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Transcriptional regulation of Nostoc uptake hydrogenase
FEMS Microbiology Letters 170 (1999) 77^81
Transcriptional regulation of Nostoc uptake hydrogenase Rikard Axelsson *, Fredrik Oxelfelt, Peter Lindblad Department of Physiological Botany, Uppsala University, Villavaëgen 6, S-752 36 Uppsala, Sweden Received 23 September 1998 ; received in revised form 3 November 1998; accepted 4 November 1998
Abstract Filamentous, heterocystous cyanobacteria may contain both an uptake hydrogenase (encoded by hupSL) and a bidirectional enzyme (encoded by hoxFUYH). The present study identifies three strains (Anabaena variabilis, Nostoc muscorum and Nostoc sp. strain PCC 73102) with a contiguous hupL in both vegetative cells and heterocysts. The two Nostoc strains differ in either containing a bidirectional enzyme (N. muscorum) or lacking this enzyme (N. PCC 73102). Transcriptional studies, using reverse transcriptase-polymerase chain reaction, demonstrated an induction of a hupL transcript approximately 24 h after a shift from non-nitrogen-fixing to nitrogen-fixing conditions (in parallel with the induction of an in vivo light-dependent H2 -uptake activity) in N. muscorum. However, the level of hoxH transcripts did not change significantly during the induction of the H2 uptake activity. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cyanobacteria; RT-PCR; Hydrogen uptake ; hox; hup ; Transcriptional regulation
1. Introduction Hydrogen (H2 ) metabolism in nitrogen-¢xing ¢lamentous cyanobacteria involves at least three enzymes [1]. H2 is produced, concomitantly with the ¢xation of atmospheric nitrogen into ammonia, through the action of nitrogenase. The evolved H2 is recycled by an unidirectional uptake hydrogenase. In addition, a bidirectional hydrogenase, with the capacity to both take up and evolve H2 , may also be present. The structural genes encoding cyanobacterial up-
* Corresponding author. Tel.: +46 (18) 471-2814; Fax: +46 (18) 471-2826; E-mail: [email protected]
take and bidirectional hydrogenases have been sequenced and characterized in only few strains. The uptake hydrogenase is a dimeric enzyme consisting of a large subunit (encoded by hupL) and a small subunit (encoded by hupS) [1^3]. The cyanobacterial bidirectional hydrogenase is a tetrameric enzyme where the diaphorase moiety is encoded by hoxFU and the hydrogenase part by hoxYH [1,4,5]. The cyanobacterial uptake hydrogenase has been shown to be predominantly active in heterocysts, the site for N2 -¢xation [6]. In support of this, it was demonstrated that, in Anabaena sp. strain PCC 7120, hupL is transcribed in heterocysts only [2]. A 10.5-kb DNA fragment is excised within hupL by the site-speci¢c recombinase XisC (encoded by xisC) during the heterocyst di¡erentiation. In contrast, recent molecular experiments showed that no such re-
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 2 7 - 8
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arrangement occurs in Nostoc sp. strain PCC 73102 [3]. Regulatory studies demonstrated that nickel ions (Ni2 ) have a stimulatory e¡ect on the hydrogen uptake activity in a number of cyanobacteria [7^9]. Moreover, molecular hydrogen has been shown to induce higher hydrogen consumption activity [6,10]. In agreement, using an H2 -electrode, addition of nickel increased the in vivo hydrogen uptake activity in N. PCC 73102 [11]. Stimulatory e¡ects were also demonstrated by the addition of both H2 and organic carbon. Addition of combined nitrogen (in the form of ammonia) resulted in reduced in vivo H2 uptake activity indicating co-regulated nitrogenase and uptake hydrogenase activities [11]. Transcriptional studies of the uptake hydrogenase have been performed in many di¡erent organisms other than cyanobacteria. lacZ:hup fusions in Bradyrhizobium japonicum demonstrated a stimulatory e¡ect of both Ni2 and H2 at the transcriptional level [12]. However, the transcription was repressed by elevated levels of O2 . The knowledge about the transcription of genes encoding cyanobacterial hydrogenases is limited. Analyzing non-nitrogen-¢xing cells of A. PCC 7120 transferred to nitrogen-¢xing conditions demonstrated that the transcription of hupL is con¢ned to mature heterocysts [2]. In the present work, we continue our characterization of hydrogenases in ¢lamentous heterocystforming cyanobacteria and concentrate on two Nostoc strains containing a non-interrupted hupL in both vegetative cells and heterocysts. We initiate studies on the expression of the structural hup and hox genes, using RT-PCR, and examine the appearance of a hup transcript in a strain containing both hydrogenases (N. muscorum).
