Growth conditions and heterocytes induction
Cyanobacterial strains were grown in BG11 medium (nitrate replete) at 30 °C under continuous illumination (30 µE m-2s-1). Cultures of recombinant strains were supplemented with neomycin (50 μg mL−1). Heterocyte formation was induced by transferring the exponentially growing cultures (OD 750 = 0.8) to BG110 (BG11 devoid of sodium nitrate) by filtration (0.2 µm pore size filters, Sigma) and resuspension of cells into the nitrate-free medium. The growth was maintained for 4 days. The presence of heterocytes was confirmed by light microscopy and their distribution within filaments was rated visually by counting the number of vegetative cells between two heterocytes. At least 400 total vegetative cells were counted for each strain.
In the H2 production experiments, the strains were grown under continuous illumination of 20 µE m-2s-1 or 60 µE m-2s-1 .
Construction of plasmids and strains
To construct the Flv3B overproducing strain, the promoter region of patB (all2512, 500 bp upstream the start codon) was amplified by PCR from Nostoc sp. PCC 7120 genomic DNA using the ppatB forward and ppatB reverse primers (Table 3). The ppatB reverse primer contained a multiple cloning site (ApaI, ClaI, BamHI, SalI, ScaI, EcoRI). The amplified promoter was cloned into BglII and EcoRI restriction sites of the pRL25T plasmid [26], yielding the pRL25T-patB plasmid. The open reading frame of flv3B gene was amplified using the flv3B forward and reverse primers (Table3), and cloned into the ApaI and ScaI restriction sites of the pRLpatB. The recombinant plasmid (pRL25T-patB-flv3B) was analyzed by sequencing (Millegen). Conjugation of Nostoc was performed as described in reference [27]. Briefly, E. coli strains (bearing the replicative pRL25T-patB-flv3B and the RP-4 conjugative plasmid) grown to exponential growth phase, were mixed to an exponentially grown Nostoc culture. The mixture was plated on BG11 plates and Neomycin was added 24 hours later for plasmid selection. Plasmid extraction was used to analyze the obtained recombinant clones.
Deletion of the hupL gene, yielding the DhetL strain, was obtained by homologous recombination replacing the hupL3’ gene (all0687C) with the gene encoding the spectinomycin/streptomycin resistance (Sp/Sm cassette hereafter). For this purpose, the upstream and downstream 1500 bp flanking the hupL3’ gene were amplified form Nostoc genomic DNA using the all0678 forward/ all0678 reverse and the Strp-all0678 forward/ Strp-all0678 forward, respectively; The Sp/Sm cassette was amplified using the Strp forward/Strp reverse primers (Table 3), using the pBAD42 plasmid (Addgen) as template. Gibson’s assembly technique (New-England Biolabs) was applied to insert the three resulting fragments into the suicide pRL271 vector linearized by SpeI. The resulting recombinant plasmid was conjugated into Nostoc as described above. The initial conjugants were selected by screening for resistance to 5 μg/mL of Sm, and the resulting cells were then grown on BG11 plates containing 5% sucrose to select double recombinants. Genomic DNA of the recombinant cells were analyzed by PCR.
The strains and plasmids used in this study are listed in Table 2.
RNA Preparation and Reverse Transcription
RNAs were prepared using the Qiagen RNA extraction kit (Qiagen) following the manufacturer instructions. An extra TURBO DNase (Invitrogen) digestion step was undergone to eliminate the contaminating DNA. The RNA quality was assessed by tape station system (Agilent). RNAs were quantified spectrophotometrically at 260 nm (NanoDrop 1000; Thermo Fisher Scientific). For cDNA synthesis, 1 µg total RNA and 0.5 μg random primers (Promega) were used with the GoScript™ Reverse transcriptase (Promega) according to the manufacturer instructions.
Quantitative Real-Time-PCR for Transcriptional Analyses
Quantitative real-time PCR (qPCR) analyses were performed on a CFX96 Real-Time System (Bio-Rad). The reaction volume was 15 μL and the final concentration of each primer was 0.5 μM. The qPCR cycling parameters were 95°C for 2 min, followed by 45 cycles of 95°C for 5 s, 55°C for 60 s. A final melting curve from 65°C to 95°C was added to determine the specificity of the amplification. To determine the amplification kinetics of each product, the fluorescence derived from the incorporation of BRYT Green® Dye into the double-stranded PCR products was measured at the end of each cycle using the GoTaq® qPCR Master Mix 2X Kit (Promega). The results were analysed using Bio-Rad CFX Maestro software, version 1.1 (Bio-Rad, France). The rnpB gene was used as a reference for normalization. A technical duplicate was performed for each point. The amplification efficiencies of each primer pairs were 80 to 100%. All of the primer pairs used for qPCR are reported in Table 3.
Western blot analysis
Proteins (75 µg) extracted from cyanobacterial strains were fractionated by performing SDS-PAGE 12%, and transferred to nitrocellulose membranes before being revealed with specific polyclonal antibodies. Immune complexes were detected with anti-rabbit peroxidase-conjugated secondary antibodies (Promega) and enhanced chemoluminescence reagents (Pierce). Anti-FlvB antibodies, developed against the FlvB protein of C. reinhardtii [18], were used at a 1: 1000 dilution. Anti-Rbcl antibodies (Agrisera) were used a 1: 5000 dilution.
H2 production assays
Nostoc wild type strain and its derivatives were grown as described above for heterocyte induction. Chlorophyll a concentration was quantified according to the following method: 1 mL of culture was centrifuged (5 min, 6700 g, 4°C), the pellet was resuspended in 1 mL of cold methanol and incubated at 4°C for 30 minutes under shaking. Cells were then harvested (5 min, 6700 g, 4°C) and absorbance of the supernatant was measured at 665 nm and 720 nm. The chlorophyll a concentration was calculated according to the formula: [Chl a] = 12,9447 (A665-A720) and expressed in µg of Chla/mL of culture [28]. A 40-mL volume of cell culture was then harvested (5 min, 6700 g, 4°C) and cells were resuspended in sterile nitrate-depleted medium yielding a concentration of 10 μg Chla mL−1. 12 mL of this cell suspension were transferred to Hungate tubes (leaving a 4.4-mL head space volume). The vials were sparged with Argon (Ar), and the samples were maintained under illumination (20 or 60 μmol photons m−2 s−1) for 96 hours. 100 μL of headspace gas was removed every 12 hours using a gastight syringe and injected into a gas chromatography system (Agilent 7820) equipped with a thermal conductivity detector and a HP-plot Molesieve capillary column (30 m, 0.53 mm, 25 µm), using argon as the carrier gas, at a flow rate of 4.2 mL/min, an oven temperature of 30 °C and a detector temperature of 150 °C. H2 was quantified according to a standard calibration curve. H2 production rate was expressed as mol of H2 produced per mg of Chlorophyll.
Nitrogenase activity
An on-line acetylene reduction assay [29] was used to measure nitrogenase activity. Briefly, cyanobacterial strains were grown in batch cultures under light/dark cycles of 12 hours/12 hours. Nitrogenase activity was monitored for 20 hours. Before the onset of nitrogenase activity, Nostoc cultures were transferred to a GF/F filter (Whatman, 47 mm) and placed in a custom-made, light and temperature-controlled gas flow-through incubator connected to the gas chromatograph. Acetylene represented 10% of the gas mixture and the total gas flow rate was 1 l h-1. Ethylene production was measured every 10 min by gas chromatography using an Agilent 7890 equipped with an auto-injector and a photoionization detector.