Analysis of the hupSL Operon of the Nonheterocystous Cyanobacterium Lyngbya majuscula CCAP 1446/4: Regulation of Transcription and Expression under a Light-Dark Regimen

Organism and growth conditions.The filamentous nonheterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4 (sourced from the Culture Collection of Algae and Protozoa, Scotland, United Kingdom) was maintained in ASW:BG medium (1) and grown in either BG11 or BG11₀ (BG11 in the absence of nitrate) (52) at 25°C, on a 12-h light (7 μmol photons m⁻² s⁻¹)/12-h dark regime. For the hydrogen uptake activity studies (see below), a defined medium was required. Since Lyngbya majuscula CCAP 1446/4 is known to be able to grow within both marine and freshwater media (7), both BG11 and BG11 supplemented with 10 g liter⁻¹ NaCl and 1 μg liter⁻¹ vitamin B₁₂ were tested. Unexpectedly, Lyngbya majuscula CCAP 1446/4 exhibited better growth in sodium-free medium; therefore, BG11 and BG11₀ were selected for further studies.

Hydrogen uptake activity.In vivo hydrogen uptake was measured using a DW1 O₂/H₂ electrode (Hansatech, Ltd., United Kingdom) according to the methods described previously (46). The filaments were transferred from BG11 to BG11₀ for adaptation 1 week prior to the experiment and subcultured into fresh medium every day for 3 days before the start of the experiment. Due to the mode of growth of Lyngbya majuscula CCAP 1446/4 (thick cohesive mats), the filaments were cut into pieces, one for each time point. H₂ uptake activity was assayed every third hour during a complete light/dark cycle (24 h). The entire experiment was repeated at least three times, showing a H₂ uptake pattern within 10% error fluctuations.

Chlorophyll a content.The total chlorophyll a content was determined by extracting the cyanobacterial cells in 90% (vol/vol) methanol, measuring the absorbance at 663 nm, and using the equation micrograms of chlorophyll a per milliliter = 12.7 × A₆₆₃, as described by Meeks and Castenholz (36).

Isolation of DNA, PCR, agarose gel electrophoresis, and DNA recovery.Genomic DNA was extracted, and PCR was carried out in the thermal cycler GeneAmp PCR system 2400 (Perkin-Elmer, Inc., Wellesley, MA) according to the methods described previously (55). Initially, primers designed against conserved regions within hydrogenase-related genes from Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413, and Nostoc punctiforme PCC 73102 were used. Furthermore, once preliminary sequences were analyzed, Lyngbya majuscula CCAP 1446/4-specific primers (LM−) were synthesized to obtain contiguous sequences. The oligonucleotides used in this study are listed in Table 1. Agarose gel electrophoresis was performed by standard protocols using either 1× TAE or 1× TBE buffer (49). DNA fragments were isolated from agarose gels using the QIAEX II Gel Extraction kit (Qiagen, Hilden, Germany) or the NucleoSpin Extract kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions.

RNA extraction.For RNA extraction, three methods were used during the course of this work to optimize the yield of DNA-free RNA. (i) The DNA isolation method described above was followed by RNA precipitation with a 1/5 volume of 10 M LiCl and 2 volumes of 100% ethanol at −20°C for at least 1 h, before being washed with 70% ethanol, dried on ice, and resuspended in water. (ii) A hot-phenol method described elsewhere (3) was used. (iii) TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) was used, following the manufacturer's instructions, after the cells were disrupted with 0.2 g of acid-washed 0.2-mm-diameter glass beads.

Generation of probes and nucleic acid hybridizations.The primer pairs LMH4′A-LMH4′B, LMS3′A-H1B, and CYA106F-CYA781R were used to obtain the homologous probes Lmhup, the hupSL intergenic region (Lm_ig), and 16S rDNA, respectively (Table 1; Fig. 1 and Fig. 2D). Nonradioactive Southern hybridizations were performed at 57°C following the protocol described previously (50). Radioactive Northern hybridizations were performed at 65°C using RNA extracted with TRIzol (see above) and following the protocol of Ausubel et al. (2). RNA was extracted from replicate samples collected at the same time points as the samples used for the determination of the hydrogen uptake activity (see above). Probes were labeled with ³²P using Ready-To-Go DNA Labeling beads (dCTP) (Amersham Biosciences, Buckinghamshire, United Kingdom), and unincorporated labeled nucleotides were removed with ProbeQuant G-50 Micro Columns (Amersham Biosciences, Buckinghamshire, United Kingdom). Stripping of the membranes was performed according to the protocol from the Hybond-N+ nylon membrane (Amersham Biosciences, Buckinghamshire, United Kingdom).

