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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY260103.
Present address: Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, 75236 Uppsala, Sweden.
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INTRODUCTION |
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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.
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METHODS |
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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 0·51 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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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 6256 °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 0·51 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 [-32P]ATP, following the instructions of the manufacturer. The end-labelled fragment was separated from non-incorporated [
-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 0·510 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).
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RESULTS AND DISCUSSION |
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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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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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ACKNOWLEDGEMENTS |
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Received 16 April 2004;
revised 28 July 2004;
accepted 10 August 2004.
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