Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

Paulo Oliveira1,{dagger}, Elsa Leitão2, Paula Tamagnini1,2, Pedro Moradas-Ferreira2,3 and Fredrik Oxelfelt2

1 Department of Botany, Faculty of Sciences, University of Porto, Rua do Campo Alegre 1191, 4150-181 Porto, Portugal
2 Institute for Molecular and Cell Biology – Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
3 Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Largo Abel Salazar 2, 4099-003 Porto, Portugal

Correspondence
Fredrik Oxelfelt
fredrik{at}ibmc.up.pt


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N2 fixation, were identified and sequenced. 3'-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at –41·5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW.


Abbreviations: RACE, rapid amplification of cDNA ends

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY260103.

{dagger}Present address: Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, 75236 Uppsala, Sweden.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Several species of bacteria and cyanobacteria are capable of N2 fixation. During the N2 fixation process H2 is formed as a by-product. This nitrogenase-dependent H2 production is often compromised by the presence of an uptake hydrogenase (encoded by hupSL) that rapidly consumes the H2 generated. In addition, a bi-directional enzyme (encoded by hoxEFUYH) may be present which, depending on the growth conditions, may display the capacity of both producing and consuming H2 (Lambert & Smith, 1981; Houchins, 1984; Schmitz et al., 2002; Tamagnini et al., 2002). All cyanobacteria examined so far contain an uptake, a bi-directional or both the hydrogenases (Schmitz et al., 1995, 2002; Boison et al., 1996; Tamagnini et al., 2000, 2002; Sheremetieva et al., 2002; Schütz et al., 2004). Moreover, the uptake-type enzyme has been found in all nitrogen fixing cyanobacterial strains studied to date (Carrasco & Golden, 1995; Oxelfelt et al., 1998; Happe et al., 2000; Tamagnini et al., 2000, 2002; Schütz et al., 2004).

The first data on cyanobacterial hupSL transcription appeared in 1995 (Carrasco & Golden, 1995). RT-PCR experiments on Anabaena/Nostoc sp. strain PCC 7120 demonstrated that hupL transcription coincides with the formation of heterocysts. Subsequent studies, in other filamentous strains, have confirmed the induction of an hupL transcript under nitrogen-fixing conditions only (Axelsson et al., 1999; Happe et al., 2000; Hansel et al., 2001). One exception is Anabaena variabilis ATCC 29413, where a low level of hupL expression has been detected in vegetative cells grown with the addition of ammonia (Boison et al., 2000). In all the cyanobacterial strains examined so far, transcriptional studies have shown that the hupSL genes constitute a single transcript, containing no additional ORFs (Happe et al., 2000; Lindberg et al., 2000).

The maturation of hydrogenases is a complex process requiring a number of accessory proteins (Menon et al., 1993; Vignais & Toussaint, 1994; Maier & Triplett, 1996; Buhrke et al., 2001; Casalot & Rousset, 2001; Vignais et al., 2001; Blokesch et al., 2002; Paschos et al., 2002). One distinct feature in the NiFe-hydrogenases maturation process is the endoproteolytic cleavage of a C-terminal peptide of the large subunit precursor, carried out by a specific C-terminal endopeptidase (Casalot & Rousset, 2001; Paschos et al., 2002). Until now, the available data on the maturation of cyanobacterial NiFe-hydrogenases are scarce. Recently, the presence and expression of endopeptidases specific for cyanobacterial hydrogenases was reported (Wünschiers et al., 2003). These authors screened three completed cyanobacterial genome sequences [Anabaena/Nostoc sp. strain PCC 7120 (www.kazusa.or.jp/cyano/Anabaena/), Nostoc punctiforme ATCC 29133/PCC 73102 (http://genome.jgi-psf.org/draft_microbes/nospu/nospu.home.html), and Synechocystis PCC 6803 (www.kazusa.or.jp/cyano/Synechocystis/)] with the purpose of identifying genes putatively encoding C-terminal specific endopeptidases. In agreement with previous nomenclature they proposed the gene name hoxW (endopeptidase specific for the bi-directional hydrogenase) for the ORFs all0770 (Anabaena/Nostoc PCC 7120) and slr1876 (Synechocystis PCC 6803), whereas the ORFs alr1423 (Anabaena/Nostoc PCC 7120) and c509/r320 (Nostoc PCC 73102) were named hupW (endopeptidase specific for the uptake hydrogenase). These ORFs are not clustered with any known hydrogenase-related gene(s).