2. Materials and methods 2.1. Strains and culture conditions Cells of Anabaena sp. strain PCC 7120, A. variabilis ATCC 29413, Nostoc sp. strain PCC 73102, and N. muscorum Agardh CCAP 1453/12 were grown either in BG 110 ([13] all strains; isolation of genomic DNA) or in BG 110 supplemented with 5 mM NH4 Cl and 10 mM HEPES (pH 7.5) (N.
PCC 73102 and N. muscorum), and a continuous irradiance of 40 Wmol m32 s31 (see [5]). To induce heterocyst formation/nitrogen-¢xing conditions, the Nostoc cultures were pelleted by centrifugation, washed once in BG 110 medium (medium lacking combined nitrogen) before being resuspended in the same medium. All cultures were sparged with air and mixed with a magnetical stirrer. 2.2. Isolation of DNA, Southern blot hybridization, and DNA sequencing Genomic DNA was isolated from nitrogen-¢xing cultures of A. PCC 7120, A. variabilis ATCC 29413, N. PCC 73102, and N. muscorum Agardh CCAP 1453/12 as described [5]. Southern blot hybridizations (using cloned xisC as the probe, see below) and DNA sequencing (PCR-generated DNA fragments of N. muscorum) were performed as detailed before [3,5]. Cloned xisC from A. PCC 7120 [2], encoding the site-speci¢c recombinase XisC responsible for the excision of a 10.5-kb DNA fragment within hupL during the heterocyst di¡erentiation process, was kindly provided by Prof. J. Golden (Texas ApM University, College Station, TX, USA). 2.3. Hydrogen uptake activity In vivo hydrogen uptake activity was determined amperometrically with a Hansatech hydrogen electrode (Hansatech, King's Lynn, Norfolk, UK) as described [11]. 2.4. RNA isolation and DNase treatment Total RNA was isolated as described [14] with the following modi¢cations: The cells were frozen in liquid N2 only once, and all precipitations were performed with 99.5% ethanol. Forty units of RNase free DNase I (Amersham-Pharmacia Biotech, Uppsala, Sweden) were added to remove contaminating DNA, followed by a phenol/chloroform extraction. The RNA was resuspended in 20^30 Wl of sterile water. The quality of the RNA was checked on a 1% agarose gel, and the concentration was determined by measuring the absorbance at 260 nm.
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2.5. RT-PCR of hupL and hoxH transcripts, respectively Total RNA was reverse transcribed (RT) into cDNA as follows. An 11 Wl RT mixture containing 1.0 Wg total RNA and 200 nM antisense primer was incubated for 5 min at 70³C before being cooled on ice. The antisense primers were: 5P-CCACTTTTCATAATCAT-3P for hupL (based on the N. PCC 73102 sequence [3]), and 5P-GGAACAACTTAAACAGGGGTCAAAAGCCCGGAT-3P for hoxH (primer OL8B in [15]), respectively. A 4-Wl amount of 2.5 mM dNTP and 4 Wl of 5URT bu¡er (Promega) were added, and the mixture was incubated for 2 min at 42³C. Ten units of avian myeloblastosis virus reverse transcriptase (Promega) were then added to the mixture to give a ¢nal volume of 20 Wl. The mixture was incubated for 60 min at 42³C, denatured for 5 min at 95³C, before being cooled on ice. All PCRs (see [5]) were 50 Wl and contained 5 Wl from the RT reaction, 200 nM of each oligonucleotide primer, 0.2 mM dNTP, 1UTaq polymerase bu¡er (Amersham-Pharmacia Biotech), and 2.5 U Taq polymerase (Amersham-Pharmacia Biotech). The hupL cDNA sense primer was 5P-CTGTTGGGCGGACAATGGCCTCA-3P (based on the N. PCC 73102 sequence [3]) and the hoxH cDNA sense primer was 5P-CCACTATGCTCGTTTAATTGAAATTCT-3P (primer OL6A in [15]), respectively. The corresponding antisense primers were the same as in the previous RT-reactions. The RT-PCR of hupL and hoxH generated DNA fragments of 383 bp (based on the N. PCC 73102 sequence [3]) and 355 bp (based on the A. variabilis sequence [4]), respectively. Negative controls included no RT reaction (each RNA sample) and no RNA. None of the negative controls resulted in any ampli¢ed DNA fragments. Genomic DNA was used as a positive control.