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FIG. 1.

(A) Physical map of the hupSL genes in Lyngbya majuscula CCAP 1446/4. +1 indicates the transcription start site. Vertical black bars and numbers represent the primers used within this study (see also Table 1). Lmhup indicates the homologous probe used in Northern and Southern hybridizations. Black arrows indicate the restriction sites. The thick gray lines indicate regions where repeats are present (the hupSL intergenic region and downstream of hupL). The 4.3-kb sequence is available from GenBank under accession number AF368526 . (B) Nucleotide sequence of the promoter region upstream hupS of L. majuscula. The following regions are highlighted: putative NtcA- and IHF-binding sites, −35 and −10 boxes, the transcriptional start point (+1), and a putative Shine-Dalgarno sequence (RBS [ribosomal binding site]). For IHF, only the most conserved part of the sequence is indicated. The start codon of hupS is indicated in boldface type, and the initial deduced amino acid sequence is given below.

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FIG. 2.

Repetitive sequences present within the hupSL intergenic region, and the region directly downstream of hupL in Lyngbya majuscula CCAP 1446/4. (A) Schematic representation of the intergenic region with the relative positions of the 30-bp and the 58-bp LRRs indicated by light and dark gray, respectively. Regions at the extremes contain no repetitive sequences (in black). The table shows the sequences and positions of the two sets of LRRs. A common consensus can be discerned from the 30-bp LRRs, while the 58-bp LRRs show higher variability. Repeats 9 to 11 (boxed) are essentially identical, except for six nucleotides represented in italics. (B) Schematic representation of the region directly downstream of hupL harboring repetitive sequences, with the relative positions of the 69-bp, 11-bp, and 66-bp repeats indicated by A, B, and C, respectively. (C) Computer-predicted RNA secondary structure with the relative positions of the 30-bp and the 58-bp LRRs indicated by shades (light and dark gray, respectively) and by numbers. (D) Schematic representation of the L. majuscula PCR-generated probe for the hupSL intergenic region (for details of primers, see Table 1), and the respective Southern blot showing hybridization to a single fragment of genomic DNA digested with HindIII or EcoRI plus HindIII.

Construction of partial genomic libraries and isolation of hup genes.Genomic DNA was digested with the restriction endonuclease HindIII and separated on a 1× TBE agarose gel. For hup gene isolation, a region containing the fragment identified by Southern blot hybridization with the probe Lmhup was cut out, and the DNA was recovered (Fig. 1). Ligation into the vector pGEM3Zf(+) (Promega Corporation, Madison, WI), transformation, and screening were performed as described previously (45).

5′ RACE.The RNA used in 5′ rapid amplification of cDNA ends (RACE) experiments was extracted using the modified DNA isolation method for RNA (see above) from cells grown under N₂-fixing conditions (BG11₀) with hydrogen uptake activity confirmed using the H₂ electrode. The transcriptional start site was located by 5′ RACE, with the FirstChoice RLM-RACE kit (Ambion, Inc., Austin, TX), following the instructions of the manufacturer, except in the outer PCR where a double volume of the reverse transcription reaction mixture was used. In both PCRs, the cycle extension was increased to 1 min. S1rev, LMSB, and LMSR1B were used as gene-specific antisense primers (Table 1). Resultant DNA fragments were cloned into pGEM-T Easy (Promega Corporation, Madison, WI) and transformed into XL1-Blue Supercompetent cells (Stratagene, La Jolla, CA). Recombinant plasmids were extracted with the GenElute Plasmid Miniprep Kit (Sigma-Aldrich Co., St. Louis, Mo.), and the target fragments were subsequently sequenced.

Genome walking and cloning of the hup promoter.The Lyngbya majuscula CCAP 1446/4 genome walking library was constructed according to the Universal GenomeWalker kit (Clontech Laboratories, Inc., Palo Alto, CA), using EcoRV for genomic DNA digestion, and GW5Lmhup1 as the hupS-specific primer in PCR amplification (Table 1). The obtained PCR product was cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI), yielding the plasmid pGW5Lmhup, and subsequently sequenced.

Band shift assay.The 342-bp HincII-MunI fragment (−317 to +25 bp) containing the Lyngbya majuscula CCAP 1446/4 putative hupSL promoter sequence was obtained from pGW5Lmhup. The 255-bp DNA fragment used as nonrelated, negative-control DNA was amplified by PCR using pBluescript SK(+) as a template and the universal M13 forward (−40) and M13 reverse primers. A cell-free protein extract of Escherichia coli BL21(pCSAM70, pREP4), which overproduces Anabaena sp. PCC 7120 NtcA protein (kindly provided by E. Flores) (40), was used as source of NtcA, while Escherichia coli BL21 cell-free protein extract was used as a negative control. Binding assays were carried out as described previously (39) with 75 to 100 ng of DNA fragment and 1.5 μg of Escherichia coli cell extract.