A strong correlation between nitrogen fixation and uptake hydrogenase activity has been demonstrated in filamentous cyanobacteria (Lambert & Smith 1981; Houchins, 1984; Wolk et al., 1994; Oxelfelt et al., 1995; Masukawa et al., 2002; Schütz et al., 2004). In cyanobacteria nitrogen control is mediated by a transcriptional regulator, NtcA, belonging to the CAP family (the catabolite gene activator or cAMP receptor protein) (Herrero et al., 2001). In response to ammonium withdrawal, NtcA binds to specific sites in the promoter region of regulated genes involved in nitrogen assimilation. The NtcA-activated promoter structure consists of a –10 box in the form TAN3T and an NtcA-binding site with the consensus sequence GTAN8TAC, usually located 20 to 23 nucleotides upstream of the –10 box, which appears to substitute for the –35 box (Luque et al., 1994; Muro-Pastor et al., 1999; Herrero et al., 2001). Other proposed consensus NtcA binding sites are TGTN9/10ACA, and TGTAN8TACA (Ramasubramanian et al., 1994; Jiang et al., 2000; Wisén, 2003).

Up to now, only limited amounts of biochemical/physiological data are available concerning uptake hydrogenases in unicellular cyanobacteria (Lambert & Smith, 1981; Houchins, 1984; Schütz et al., 2004). Recently, in the unicellular cyanobacterium Gloeothece sp. strain ATCC 27152, the unequivocal presence of an uptake hydrogenase was reported, in contrast with the lack of hybridization signals when probes for hox genes were used (Schütz et al., 2004). However, a residual level of methyl-viologen-dependent H2 evolution could be detected, therefore the presence of a bi-directional hydrogenase in Gloeothece sp. ATCC 27152 cannot be excluded.

This study presents the first comprehensive molecular data on an uptake hydrogenase being present in a unicellular cyanobacterium, and provides new information on how oxygen-evolving photosynthesis and an essentially anaerobic process like hydrogen uptake can occur within a single cell. The structural genes (hupSL) encoding this enzyme in Gloeothece sp. ATCC 27152 were identified, sequenced and characterized. Moreover, a gene encoding a cyanobacterial hydrogenase specific endopeptidase – hupW – was found immediately downstream of hupL, and was shown to be co-transcribed with hupSL. The three genes are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. Evidence for the involvement of NtcA in the transcriptional regulation of hupSLW is also presented.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Organism and growth conditions.
Gloeothece sp. strain ATCC 27152 was cultured at 25 °C in BG110 or BG11 for the Northern blot experiments (Rippka et al., 1979), under a 12 h light (10 µmol photons m–2 s–1)/12 h dark cycle regime.

Hydrogen uptake activity.
In vivo hydrogen uptake was measured using a Hansatech DW1 O2/H2 electrode (Hansatech) according to the methods described previously (Oxelfelt et al., 1995).

Nucleic acid extraction and analysis.
Genomic DNA was isolated from Gloeothece sp. ATCC 27152 cells by phenol/chloroform extraction as described elsewhere (Tamagnini et al., 1997). In order to obtain clean DNA (e.g. free from extracellular polysaccharides), additional washing steps were required: the cells were collected by centrifugation and resuspended in washing buffer [50 mM NaCl, 5 mM EDTA and 50 mM Tris/HCl pH 8·0 (Fiore et al., 2000)], followed by the addition of 0·5–1 g acid-washed 0·6 mm diameter glass beads and vortexing. The supernatant was collected and the washing step was repeated twice without the presence of glass beads. The DNA was then extracted following the protocol referred to above. After the precipitation step, the pellet was again resuspended in washing buffer and the DNA was precipitated. The DNA was recovered using a sterile micropipette tip, washed with 70 % (v/v) ethanol, dried and dissolved in sterile water.