3. Results and discussion Previous Southern blot hybridizations using a PCR-ampli¢ed DNA-fragment of hupL of Anabaena PCC 7120 as the probe [2] demonstrated the presence of homologous sequences in A. variabilis, Nostoc PCC 73102, and N. muscorum [5]. The A. PCC 7120 hupL undergoes a developmental rearrange-
Fig. 1. Presence/absence of the structural genes encoding an uptake hydrogenase (hup) and a bidirectional hydrogenase (hox) as well as the rearrangement within the large subunit of the uptake hydrogenase in two Anabaena and two Nostoc strains. (A) Southern blot hybridization demonstrating the presence/absence of DNA sequences homologous to cloned xisC from Anabaena PCC 7120 using genomic DNA, digested with HindIII, isolated from Nostoc PCC 73102 (N.73102), Anabaena PCC 7120 (A.7120), A. variabilis (A.v.), and N. muscorum (N.m.). (B) Summary of the present knowledge of the presence/absence of DNA sequences homologous to hup, xisC, and hox in Anabaena PCC 7120, A. variabilis, Nostoc PCC 73102, and N. muscorum. The two Nostoc strains were selected for further transcriptional analyzes.
ment in which approximately 10.5 kb are excised by the site-speci¢c recombinase XisC (encoded by xisC) late during the di¡erentiation process [2]. However, a non-interrupted hupL homolog was recently cloned and characterized in N. PCC 73102 [3]. In addition, both molecular and physiological experiments veri¢ed the presence of a bidirectional hydro-
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genase in A. PCC 7120, A. variabilis, and N. muscorum, whereas N. PCC 73102 lacks this enzyme [5]. Further Southern blot hybridizations (using cloned A. PCC 7120 xisC as the probe) demonstrated noninterrupted hupL genes (absence of xisC) in both A. variabilis and N. muscorum (Fig. 1). It also con¢rmed the absence of a developmental rearrangement in N. PCC 73102. Negative results (e.g. the absence of a speci¢c enzyme or a developmental rearrangement) are always more di¤cult to interpret (see [5]). The absence of any interrupting DNA fragment(s) within hupL of N. PCC 73102 and N. muscorum was con¢rmed by sequencing and Southern blot hybridizations ([3], data not shown). Interestingly, the nucleotide sequence of the recombination site in A. PCC 7120 and the corresponding sequence in N. muscorum were found to be identical, whereas they di¡er considerably with the sequence in N. PCC 73102 (Fig. 2). However, there is no correlation between the 16-bp nucleotide sequence, which in all three strains examined will be translated into the same deduced amino acid sequence, and the presence, or absence, of either an interrupting DNA fragment or xisC. The non-coding regions between hupSL in A. PCC 7120 and N. muscorum were of the same length and showed sequence identity (data not shown). The intergenic sequence in N. PCC 73102 is longer and shows little sequence identity [3]. The two Nostoc strains with non-interrupted hupL, and either containing a bidirectional enzyme (N. muscorum) or lacking this enzyme (N. PCC 73102) (see Fig. 1), were selected for further transcriptional analyses. Non-nitrogen-¢xing cultures (cells grown with the addition of ammonia) exhibited no in vivo light-dependent H2 -uptake activity (Fig. 3, [11]). Cells of N. muscorum were further analyzed in detail since they contain both an uptake hydrogenase and a bidirectional enzyme (Fig. 1B). A transfer to media lacking combined nitrogen induced the presence of an in
Fig. 2. Comparison of the nucleotide sequence of the recombination site within hupL of Anabaena PCC 7120 [2] with the corresponding potential sequences in Nostoc PCC 73102 [3] and Nostoc muscorum.