RT-PCR.RNA from Lyngbya majuscula CCAP 1446/4 was isolated from cells grown in BG11₀ and collected 5 h into the dark phase, using the hot-phenol method (see above). Reverse transcription (RT) reactions were performed following the protocol of the ThermoScript RT-PCR System (Invitrogen Corporation, Carlsbad, CA, USA) using 1 μg total RNA. For the detection of hupSL cotranscription, LMH2B was used as primer for the cDNA synthesis, and the primer pair used in the PCR was LMS3′A-LMH2B (Table 1). The PCR program profile was 95°C for 2 min; followed by 35 cycles, each consisting of 45 s of denaturation at 95°C, 45 s of annealing at 55°C, and 1 to 2 min of elongation at 72°C; and concluding with 7 min at 72°C. Negative controls included the omission of reverse transcriptase in the RT reaction mixture prior to the PCR and a PCR to which no template was added. Genomic DNA was used as a positive control.

Production of polyclonal antibodies against recombinant HupL.To raise antibodies against the large subunit of the cyanobacterial uptake hydrogenase, a recombinant protein comprising the amino acids 1 to 521 of HupL (lacking the C-terminal part, presumably cleaved off by an uptake hydrogenase-specific endopeptidase) of Lyngbya majuscula CCAP 1446/4 was overexpressed in Escherichia coli and purified. The corresponding genomic sequence was amplified by PCR using the primer pair ExLmhupLF2-ExLmhupLR (Table 1). The resulting PCR product was digested with BamHI and SalI and cloned into the pQE30 plasmid (QIAGEN, Hilden, Germany), and its identity was established by sequencing. The recombinant HupL, containing a amino-terminal six-histidine tag, was overexpressed in Escherichia coli M15(pREP4) cultures induced for 3 h at 37°C with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside).

The His-tagged recombinant HupL was purified from the insoluble proteins fraction under denaturing conditions. The induced cells were pelleted and lysed by sonication in 1× phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4), and the inclusion bodies were solubilized in 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, and 8 M urea, pH 8.0 (solubilization buffer). The purification was performed by loading the extract to columns packed with Ni-nitrilotriacetic acid agarose (QIAGEN, Hilden, Germany) and by applying wash and elution buffers (solubilization buffer with 20 mM and 250 mM imidazole, respectively). The fractions with the recombinant protein were further purified by eluting the protein from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by lyophilization and precipitation with acetone.

For the production of polyclonal antibodies against the purified His-tagged recombinant HupL, Wistar rats were subjected to a first subcutaneous injection of 100 μg of purified protein in 1× PBS suspended in Freund's complete adjuvant (Sigma-Aldrich Co., St. Louis, Mo), followed by five boosts, each consisting of 50 μg of purified protein in 1× PBS suspended in Freund's incomplete adjuvant (Sigma-Aldrich Co., St. Louis, Mo). The specificity of the HupL antibody was confirmed by testing it against the recombinant purified HupL.

Protein extraction, SDS-PAGE, and Western blotting analysis. Lyngbya majuscula CCAP 1446/4 proteins were extracted from replicate samples collected at the same time points as the samples used for the determination of the hydrogen uptake activity (see above). Protein extracts were prepared in lysis buffer (10 mM HEPES, 0.5% [wt/vol] Triton X-100, 10 mM EDTA, 2 mM dithiothreitol, pH 8.0, supplemented with protease inhibitor cocktail [Roche Diagnostics GmbH, Penzberg, Germany]) by sonication on ice and centrifuged at maximum speed for 10 min at 4°C. Protein content was determined by using the BCA Protein assay (Pierce Biotechnology, Inc., Rockford, IL) with bovine serum albumin as a standard. Protein samples (25 μg per lane) were separated by discontinuous SDS-PAGE (26) with a 10% resolving gel and a 3% stacking gel. After electrophoresis, proteins were either stained with Coomassie blue or transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, United Kingdom) in a semidry system during 1 h at 0.8 mA cm⁻¹, with 39 mM glycine, 48 mM Tris, 0.0375% SDS, and 20% methanol as the transfer buffer. Membranes were probed with polyclonal rat anti-HupL antiserum at a 1:3,000 dilution, followed by goat anti-rat immunoglobulin G linked to horseradish peroxidase (Amersham Biosciences, Buckinghamshire, United Kingdom) at a 1:4,000 dilution. Immunodetection was performed by chemiluminescence using ECL Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, United Kingdom).