For RNA isolation, cells of Gloeothece sp. ATCC 27152 were harvested by centrifugation at 4 °C, frozen in liquid nitrogen and left to thaw on ice. This freezing and thawing was repeated twice. Total RNA was then isolated following the protocol of Axelsson et al. (1999), with the exception that 20 U DNase I FPLCpure (Amersham Biosciences) was added during the hot phenol treatment. RNA used for the Northern hybridizations was isolated using TRIZOL reagent (Invitrogen), following the manufacturer's instructions. In the homogenization step, 0·5 g acid-washed 0·2 mm diameter glass beads were added to the samples, and the disruption of the cells was accomplished using a Mini-Beadbeater (Biospec Products). DNA and RNA were analysed by agarose gel electrophoresis using 1x TAE or TBE buffer (Sambrook et al., 1989).

PCR, DNA sequencing and sequence analysis.
All oligonucleotides used in this study are listed in Table 1 (see also Fig. 1a). PCR amplifications were carried out in a Gene Amp PCR System 2400 (Perkin-Elmer) thermal cycler with Taq DNA polymerase (Amersham Biosciences) as previously described (Tamagnini et al., 1997).


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Table 1. Oligonucleotide primers used in the present study

For the specific positions within the hupSLW sequence see Fig. 1(a).

 


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Fig. 1. (a) Physical map of hupSLW in Gloeothece sp. ATCC 27152. The transcriptional start point is indicated by +1, and is located 238 bp upstream from the hupS start codon. The numbered grey arrowheads represent the oligonucleotide primers used in this study (see also Table 1). GhupS indicates the homologous probe used in Southern hybridization for the identification of the HindIII fragment (clone GhSL1, dashed line not to scale). Black vertical arrows indicate the restriction sites. The sequence harbouring the hupSLW (3891 kb) is available from GenBank under accession no. AY260103. (b) Nucleotide sequence of the promoter region upstream of hupS in Gloeothece sp. ATCC 27152. A putative NtcA binding site is boxed. The transcription start point is indicated by +1 and a putative –10 consensus sequence is underlined. The start codon of hupS is shown in bold and given below is the deduced N-terminal amino acid sequence is given below.

 
DNA fragments were isolated from agarose gels using the QIAEX II gel extraction kit (Qiagen) or the NucleoSpin Extract kit (Macherey-Nagel), following the manufacturer's instructions. Sequencing reactions were performed with an ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems), the thermal cycler mentioned above, and the ABI 377 DNA automated sequencer system (Applied Biosystems). Published sequences were retrieved from GenBank and computer-assisted sequence analyses were performed using CLUSTAL W (Thompson et al., 1994). Novel sequences associated with this study (Gloeothece sp. ATCC 27152 hupSLW) are available in GenBank under the accession number AY260103.

Southern blot analysis.
The probe used for Southern hybridization was obtained by PCR using genomic DNA from Gloeothece sp. ATCC 27152 and the primer pair GloS1A/HS1B [probe GhupS (Fig. 1a) Schütz et al. (2004)]. The identity of the probe was confirmed by sequencing.

Radioactive and non-radioactive Southern hybridizations were carried out at 57 °C, following previously described protocols (Tamagnini et al., 1997; Schütz et al., 2004).

Construction of a partial genomic library of Gloeothece sp. ATCC 27152 and identification of hupSL.
Genomic DNA was digested by the restriction endonuclease HindIII and separated on a 1 % (w/v) agarose gel. A region between 3·5 and 4·5 kb was cut out, and the DNA was extracted from the gel as described above. Ligation into the vector pGEM 3Zf(+) (Promega), transformation and screening (using the probe GhupS; see also Fig. 1a) were performed as described previously (Oxelfelt et al., 1998). Positive clones were detected using a Typhoon 8600 Variable Mode imager (Amersham Biosciences).