Fig. 3. Induction of an in vivo light-dependent H2 -uptake in non-nitrogen-¢xing cells of Nostoc muscorum transferred to a medium lacking combined nitrogen. Ammonia grown cells were transferred to nitrogen-¢xing conditions (time=0) and analyzed for the appearance of an in vivo light-dependent H2 -uptake (A), and the presence of hupL (B) and hoxH (C) transcripts after 14, 24, 41, and 61 h, respectively. (A) In vivo light-dependent H2 -uptake measured using an H2 -electrode. (B,C) Presence of hupL (B, left panel) and hoxH (C, left panel) transcripts visualized using RNA prepared from cells sampled at di¡erent time intervals (0, 14, 24, 41, and 61 h) and RT-PCR as detailed in Section 2 (negative images). M, Marker, 100-bp DNA-ladder. , Primer artefacts (see [16]). Controls (right panel) include only a PCR (-RT; all RNA samples, only t=0 h shown, negative control), replacing RNA with water (H2 O, negative control), and using genomic DNA instead of RNA (DNA, positive control).
vivo light-dependent H2 -uptake activity after 24^41 h (Fig. 3A). Similarly, a hupL transcript could be detected after 24 h (Fig. 3B), and the relative quantity increased in parallel with an increase in the in vivo light-dependent H2 -uptake activity (Fig. 3A,B). This is in agreement with an earlier observation using the heterocystous strain A. PCC 7120 in which a 10.5 DNA fragment prevents the transcription of hupL in vegetative cells [2]. However, in N. muscorum hupL is contiguous in both vegetative cells and heterocysts. Recently, the cellular pools of transcripts for both hydrogenases in Alcaligenes eutro-
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phus were shown to correlate with the activities of the respective promoters [17]. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Further studies are needed in order to identify the external/internal condition(s)/signal(s) initiating the transcription of hupL in heterocystous strains with a contiguous gene in both vegetative cells and heterocysts. hoxH transcripts (structural gene encoding the large subunit of the bidirectional hydrogenase) were detected throughout the induction of the in vivo light-dependent H2 -uptake activity with no apparent increase, or decrease, during nitrogen-¢xing conditions (Fig. 3C). The physiological function(s) of the bidirectional enzyme remains to be studied. Speci¢cally, the presence during nitrogen-¢xing conditions and the cellular localization of transcript, and corresponding polypeptide/enzyme, deserve additional attention.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments We thank P. Tamagnini for excellent technical assistance and Prof. J. Golden for providing the Anabaena sp. strain PCC 7120 xisC clone. This work was ¢nancially supported by grants from the Swedish National Energy Administration, and the Swedish Natural Science Research Council (P. Lindblad).
References
[12]
[13]
[14]
[15]
[1] Hansel, A. and Lindblad, P. (1998) Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source. Appl. Microbiol. Biotechnol. 50, 153^160. [2] Carrasco, C.D., Buettner, J.A. and Golden, J.W. (1995) Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts. Proc. Natl. Acad. Sci. USA 92, 791^795. [3] Oxelfelt, F., Tamagnini, P. and Lindblad, P. (1998) Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue. Arch. Microbiol. 169, 267^ 274. [4] Schmitz, P., Boison, G., Hilscher, R., Hundeshagen, B.,
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Zimmer, W., Lottspeich, F. and Bothe, H. (1995) Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria. Eur. J. Biochem. 233, 266^276. Tamagnini, P., Troshina, O., Oxelfelt, F., Salema, R. and Lindblad, P. (1997) Hydrogenases in Nostoc sp. strain PCC 73102, a strain lacking a bidirectional enzyme. Appl. Environ. Microbiol. 63, 1801^1807. Houchins, J.P. (1984) The physiology and biochemistry of hydrogen metabolism in cyanobacteria. Biochim. Biophys. Acta 768, 227^255. Daday, A., Mackerras, A. and Smith, G.D. (1985) The e¡ects of nickel on hydrogen metabolism and nitrogen ¢xation in the cyanobacterium Anabaena cylindrica. J. Gen. Microbiol. 131, 231^238. Papen, H., Kentemich, T., Schmuëlling, T. and Bothe, H. (1986) Hydrogenase activities in cyanobacteria. Biochimie 68, 121^132. Xiankong, Z., Tabita, F. and Van Baalen, C. (1984) Nickel control of hydrogen production and uptake in Anabaena spp. strains CA and 1F. J. Gen. Microbiol. 130, 1815^1818. Eisbrenner, G., Distler, E., Floener, L. and Bothe, H. (1978) The occurrence of the hydrogenase in some blue-green algae. Arch. Microbiol. 118, 177^184. Oxelfelt, F., Tamagnini, P., Salema, R. and Lindblad, P. (1995) Hydrogen uptake in Nostoc strain PCC 73102: e¡ects of nickel, hydrogen, carbon and nitrogen. Plant Physiol. Biochem. 33, 617^623. Kim, H., Yu, C. and Maier, R.J. (1991) Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen. J. Bacteriol. 173, 3993^3999. Stanier, R.Y., Kunisawa, P., Mandel, M. and Cohen-Bazir, G. (1971) Puri¢cation and properties of unicellular blue-green algae (order Chrococcales). Bacteriol. Rev. 35, 171^205. Mohammed, A. and Jansson, C. (1989) In£uence of light on accumulation of photosynthesis-speci¢c transcripts in the cyanobacterium Synechocystis 6803. Plant Mol. Biol. 13, 693^ 700. Lindblad, P., Oxelfelt, F., Tamagnini, P. and Troshina, O. 1998. Cyanobacterial biotechnology. Nostoc PCC 73102 and H2 : Knowledge, research and biotechnological challenges. In: Cyanobacterial Biotechnology (Subramanian, G., Kaushik, B.D. and Venkataraman, G.S., Eds.), pp. 1^5. Oxford and IBH, New Delhi, India. Chandler, D.P., Wagnon, C.A. and Bolton, Jr., H. (1998) Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RTPCR. Appl. Environ. Microbiol. 64, 669^677. Schwartz, E., Gerischer, U. and Friedrich, B. (1998) Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes. J. Bacteriol. 180, 3197^3204.