Sequencing and sequence analysis.Sequencing reactions were performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA), following the manufacturer's protocol, the thermal cycler mentioned above, and an ABI Prism Genetic Analyzer (Applied Biosystems, Foster City, CA). Computer-assisted sequence comparisons were performed using CLUSTAL W (57). RNA secondary structure was predicted with mfold, version 3.1 (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/ ) (34, 66). Novel sequences associated with this study are available from GenBank under the accession number AF368526 (hupSL). Other novel related sequences (GenBank accession numbers in parentheses) are 16S rDNA of Lyngbya majuscula CCAP 1446/4 (AF368300 ) and hupSL intergenic regions of Nostoc muscorum PCC 7906 (AF455565 ), Nostoc sp. HCC 1048 (AF455566 ), Nostoc sp. HCC 1061 (AF455567 ), and Nostoc sp. HCC 1075 (AF455568 ).

Uptake hydrogenase structural genes.The unequivocal presence of an uptake hydrogenase in Lyngbya majuscula CCAP 1446/4 was recently demonstrated by Schütz et al. (50). To identify the structural genes encoding the uptake hydrogenase (hupSL) in this strain, oligonucleotide primers designed against conserved regions within hupSL of Anabaena sp. PCC 7120 were used. Furthermore, once preliminary sequences were analyzed, Lyngbya majuscula CCAP 1446/4-specific primers were synthesized, and the PCR products obtained were sequenced. The downstream part of hupL, the flanking 3′ untranslated region (UTR), and part of an open reading frame (ORF) were obtained from construction of a partial genomic library that led to the cloning of the 2-kb HindIII fragment identified with the homologous probe Lmhup (Fig. 1). Multiple sequence alignments were performed to obtain the contiguous 4,347-bp sequence encompassing hupSL, the 643-bp intergenic region, the promoter region, the 3′ UTR, and part of an ORF in the direction opposite to hupSL, showing a high degree of homology to an uncharacterized conserved gene present in cyanobacteria; see also the sequence discussed below, with GenBank accession number AF368526 .

hupSL intergenic region and 3′ UTR repeats.The hupSL intergenic regions of the heterocystous cyanobacteria sequenced to date consist largely of 7-bp repeated sequences (see Materials and Methods and the sequences related to this work) (28, 56) belonging to different families of short tandemly repeated repetitive sequences (12, 22, 35, 37, 61). Within the 643-bp intergenic region of Lyngbya majuscula CCAP 1446/4, although no short tandemly repeated repetitive sequences were discerned, we could identify long repeated repetitive sequences (LRRs) (Fig. 2): a cluster of four 30-bp LRRs and eight 58-bp LRRs (one incomplete, repeat no. 7). Furthermore, four combined repeats of 88 bp (30 plus 58 bp) could be observed (repeats 1 and 2, 3 and 4, 5 and 6, and 8 and 9) (Fig. 2A). A common consensus could be discerned from the 30-bp LRRs, while the 58-bp LRRs showed higher variability. Interestingly, within the 643-bp intergenic region, only sequences from positions 1 to 34 and 617 to 643, totaling 60 bp, were nonrepetitive nucleotides (Fig. 2A), with positions 628 to 643 identified as a putative ribosomal binding site preceding the hupL start codon. Sequence analysis of the hupSL intergenic region of Lyngbya majuscula CCAP 1446/4 revealed a putative stem-loop structure in the transcribed RNA (Fig. 2C).

To determine the abundance of hupSL intergenic region LRRs within the Lyngbya majuscula CCAP 1446/4 genome, Southern hybridization experiments were performed. Initially, the entire hupSL intergenic region (Lm_ig) was used as a probe, resulting in the detection of only one hybridization signal (Fig. 2D). Subsequently, the consensus of the 30-bp repeats, the most conserved part of the 58-bp repeat (initial 20 bp), and an assemblage of both were used as probes. These Southern blotting experiments, with low to moderate stringency, confirmed the initial findings that utilized the entire region (data not shown).

The 3′ UTR immediately downstream of hupL (Fig. 2B) also contained several repetitive sequences: three sets of 69 bp each (the last of these truncated at 50 bp), three sets of 11 bp each, and four sets of 66 bp each (the last of these truncated at 14 bp).