Transcription start point identification and the use of 3'-RACE to obtain the 3' end of hupL.
To locate the transcription start point, 5'-RACE experiments were carried out, using a commercially available kit (FirstChoice RLM-Race kit; Ambion). The instructions of the manufacturer were followed, except that a double volume (2 µl) of the reverse transcription (RT) reaction was used in the outer PCR and that the extension was prolonged to 1 min in both the outer and inner PCR amplifications. The gene-specific antisense primers from the 5' end of hupS of Gloeothece sp. ATCC 27152 used were: S1rev, GloS1B and GloSR1B.

To identify the 3' end of hupL in Gloeothece sp. ATCC 27152, 3'-RACE was performed essentially following the manufacturer's protocol (FirstChoice RLM-Race kit; Ambion). As in the 5'-RACE, 2 µl of the RT reaction was used for the outer PCR. For both the outer and inner PCR amplification a touch-down PCR was carried out with the following profile: 95 °C for 3 min, followed by eight cycles of 30 s denaturation at 95 °C, 45 s annealing at 62–56 °C (decreasing 2 °C every second cycle), and 1 min elongation at 72 °C, then 32 identical cycles with the exception that the annealing temperature was set to 55 °C, and concluding with 7 min at 72 °C. The gene-specific sense primers for the 3' end of hupL of Gloeothece sp. ATCC 27152 used were: GloH4A and GloH4A2.

PCR products were cloned, using the pGEM-T Easy vector system (Promega), into XL-1 Blue supercompetent cells (Stratagene). Plasmid DNA was isolated from Escherichia coli using the GenElute plasmid miniprep kit (Sigma-Aldrich). Sequencing was performed as detailed above.

Transcriptional studies.
RT reactions (with 0·5–1 µg total RNA) were performed essentially following the protocol of the ThermoScript RT-PCR system (Invitrogen), using the antisense primer GhupW1R. A double volume of the RT reaction (compared to the manufacturer's protocol) was used in PCR amplifications with the primer pairs GloS3'A/GloH1R (hupShupL detection) and GloH6A/GhupW1R (hupLhupW detection). Negative controls included the omission of reverse transcriptase in the RT reaction prior to the PCR, and a PCR to which no template was added. Genomic DNA from Gloeothece sp. ATCC 27152 was used as a positive control. The PCR program profile was: 95 °C for 2 min followed by 35 cycles of 45 s denaturation at 95 °C, 45 s annealing at 55 °C and 1 min elongation at 72 °C, concluding with 7 min at 72 °C. Generated PCR products were analysed on a 1 % agarose gel.

Northern hybridizations were performed at 65 °C following the protocol of Ausubel et al. (1993). The probes used were obtained by PCR using genomic DNA from Gloeothece sp. ATCC 27152, and the primer pairs GloS1A/GloSR1B (hupS-specific probe) and 106F/781R [16S rRNA gene-specific probe; Nübel et al. (1997)]. Stripping of the membranes was performed following the protocol provided with the Hybond-N+ nylon membrane (Amersham Biosciences).

Gel mobility shift DNA assay.
A 366 bp (–101 to +265) fragment harbouring the promoter region upstream of hupS in Gloeothece sp. ATCC 27152 was amplified by PCR using the oligonucleotides NtcA1F and S1rev, and the clone GhSL1 as template. The amplified fragment was purified from the gel as described above. T4 polynucleotide kinase (Amersham Biosciences) was used to end-label the fragment with [{gamma}-32P]ATP, following the instructions of the manufacturer. The end-labelled fragment was separated from non-incorporated [{gamma}-32P]ATP using a ProbeQuant G-50 micro column (Amersham Biosciences).