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Transcriptional regulation of Nostoc uptake hydrogenase Rikard Axelsson *, Fredrik Oxelfelt, Peter Lindblad Department of Physiological Botany, Uppsala University, Villavaëgen 6, S-752 36 Uppsala, Sweden Received 23 September 1998 ; received in revised form 3 November 1998; accepted 4 November 1998
Abstract Filamentous, heterocystous cyanobacteria may contain both an uptake hydrogenase (encoded by hupSL) and a bidirectional enzyme (encoded by hoxFUYH). The present study identifies three strains (Anabaena variabilis, Nostoc muscorum and Nostoc sp. strain PCC 73102) with a contiguous hupL in both vegetative cells and heterocysts. The two Nostoc strains differ in either containing a bidirectional enzyme (N. muscorum) or lacking this enzyme (N. PCC 73102). Transcriptional studies, using reverse transcriptase-polymerase chain reaction, demonstrated an induction of a hupL transcript approximately 24 h after a shift from non-nitrogen-fixing to nitrogen-fixing conditions (in parallel with the induction of an in vivo light-dependent H2 -uptake activity) in N. muscorum. However, the level of hoxH transcripts did not change significantly during the induction of the H2 uptake activity. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cyanobacteria; RT-PCR; Hydrogen uptake ; hox; hup ; Transcriptional regulation
1. Introduction Hydrogen (H2 ) metabolism in nitrogen-¢xing ¢lamentous cyanobacteria involves at least three enzymes [1]. H2 is produced, concomitantly with the ¢xation of atmospheric nitrogen into ammonia, through the action of nitrogenase. The evolved H2 is recycled by an unidirectional uptake hydrogenase. In addition, a bidirectional hydrogenase, with the capacity to both take up and evolve H2 , may also be present. The structural genes encoding cyanobacterial up-
* Corresponding author. Tel.: +46 (18) 471-2814; Fax: +46 (18) 471-2826; E-mail: [email protected]
take and bidirectional hydrogenases have been sequenced and characterized in only few strains. The uptake hydrogenase is a dimeric enzyme consisting of a large subunit (encoded by hupL) and a small subunit (encoded by hupS) [1^3]. The cyanobacterial bidirectional hydrogenase is a tetrameric enzyme where the diaphorase moiety is encoded by hoxFU and the hydrogenase part by hoxYH [1,4,5]. The cyanobacterial uptake hydrogenase has been shown to be predominantly active in heterocysts, the site for N2 -¢xation [6]. In support of this, it was demonstrated that, in Anabaena sp. strain PCC 7120, hupL is transcribed in heterocysts only [2]. A 10.5-kb DNA fragment is excised within hupL by the site-speci¢c recombinase XisC (encoded by xisC) during the heterocyst di¡erentiation. In contrast, recent molecular experiments showed that no such re-
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 2 7 - 8
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arrangement occurs in Nostoc sp. strain PCC 73102 [3]. Regulatory studies demonstrated that nickel ions (Ni2 ) have a stimulatory e¡ect on the hydrogen uptake activity in a number of cyanobacteria [7^9]. Moreover, molecular hydrogen has been shown to induce higher hydrogen consumption activity [6,10]. In agreement, using an H2 -electrode, addition of nickel increased the in vivo hydrogen uptake activity in N. PCC 73102 [11]. Stimulatory e¡ects were also demonstrated by the