Promoter region.5′ RACE permitted the identification and localization of the transcriptional start site 59 bp upstream from the hupS start codon and the acquisition of the sequence of the 5′ UTR, where a putative Shine-Dalgarno sequence (ribosome-binding site) (AGGAGA) could be identified (Fig. 1B). Genome walking was used to obtain the nucleotide sequence upstream of the transcriptional start site. The analysis of this region revealed the presence of a −10 (TTAGAT) and a −35 box (TTATCA) separated by 17 bp, a structure similar to that of the Escherichia coli σ⁷⁰-type promoter. In addition, the sequence positioned between −240 and −227 bp (GTATTATCTGTAC) was remarkably similar to the highly conserved palindromic NtcA-binding region signature, GTAN₈TAC (20, 31). Furthermore, a region showing good homology to the consensus integration host factor (IHF)-binding motif WATCAAN₄TTR (13, 16, 17) could be identified between −165 to −153 bp (TATCAAGTCCATA) and was preceded by an AT-rich stretch of nucleotides.

Binding of the transcriptional regulator NtcA to the promoter region of hupSL.The possible interaction between NtcA and the hupSL promoter region of Lyngbya majuscula CCAP 1446/4 containing the putative NtcA-binding site was assessed by carrying out a band shift assay. Electrophoretic retardation of a 342-bp hupSL promoter fragment (−317 and +25) was effected by Escherichia coli protein extract containing NtcA, while no retardation was observed in binding assays carried out with extracts of E. coli not expressing NtcA. Moreover, no retardation was observed for nonrelated control DNA after incubation with any of the E. coli extracts (Fig. 3). These experiments indicate a specific binding of NtcA to DNA sequences of Lyngbya majuscula CCAP 1446/4 hupSL promoter between positions −317 and +25, relative to the transcriptional start point.

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FIG. 3.

Band shift assay of NtcA binding to the HincII-MunI DNA fragment (−317 to +25 bp) containing the putative hupSL promoter of Lyngbya majuscula CCAP 1446/4. Lane 1, hupSL promoter, no NtcA; lane 2, hupSL promoter and NtcA-containing extract; lane 3, nonrelated DNA fragment, no NtcA; lane 4, nonrelated DNA fragment and NtcA-containing extract. The arrowhead indicates NtcA-bound retarded DNA fragments.

Hydrogen uptake activity.Hydrogen uptake activity in Lyngbya majuscula CCAP 1446/4 was evaluated using a H₂ electrode. Grown under alternating 12-h light/12-h dark cycles and N₂-fixing conditions, Lyngbya majuscula CCAP 1446/4 exhibited a daily pattern with a clear maximum towards the end of the dark period, at about 9 h into the dark phase. The activity declined rapidly after the transition to light but remained at a detectable level (see Fig. 5A). In contrast, only residual H₂ uptake activity could be discerned when combined nitrogen- NaNO₃ was present in the medium (data not shown). It should be mentioned that the H₂ electrode measurements were net uptake values and therefore might ultimately be due to the activity of both an uptake and a bidirectional hydrogenase. In fact, the genome of Lyngbya majuscula CCAP 1446/4 harbors the genes encoding a bidirectional hydrogenase (hox; GenBank accession no. AY536043 ). Moreover, accessory genes suggested to be involved in the maturation process of the uptake hydrogenase and/or the bidirectional enzyme are also present and have been partially sequenced (hyp; GenBank accession no. AY536041 and AY536042 ).

Transcriptional studies.To assess hupSL cotranscription in Lyngbya majuscula CCAP 1446/4, RT-PCR experiments were performed with total RNA extracted from cells grown under nitrogen-fixing conditions and harvested 5 h into the dark period. A PCR fragment comprising the 3′ end of hupS, the intergenic region, and the 5′ of hupL was amplified (Fig. 4, lane 1), confirming that hupS and hupL are indeed transcribed together.

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FIG. 4.

RT-PCR detection of hupSL cotranscription in Lyngbya majuscula CCAP 1446/4. Total RNA isolated from cells grown under nitrogen-fixing conditions was used in the RT reaction mixture, with LMH2B as the specific primer. cDNA produced was used in PCR amplifications with the primer pair LMHS3′A-LMH2B (1,007-bp fragment). Lane 1, RT-PCR; lane 2, negative control without reverse transcriptase; lane 3, PCR-negative control (no template added); lane 4, PCR-positive control (genomic DNA added); M, GeneRuler DNA Ladder mix (Fermentas).