A cell-free extract was prepared from E. coli BL21(DE3) (pREP4, pCSAM70) (kindly provided by E. Flores), a strain expressing His-tagged NtcA, according to Muro-Pastor et al. (1999). The overexpression of NtcA was confirmed by SDS-PAGE (data not shown). Five nanograms of labelled DNA fragment was incubated with 0·5–10 µg of the E. coli crude extract in a binding buffer containing 10 mM Tris/HCl (pH 8·0), 50 mM NaCl, 5 % (v/v) glycerol, 10 mM EDTA and 500 ng salmon sperm DNA. The same unlabelled DNA fragment, or an unrelated unlabelled DNA fragment (generated by PCR using pBluescript as template and the M13 reverse/forward primers), was used in the competition assays. A cell-free extract from E. coli XL-1 Blue (not carrying a cloned ntcA gene) was used as a negative control. After incubation at room temperature for 1 h, the reaction mixtures were separated by electrophoresis on a 7·5 % (w/v) native polyacrylamide gel. The radioactive gels were visualized using a Typhoon 8600 Variable Mode imager (Amersham Biosciences).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification and characterization of hupSL
The presence of an uptake hydrogenase in Gloeothece sp. ATCC 27152 was recently demonstrated by Schütz et al., 2004. These authors showed that Gloeothece sp. ATCC 27152 is able to fix nitrogen both under aerobic and anaerobic conditions, yet no nitrogenase-mediated H2 evolution was observed, most likely due to consumption by an active-uptake hydrogenase. Subsequently, these results were confirmed using additional homologous probes (generated using oligonucleotide primers designed against conserved regions within other cyanobacteria) in Southern hybridizations, and by measuring hydrogen uptake activity using a H2 electrode (this work, data not shown). Moreover, to obtain a contiguous sequence Gloeothece sp. ATCC 27152-specific primers were designed, and the respective PCR products sequenced. This revealed the majority of the hupSL sequence, disclosing the presence of a 259 bp intergenic region. Concomitantly, a partial genomic library was constructed, and a 4 kb HindIII fragment (clone GhSL1) was isolated and sequenced (see Fig. 1a). To obtain the remaining 3'-end of hupL, 3'-RACE reactions were carried out, and a number of cDNA clones of different lengths were obtained (most likely due to the fact that the mRNA polyadenylation in prokaryotes appears to be a relatively indiscriminate process, able to occur at all mRNA's 3'-ends, and does not require a specific consensus sequence as in eukaryotes; see Sarkar, 1997; Rauhut & Klug, 1999; Wagner, 2000). Sequencing revealed that all these clones consisted of the 3'-end of hupL. Multiple sequence alignments were carried out to obtain the sequence encompassing the region upstream of hupS, hupSL and the intergenic region. The hupS and hupL of Gloeothece sp. ATCC 27152 are the first structural genes encoding an uptake hydrogenase to be cloned and sequenced for a unicellular cyanobacterium, and they show a high degree of identity (ranging from 75 to 79 % and 74 to 76 %, respectively) with the corresponding genes from Trichodesmium erythraeum, Lyngbya majuscula, Anabaena/Nostoc PCC 7120, A. variabilis and Nostoc PCC 73102. Although Gloeothece sp. ATCC 27152 HupSL cluster together with non-heterocystous cyanobacteria, HupL is exactly the same length as in heterocystous strains, lacking the extra amino acids found in the filamentous non-heterocystous T. erythraeum and L. majuscula (Leitão, E., Oxelfelt, F., Oliveira, P., Ferreira, D. & Tamagnini, P., unpublished results). Overall, the Gloeothece sp. ATCC 27152 HupSL share the distinctive characteristics of cyanobacterial uptake hydrogenases (Tamagnini et al., 2002), including all the conserved cysteine residues involved in the formation of the [FeS] clusters and Ni-binding sites. Since no transmembrane domains were found in Gloeothece sp. ATCC 27152 HupSL, it is probable that a membrane anchoring protein/subunit exists, as was previously predicted for other cyanobacterial strains (Leitão et al., unpublished results; Lindberg, 2003).

