in Synechococcus PCC7002 [29] we found that that the inactivation (deletion in our study) of the Synechocystis ureC gene, normally encoding a catalytic subunits of urease, impaired urease activity, as well as cell growth on urea and subsequent urea-induced chlorosis and cell death (Fig 3). We also found that lowering the activity of the urease enzyme (for example with the UreG A85V mutation) can sustain Synechocystis growth on media containing urea as the sole nitrogen source. Furthermore, we found that the urea tolerance mutation had no detrimental effect on hydrogenase activity. The urea-tolerant hydrogenase-overproduction strain displayed the same hydrogenase activity level in both media containing either nitrate or urea as the sole nitrogen sources. Altogether the present findings imply that it should be possible to reduce the cost of hydrogen production by combining it with water treatment (urea depollution) in the future.

Conclusion
The presently reported cyanobacterial factories with a large hydrogenase activity and healthy growth on urea will be very useful for the purification of large hydrogenase quantities for biochemical and structural analyses, in order to better understand this important enzyme and improve its (weak) tolerance to O 2 . Such work has interesting implications for future economically viable industrial production of H 2 from solar energy, CO 2 and urea-polluted waters.  Table) [13]was digested by PsiI cand religated to inactivate the temperaturedependent λcI 857 repressor gene, which normally controls the strong λp R promoter (red triangle), yielding pFC1ΔcI 857 (also called pCE, CE for constitutive expression). Meanwhile, ureG was amplified with oligonucleotides primers that introduced a NdeI site (embedding its ATG start codon) and an EcoRI site (behind its stop codon). After NdeI/EcoRI double digestion ureG was cloned between the NdeI and EcoRI sites of pCE, yielding the pCE-ureG plasmid. (B) UV-light image of the agarose gel showing the PCR products typical of the pCE-ureG plasmid replicating in the WT, CE1, CE4 and CE4u strains. Marker (M) = GeneRuler™ 1Kb plus DNA Ladder (Fermentas). The lane noted H 2 O served as a negative control (no DNA template) while those noted pFC1ΔcI 857 and pCE-ureG served as a positive control of the presence of the corresponding plasmid in the studied Synechocystis strains (two clones analyzed in every case). (TIFF) S1 Table. Characteristics of the plasmids used in this study. CS, Protein Coding Sequence; Δ, deletion; TT, transcriptional terminator. (DOCX) S2 Table. List of the PCR primers used in this study. The restriction sites are written in bold letters; CS, coding sequence; RBS, ribosome binding site. Fw and Rv in the primers names stand for "forward" and "reverse", respectively. (DOCX)