RNA blot analysis was performed to examine the levels of hupSL transcripts throughout a 24-h period (Fig. 5B). After hybridization with the hupL-specific probe Lmhup (Fig. 1A), a large transcript of approximately 3.5 kb was detected, most likely corresponding to the complete hupSL. Additional hybridization signals could also be observed, possibly due to mRNA processing or specific degradation or RNA polymerase pausing. An increase in hupSL transcription was clearly observed in cells collected 3 h prior to entering the dark period and reached its maximum level in the transition between the light and the dark phase (Fig. 5B, lane D0). Three hours later (lane D3), a decrease in the intensity of the hybridization signal was observed, and the levels remained relatively constant until the end of the dark phase. No signal could be detected at the beginning of the light phase.

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FIG. 5.

Daily pattern of H₂ uptake activity, levels of uptake hydrogenase transcript(s), and HupL in Lyngbya majuscula CCAP 1446/4. Cells were grown under N₂-fixing conditions (BG11₀) and alternating 12-h light/12-h dark cycles. Samples were collected at 3-h intervals during a complete 24-h cycle; L0 to L9 and D0 to D9, samples taken during the light and dark periods, respectively. (A) In vivo H₂ uptake activity. Each time point is the mean of two consecutive measurements. The data shown are from a single experiment, but the observed pattern is typical (three separate observations). White and black bars represent light and dark periods, respectively. (B) Northern blot analysis using the probe Lmhup (see Fig. 1). For the RNA loading control, the membrane was stripped and rehybridized to the 16S rDNA probe (see Materials and Methods). The size of the putative transcript is indicated by an arrow. (C) Western blot analysis of protein extracts separated by SDS-PAGE. The membrane was probed with polyclonal rat antiserum raised against HupL of L. majuscula CCAP 1446/4 (anti-HupL). The molecular mass of the recognized polypeptide is indicated.

Immunoblot analysis.Protein extracts from Lyngbya majuscula CCAP 1446/4 cells grown under N₂-fixing conditions and alternating 12-h light/12-h dark cycles were fractionated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated with polyclonal rat antiserum raised against Lyngbya majuscula CCAP 1446/4 HupL (Fig. 5C). A single polypeptide of about 60 kDa was recognized in all samples. The lowest protein level was detected 3 h before the end of the light phase (Fig. 5C, lane L9), followed by an increase during the dark period and a subsequent decrease during the light phase.

Uptake hydrogenase structural genes and proteins.As previously reported for cyanobacteria, the genes encoding an uptake hydrogenase in Lyngbya majuscula CCAP 1446/4 are arranged in a contiguous manner with hupS upstream of hupL and with a transcriptional start site located 59 bp upstream from the hupS start codon. RT-PCR experiments confirmed that hupS and hupL are cotranscribed (Fig. 4), as previously described for other cyanobacterial strains (18, 28, 42). The presence of an unknown ORF downstream of hupL and in the opposite direction contrasts with other nonheterocystous cyanobacteria (Gloeothece sp. ATCC 27152 and Trichodesmium erythraeum IMS101), in which a gene encoding an uptake hydrogenase-specific endopeptidase (hupW) is located downstream of hupL and was shown to be cotranscribed with hupSL in Gloeothece sp. ATCC 27152 (42). The presence of different hydrogenase nonrelated ORFs downstream of hupSL was reported for several heterocystous strains (56). However, the ORF located downstream of hupSL in Lyngbya majuscula CCAP 1446/4 shows no homology with the ORFs found in those strains.

Analysis of the predicted proteins demonstrates that HupS in Lyngbya majuscula CCAP 1446/4 has the same number of residues as the corresponding subunit within other cyanobacteria (320 amino acids), while HupL (537 amino acids) has 3 additional amino acids compared to Trichodesmium erythraeum IMS101 and 6 extra amino acids compared to the known sequences of unicellular and heterocystous strains (one at position 344 and the others at the N terminus). Nevertheless, the general features of Lyngbya majuscula CCAP 1446/4 HupSL are in agreement with those previously described for cyanobacteria (53, 56, 59, 60). HupS lacks both the twin-arginine signal peptide at the N terminus and the hydrophobic motif at the C terminus, proposed to be involved in translocation and anchorage to the membrane, respectively. In agreement, HupL contains the C-terminal region that is presumably cleaved off during the maturation process of the large subunit (53, 64). Moreover, in Lyngbya majuscula CCAP 1446/4, as in all cyanobacteria for which uptake hydrogenase sequences are available, no transmembrane domains were identified within HupS and HupL (53).

The HupSL of Lyngbya majuscula CCAP 1446/4 contains all conserved cysteine residues putatively involved in the formation of the [FeS] clusters and the Ni-binding sites in HupS and HupL, respectively (9, 18, 45, 62). However, due to the presence of additional amino acids (1 to 5 and a glycine at 344) in HupL, the position of the cysteine residues are slightly shifted compared to other cyanobacteria: Cys₆₇, Cys₇₀, Cys₅₁₅, and Cys₅₁₈ instead of Cys₆₂, Cys₆₅, Cys₅₀₉, and Cys₅₁₂, respectively.