addition of both H2 and organic carbon. Addition of combined nitrogen (in the form of ammonia) resulted in reduced in vivo H2 uptake activity indicating co-regulated nitrogenase and uptake hydrogenase activities [11]. Transcriptional studies of the uptake hydrogenase have been performed in many di¡erent organisms other than cyanobacteria. lacZ:hup fusions in Bradyrhizobium japonicum demonstrated a stimulatory e¡ect of both Ni2 and H2 at the transcriptional level [12]. However, the transcription was repressed by elevated levels of O2 . The knowledge about the transcription of genes encoding cyanobacterial hydrogenases is limited. Analyzing non-nitrogen-¢xing cells of A. PCC 7120 transferred to nitrogen-¢xing conditions demonstrated that the transcription of hupL is con¢ned to mature heterocysts [2]. In the present work, we continue our characterization of hydrogenases in ¢lamentous heterocystforming cyanobacteria and concentrate on two Nostoc strains containing a non-interrupted hupL in both vegetative cells and heterocysts. We initiate studies on the expression of the structural hup and hox genes, using RT-PCR, and examine the appearance of a hup transcript in a strain containing both hydrogenases (N. muscorum).
2. Materials and methods 2.1. Strains and culture conditions Cells of Anabaena sp. strain PCC 7120, A. variabilis ATCC 29413, Nostoc sp. strain PCC 73102, and N. muscorum Agardh CCAP 1453/12 were grown either in BG 110 ([13] all strains; isolation of genomic DNA) or in BG 110 supplemented with 5 mM NH4 Cl and 10 mM HEPES (pH 7.5) (N.
PCC 73102 and N. muscorum), and a continuous irradiance of 40 Wmol m32 s31 (see [5]). To induce heterocyst formation/nitrogen-¢xing conditions, the Nostoc cultures were pelleted by centrifugation, washed once in BG 110 medium (medium lacking combined nitrogen) before being resuspended in the same medium. All cultures were sparged with air and mixed with a magnetical stirrer. 2.2. Isolation of DNA, Southern blot hybridization, and DNA sequencing Genomic DNA was isolated from nitrogen-¢xing cultures of A. PCC 7120, A. variabilis ATCC 29413, N. PCC 73102, and N. muscorum Agardh CCAP 1453/12 as described [5]. Southern blot hybridizations (using cloned xisC as the probe, see below) and DNA sequencing (PCR-generated DNA fragments of N. muscorum) were performed as detailed before [3,5]. Cloned xisC from A. PCC 7120 [2], encoding the site-speci¢c recombinase XisC responsible for the excision of a 10.5-kb DNA fragment within hupL during the heterocyst di¡erentiation process, was kindly provided by Prof. J. Golden (Texas ApM University, College Station, TX, USA). 2.3. Hydrogen uptake activity In vivo hydrogen uptake activity was determined amperometrically with a Hansatech hydrogen electrode (Hansatech, King's Lynn, Norfolk, UK) as described [11]. 2.4. RNA isolation and DNase treatment Total RNA was isolated as described [14] with the following modi¢cations: The cells were frozen in liquid N2 only once, and all precipitations were performed with 99.5% ethanol. Forty units of RNase free DNase I (Amersham-Pharmacia Biotech, Uppsala, Sweden) were added to remove contaminating DNA, followed by a phenol/chloroform extraction. The RNA was resuspended in 20^30 Wl of sterile water. The quality of the RNA was checked on a 1% agarose gel, and the concentration was determined by measuring the absorbance at 260 nm.