hupW is localized directly downstream of hupL
Sequencing the longer clones obtained in the 3'-RACE experiments (see above) revealed the presence of an additional ORF located 184 bp downstream of hupL. Comparative analysis suggested that this ORF encodes a hydrogenase maturation protease (endopeptidase), involved in the C-terminal cleavage of the hydrogenase large subunit precursor protein (Casalot & Rousset, 2001; Vignais et al., 2001; Paschos et al., 2002). In a recent study, a gene encoding an uptake-hydrogenase-specific endopeptidase (named hupW in the referred work) was identified while screening the available genome sequences of Anabaena/Nostoc PCC 7120 and N. punctiforme ATCC 29133/PCC 73102 (Wünschiers et al., 2003). However, the identified gene was not part of any known hydrogenase-related gene cluster. Therefore, the location of hupW immediately downstream of hupSL in Gloeothece sp. ATCC 27152, and oriented in the same direction, contrasts with the position of the corresponding gene in the two heterocystous cyanobacteria. A putative hupW is also present in the draft genome of the heterocystous cyanobacterium A. variabilis (contig 249), while hupSL are located on contig 240 (http://genome.jgi-psf.org/draft_microbes/anava/anava.home.html). Screening the genome sequence of the marine filamentous non-heterocystous T. erythraeum (http://genome.jgi-psf.org/draft_microbes/trier/trier.home.html) also revealed the presence of an ORF showing a high degree of identity with hupW, and located 614 bp downstream of hupL. Deduced amino acid sequence alignments of the putative endopeptidase from Gloeothece sp. ATCC 27152 and the HupW from Anabaena/Nostoc PCC 7120, Nostoc PCC 73102 and T. erythraeum revealed an overall high identity (Fig. 2). The HupW protein of Gloeothece sp. ATCC 27152 contains the two conserved aspartic acid residues and the conserved histidine residue which, in HypD of E. coli, form a nickel-binding site (Fritsche et al., 1999). In Gloeothece sp. ATCC 27152, the C-terminal end of HupW, harbours six to ten additional amino acid residues in comparison to the corresponding protein in the other cyanobacterial strains.



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Fig. 2. Deduced amino acid sequence alignment of the putative hydrogenase-specific protease of Gloeothece sp. ATCC 27152 (HupW G27152) and the corresponding protein in Anabaena/Nostoc sp. PCC 7120 (HupW A7120), N. punctiforme PCC 73102 (HupW N73102) and T. erythraeum IMS 101 (HupW Te). Conserved nickel-coordinating amino acid residues are indicated by grey bars.

 
The hupSLW genes in Gloeothece sp. ATCC 27152 are co-transcribed
RT-PCR experiments, using total RNA extracted from Gloeothece sp. ATCC 27152 cells grown under nitrogen fixing conditions, were performed for the transcriptional analysis of the hupS, hupL and hupW genes. The culture was harvested 5 h into the dark phase, hydrogen uptake activity was confirmed using a hydrogen electrode (data not shown), and RNA was extracted. An RT reaction using GhupW1R as the hupW-specific antisense primer was performed, and the resulting cDNA was used as template in PCR amplifications for the detection of hupShupL and hupLhupW co-transcription. The results showed that the three genes (hupSLW) are indeed transcribed together (Fig. 3). The 3'-RACE results described above already indicated that hupW could be transcribed along with hupL. In the cyanobacterial strains studied so far, the hupSL genes constitute a single transcript with no additional ORFs (Happe et al., 2000; Lindberg et al., 2000). Moreover, the transcription of hupW in Anabaena/Nostoc PCC 7120 and N. punctiforme ATCC 29133/PCC 73102 has been shown to occur independently of that of hupSL (Wünschiers et al., 2003). Thus, this is the first time that the gene (hupW) encoding an uptake-hydrogenase-specific endopeptidase has been reported to be co-transcribed with the uptake hydrogenase structural genes (hupSL) in cyanobacteria.



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Fig. 3. RT-PCR detection of the hupSLW transcript in Gloeothece sp. ATCC 27152. Total RNA isolated from cells grown under nitrogen-fixing conditions was used in the RT reaction, with GhupW1R as the hupW-specific primer. cDNA produced in this reaction was used in PCR amplifications with the primer pairs GloS3'A/GloH1'R (544 bp fragment) and GloH6A/GhupW1R (511 bp fragment), for hupSL and hupLW detection, respectively. Lanes: 1, RT-PCR; 2, negative control without RT; 3, PCR negative control (no template added); 4, PCR positive control (genomic DNA added); M, 100 bp ladder (Amersham Biosciences).