The deduced amino acid sequence of the Lyngbya majuscula CCAP 1446/4 uptake hydrogenase show 79 to 89% identity and 87 to 98% similarity to the corresponding sequences of other cyanobacteria and has been reported to cluster with the corresponding enzyme of other nonheterocystous cyanobacteria (53).

hupSL intergenic region and repetitive sequences.Cyanobacterial genomes generally have a variety of repeated repetitive sequences (37) with a currently unknown function(s). In the case of hupSL and as discussed previously for nitrogen fixation genes (nif) (22, 35), since the repetitive sequences in the intergenic regions are highly variable or even not present, it is unlikely that these repeats play a direct role in the regulation of gene expression. However, sequence analysis of the hupSL intergenic regions of heterocystous cyanobacterial strains revealed possible hairpin formation in the transcribed RNA (28, 56). Indeed, in the corresponding region of Lyngbya majuscula CCAP 1446/4, a similar stem-loop structure, derived via two-dimensional computer modeling, could be discerned (Fig. 2C). In view of the possible hairpin formation and as pointed out by Lindberg et al. (28), the actual sequence of the repeat may not be important, only the three-dimensional structure that these sequences form when transcribed into mRNA. It has been suggested that these hairpins may increase the stability of the transcript and/or confer a translational coupling between hupS and hupL, ensuring the synthesis of the two subunits in equal amounts (28). Yet, only the construction of specific mutants will help to clarify the function of these intergenic regions.

It has previously been reported that various long repetitive sequences occur in heterocystous and unicellular strains (32, 65). However, the long repetitive sequences present within Lyngbya majuscula CCAP 1446/4 exhibit a different pattern with no sequence homology to those repeats. Moreover, the LRRs present within the hupSL intergenic region of Lyngbya majuscula CCAP 1446/4 were shown on Southern hybridization to be infrequent within the genome. In contrast, the number of contiguous repeated sequences is surprisingly high in the genome of Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120. For example, sequences where the repeat region is at least 20 bp account for about 1.5% of the total DNA and approximately 7.5% of the total DNA in intergenic sequences of Nostoc punctiforme ATCC 29133 (35).

hupSL promoter region and NtcA.The TSP of the hupSL operon was located in the 5′ UTR of hupS. The analysis of the sequence upstream the TSP revealed the presence of an Escherichia coli σ⁷⁰-type promoter carrying a −10 and a −35 box. A NtcA-binding site was found to be centered at position −233.5 with respect to the TSP (Fig. 1B). Gel shift assays demonstrated that NtcA binds specifically to DNA sequences of the Lyngbya majuscula CCAP 1446/4 hupSL promoter (positions −317 to +25), suggesting its involvement in the transcriptional regulation of the uptake hydrogenase structural genes (Fig. 3).

In the canonical NtcA-activated promoters, the binding site is located in the place of the −35 box (centered at about −41.5), separated by ca. 22 nucleotides from the −10 box, a structure similar to that of class II bacterial promoters activated by CAP (8, 19). Since the NtcA-binding site in the Lyngbya majuscula CCAP 1446/4 hupSL promoter is located further upstream, it resembles a class I CAP-dependent promoter (8, 19). Similarly, in the filamentous heterocystous Nostoc punctiforme PCC 73102, the NtcA-binding motif in the hupSL promoter is centered at −258.5 (28). In contrast, hupSL in the unicellular Gloeothece sp. ATCC 27152 is preceded by a class II-type NtcA-activated promoter (42). Hence, it seems that the type of NtcA-activated promoter (class I versus II) is not correlated to the strategies used by heterocystous and nonheterocystous cyanobacteria to separate N₂ fixation and photosynthesis.

In the class I-type NtcA-activated hupSL promoters of Lyngbya majuscula CCAP 1446/4 (Fig. 1B) and Nostoc punctiforme PCC 73102 (28), a putative IHF-binding site was identified between the NtcA-binding motif and the −35 box. It is possible that the binding of IHF to that region of the promoter, bending the DNA (15), could mediate the contact of the upstream binding activator protein (NtcA) with the RNA polymerase complex and activate hupSL transcription.