 
Transcription start point identification and characterization of the hupS promoter region
5'-RACE permitted the identification of the transcription start point (tsp), 238 bp upstream from the hupS start codon (Fig. 1). In Gloeothece sp. ATCC 27152, a putative –10 box (TAATGT) is located six nucleotides upstream of the tsp, matching well with the consensus {sigma}70-like –10 box sequence (TAN3T) found in other cyanobacteria (Luque et al., 1994; Herrero et al., 2001). A putative NtcA binding site (GTAACCAAGATTAC) was also identified 22 nucleotides further upstream of the –10 region. This sequence contains the highly conserved palindromic NtcA-binding region signature GTAN8TAC (Luque et al., 1994; Herrero et al., 2001), and is flanked by T-rich sequences both upstream and downstream.

hupSLW transcription under different growth conditions
Northern blot experiments were performed with total RNA extracted from Gloeothece sp. ATCC 27152 cells grown under nitrogen-fixing or non-nitrogen fixing conditions, and 12 h light/12 h dark cycles. Samples were collected at four different time-points from both growth conditions (Fig. 4). The hybridizations were carried out using a hupS-specific probe. The results clearly show that the transcript(s) is present when Gloeothece sp. ATCC 27152 cells are grown under nitrogen-fixing conditions, but totally absent under non-nitrogen fixing conditions (NaNO3, Fig. 4). In addition, there is an evident light/dark regulation, with the highest transcript levels detected during the light cycle. This is interesting since Gloeothece sp. ATCC 27152 has been shown to fix nitrogen mainly in the dark (Reade et al., 1999), and consequently displays a higher hydrogen-uptake activity during the dark cycle (confirmed in this study using a hydrogen electrode, data not shown). The appearance of the transcript(s) prior to a detectable hydrogen uptake activity may be due to the fact that the uptake hydrogenase requires a complex maturation process. In contrast to heterocystous strains (Anabaena/Nostoc PCC 7120 and Nostoc PCC 73102), in which hupW is transcribed independently from hupSL (Wünschiers et al., 2003), hupSLW in Gloeothece sp. ATCC 27152 appear to be co-transcribed. This difference, together with the fact that there is a temporal separation between photosynthesis and nitrogen fixation/hydrogen-uptake activity in Gloeothece sp. ATCC 27152, may result in an extended period between transcription and activity. Furthermore, the Northern hybridizations revealed the presence of at least three different transcripts [or possibly a combination of transcript(s) and degradation products]. The largest transcript being approximately 3800 nt, and therefore probably corresponding to hupSLW, a smaller transcript of about 2000 nt (possibly a degradation product), and a third transcript of about 1200 nt possibly corresponding to hupS alone. These results corroborate the RT-PCR data (co-transcription of hupSLW), but also suggest the possibility of multiple transcripts.



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Fig. 4. hupSLW transcript(s) levels in Gloeothece sp. ATCC 27152 cells grown under a 12 h light/12 h dark cycle regime, and under N2-fixing (BG110) and non-N2-fixing (BG11) conditions. The probe used was generated with the primer pair GloS1A/GloSR1B. Fifteen micrograms total RNA was loaded in each lane. RNA was extracted from samples collected: 1 h prior to entering the dark cycle (lane L11); 6 h into the dark cycle (D6); 1 h prior to entering the light cycle (D11); 6 h into the light cycle (L6). The membrane was stripped and subsequently rehybridized to the RNA component of 16S rRNA (loading control). Positions of the Invitrogen 0·24–9·50 kb RNA ladder are indicated.