Hydrogen uptake activity and levels of uptake hydrogenase transcripts and proteins. Lyngbya majuscula CCAP 1446/4 exhibits a daily pattern of hydrogen uptake activity with a clear maximum towards the end of the dark period, when grown under alternating light/dark cycles and N₂-fixing conditions (Fig. 5A). The temporal separation between N₂ fixation and oxygenic photosynthesis is the most common strategy adopted by nonheterocystous cyanobacteria to minimize nitrogenase inactivation by oxygen (4). Recently, the nitrogen fixation behavior of a Lyngbya majuscula strain isolated from subtidal zones of Zanzibar was reevaluated. In contrast with previous reports indicating that Lyngbya spp. fix N₂ in light (23, 47, 48, 51), it was shown that this strain fixes N₂ during the dark phase of a daily cycle, with a maximum after 4 to 8 h into the dark phase (30). It was also demonstrated that nitrogenase synthesis occurs in all cells in this strain of Lyngbya majuscula, in contrast to the filamentous nonheterocystous Trichodesmium erythraeum IMS101, where the nitrogenase was shown to be active only within the diazocytes (5). In addition, it was also shown that the nitrogenase activity for Lyngbya sp. and Microcoleus chthonoplastes mats follows a diurnal pattern, with the highest rates occurring during the dark period (43).

A strong correlation between the activity of the uptake hydrogenase and N₂ fixation has been demonstrated for several filamentous heterocystous cyanobacteria (21, 46, 58, 63). Analyzing the N₂ fixation and nitrogenase expression data recently obtained (30, 43) and the hydrogen uptake pattern observed in the present study (although not in the same strain), it seems probable that both processes are indeed correlated and confined to the dark period in Lyngbya majuscula.

To determine the correlation of the H₂ uptake activity with hupSL transcription and levels of HupL protein, Northern and Western blot analyses were performed. The maximum H₂ uptake activity in Lyngbya majuscula CCAP 1446/4 is reached late within the dark period (Fig. 5A, lane D9), whereas the peak of hupSL transcription occurs in the transition between the light and the dark phase (Fig. 5B, lane D0), synchronized with an increase of HupL protein levels (Fig. 5C). Similarly, in the unicellular Gloeothece sp. ATCC 27152, the highest transcription was detected towards the end of the light period (42); as described here for Lyngbya majuscula CCAP 1446/4, a decrease in the levels of transcription was evident a few hours into the dark period. In Lyngbya majuscula CCAP 1446/4, the transcription is then kept relatively constant during the rest of the dark period, but there is an increase in the levels of HupL and concomitantly in the H₂ uptake activity. In the beginning of the light phase, no hupSL transcription is detectable, and the levels of HupL and H₂ uptake activity begin to decline. Although the H₂ uptake activity gradually diminishes during the light period, an evident decrease in HupL levels is only observed once the uptake activity reaches its minimum (Fig. 5C, lane L9). The time difference between the initiation of hupSL transcription and H₂ uptake activity observed for Lyngbya majuscula CCAP 1446/4 might, as proposed previously for Gloeothece sp. ATCC 27152 (42), be due to the complexity of the maturation process of the uptake hydrogenase (10). In addition, the temporal separation between the photosynthesis and nitrogen fixation/hydrogen uptake activity (O₂-sensitive processes) may also influence the time lag between transcription and activity. The decrease of the HupL levels at the end of the light period and its increase during the dark phase suggest that protein turnover occurs and that degradation takes place during the light period, while during the dark phase there is de novo synthesis of protein (coinciding with the rise in the uptake hydrogenase activity).

In conclusion, from the data presented here it is clear that an active uptake hydrogenase is present in Lyngbya majuscula CCAP 1446/4 and that a daily pattern of hydrogen uptake activity, with a clear maximum towards the end of the dark period, is observed in cells grown under alternating light/dark cycles and N₂-fixing conditions. Our results suggest the involvement of NtcA in the transcriptional regulation of the uptake hydrogenase structural genes hupSL. It is also reported the presence of repetitive sequences both in the hupSL intergenic region and in the region downstream of hupL. Still, the construction of specific mutants is needed to understand the function of these repeats. The first antibodies directed against a cyanobacterial uptake hydrogenase were produced during the course of this work and will allow further studies, notably the cellular/subcellular localization of the enzyme (currently a controversial subject), and its purification.

To be able to use Lyngbya majuscula CCAP 1446/4 for biological hydrogen production, it is necessary to inactivate the uptake hydrogenase, since this enzyme scavenges H₂. The development of a method for gene transfer to Lyngbya majuscula CCAP 1446/4, enabling the production of an H₂ uptake mutant, is currently under way. The gene transfer method could subsequently be adjusted for other biotechnological applications, such as the use of environmental strains of Lyngbya majuscula, for the production of natural biochemically active compounds.