 
Binding of NtcA to the promoter region of the hupS gene
The potential NtcA binding site, identified in the Gloeothece sp. ATCC 27152 hupS promoter region, is centred at –41·5 bp with respect to the transcription start point. In order to confirm binding of NtcA to the promoter region, gel mobility shift DNA-binding assays were performed. Cell-free extracts were prepared from E. coli BL21(DE3) (pREP4, pCSAM70), where the overexpression of NtcA had been induced by 1 mM IPTG (Muro-Pastor et al., 1999). The mobility shift assays were carried out with a 366 bp, 32P-labelled DNA fragment covering the promoter region of hupS (–101 to +265). Fig. 5 shows that electrophoretic retardation of the labelled DNA fragment was significantly effected by the E. coli cell-free extract containing the overexpressed NtcA, whereas no retardation could be detected when a non-related E. coli cell-free extract (not carrying a cloned ntcA gene) was used. Band retardation of the 32P-labelled fragment was successfully outcompeted by the identical unlabelled DNA fragment (Fig. 5, lane 6). These results suggest that NtcA binds specifically to the identified putative site (GTAACCAAGATTAC) in the promoter region of hupS of Gloeothece sp. ATCC 27152, and thus may be involved in the transcriptional regulation of hupSLW.



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Fig. 5. Gel mobility shift analysis of NtcA binding to the target sequence in the promoter region upstream of hupS in Gloeothece sp. ATCC 27152. Five nanograms of the 366 bp labelled DNA fragment (–101 to +265) was used throughout the experiment. Lanes: 1, labelled DNA fragment only; 2–4, assays carried out with 500 ng, 1 µg and 10 µg total protein of E. coli cell-free extracts expressing NtcA, respectively; 5 and 6, assays carried out with 1 µg total protein, and in the presence of fivefold (lane 5) or 50-fold (lane 6) molar excess of the corresponding unlabelled fragment as competitor DNA; 7 and 8, 1 µg total protein and the presence of fivefold (lane 7) or 50-fold (lane 8) molar excess of an unrelated, unlabelled fragment; 9 and 10, assays carried out with 1 or 10 µg total protein of a E. coli cell-free extract not expressing NtcA, respectively.

 
A variety of sequence motifs involved in the binding of NtcA have been reported (Jiang et al., 2000; Herrero et al., 2001; Lindberg, 2003). TGT(N9/10)ACA has been suggested as a consensus recognition site in Anabaena/Nostoc PCC 7120 (Ramasubramanian et al., 1994), whereas the motif GTAN8TAC was described as being the NtcA binding site in a number of other cyanobacterial strains (Luque et al., 1994; Herrero et al., 2001). In a study examining the binding of NtcA to a library consisting of DNA fragments with 13 random nucleotides followed by ACA, an eight-base palindromic sequence (TGTAN8TACA) was found to be the optimal binding site (Jiang et al., 2000). The NtcA motif upstream of hupS in Gloeothece sp. ATCC 27152 matches well with the GTAN8TAC sequence signature, while the corresponding motif identified in N. punctiforme (Lindberg, 2003) is more similar to the consensus sequence from Anabaena/Nostoc PCC 7120. N. punctiforme is a heterocystous cyanobacterium and thus able to carry out nitrogen fixation concomitantly with photosynthesis (Whitton & Potts, 2000). The hupSL NtcA binding site in N. punctiforme falls into the same group of binding sites found in promoter regions of several genes regulated during heterocyst differentiation (Lindberg, 2003). Being a unicellular cyanobacterium, Gloeothece sp. ATCC 27152 fixes N2 almost exclusively during the dark, effecting a temporal separation between photosynthesis and N2 fixation (Reade et al., 1999). Therefore it is possible that the NtcA binding motif upstream of hupS in Gloeothece sp. ATCC 27152 has a different signature compared to N. punctiforme, due to the lack of cells specialized in N2 fixation.


   ACKNOWLEDGEMENTS
 
This work was financially supported by FCT (PRAXIS/P/BIA/13238/98, SFRH/BD/4912/2001, PRAXISXXI/BPD/20225/99). We gratefully acknowledge the late Professor John Gallon for supplying Gloeothece sp. strain ATCC 27152, Professor Enrique Flores for providing the NtcA clone, Professor Peter Lindblad for initial help with RT-PCRs, Dr Paula Coelho for skilful technical assistance with the gel mobility shift assays, and Joana Loureiro and Lígia Almeida for preliminary studies on the Gloeothece sp. ATCC 27152 uptake hydrogenase.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 16 April 2004; revised 28 July 2004; accepted 10 August 2004.



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