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2.5. RT-PCR of hupL and hoxH transcripts, respectively Total RNA was reverse transcribed (RT) into cDNA as follows. An 11 Wl RT mixture containing 1.0 Wg total RNA and 200 nM antisense primer was incubated for 5 min at 70³C before being cooled on ice. The antisense primers were: 5P-CCACTTTTCATAATCAT-3P for hupL (based on the N. PCC 73102 sequence [3]), and 5P-GGAACAACTTAAACAGGGGTCAAAAGCCCGGAT-3P for hoxH (primer OL8B in [15]), respectively. A 4-Wl amount of 2.5 mM dNTP and 4 Wl of 5URT bu¡er (Promega) were added, and the mixture was incubated for 2 min at 42³C. Ten units of avian myeloblastosis virus reverse transcriptase (Promega) were then added to the mixture to give a ¢nal volume of 20 Wl. The mixture was incubated for 60 min at 42³C, denatured for 5 min at 95³C, before being cooled on ice. All PCRs (see [5]) were 50 Wl and contained 5 Wl from the RT reaction, 200 nM of each oligonucleotide primer, 0.2 mM dNTP, 1UTaq polymerase bu¡er (Amersham-Pharmacia Biotech), and 2.5 U Taq polymerase (Amersham-Pharmacia Biotech). The hupL cDNA sense primer was 5P-CTGTTGGGCGGACAATGGCCTCA-3P (based on the N. PCC 73102 sequence [3]) and the hoxH cDNA sense primer was 5P-CCACTATGCTCGTTTAATTGAAATTCT-3P (primer OL6A in [15]), respectively. The corresponding antisense primers were the same as in the previous RT-reactions. The RT-PCR of hupL and hoxH generated DNA fragments of 383 bp (based on the N. PCC 73102 sequence [3]) and 355 bp (based on the A. variabilis sequence [4]), respectively. Negative controls included no RT reaction (each RNA sample) and no RNA. None of the negative controls resulted in any ampli¢ed DNA fragments. Genomic DNA was used as a positive control.
3. Results and discussion Previous Southern blot hybridizations using a PCR-ampli¢ed DNA-fragment of hupL of Anabaena PCC 7120 as the probe [2] demonstrated the presence of homologous sequences in A. variabilis, Nostoc PCC 73102, and N. muscorum [5]. The A. PCC 7120 hupL undergoes a developmental rearrange-
Fig. 1. Presence/absence of the structural genes encoding an uptake hydrogenase (hup) and a bidirectional hydrogenase (hox) as well as the rearrangement within the large subunit of the uptake hydrogenase in two Anabaena and two Nostoc strains. (A) Southern blot hybridization demonstrating the presence/absence of DNA sequences homologous to cloned xisC from Anabaena PCC 7120 using genomic DNA, digested with HindIII, isolated from Nostoc PCC 73102 (N.73102), Anabaena PCC 7120 (A.7120), A. variabilis (A.v.), and N. muscorum (N.m.). (B) Summary of the present knowledge of the presence/absence of DNA sequences homologous to hup, xisC, and hox in Anabaena PCC 7120, A. variabilis, Nostoc PCC 73102, and N. muscorum. The two Nostoc strains were selected for further transcriptional analyzes.
ment in which approximately 10.5 kb are excised by the site-speci¢c recombinase XisC (encoded by xisC) late during the di¡erentiation process [2]. However, a non-interrupted hupL homolog was recently cloned and characterized in N. PCC 73102 [3]. In addition, both molecular and physiological experiments veri¢ed the presence of a bidirectional hydro-
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genase in A. PCC 7120, A. variabilis, and N. muscorum, whereas N. PCC 73102 lacks this enzyme [5]. Further Southern blot hybridizations (using cloned A. PCC 7120 xisC as the probe) demonstrated noninterrupted hupL genes (absence of xisC) in both A. variabilis and N. muscorum (Fig. 1). It also con¢rmed the absence of a developmental rearrangement in N. PCC 73102. Negative results (e.g. the absence of a speci¢c enzyme or a developmental rearrangement) are always more di¤cult to interpret (see [5]). The absence of any interrupting DNA fragment(s) within hupL of N. PCC 73102 and N. muscorum was con¢rmed by sequencing and Southern blot hybridizations ([3], data not shown). Interestingly, the nucleotide sequence of the recombination site in A. PCC 7120 and the corresponding sequence in N. muscorum were found to be identical, whereas they di¡er considerably with the sequence in N. PCC 73102 (Fig. 2). However, there is no correlation between the 16-bp nucleotide sequence, which in all three strains examined will be translated into the same deduced amino acid sequence, and the presence, or absence, of either an interrupting DNA fragment or xisC. The non-coding regions between hupSL in A. PCC 7120 and N. muscorum were of the same length and showed sequence identity (data not shown). The intergenic sequence in N. PCC 73102 is longer and shows little sequence identity [3]. The two Nostoc strains with non-interrupted hupL, and either containing a bidirectional enzyme (N. muscorum) or lacking this enzyme (N. PCC 73102) (see Fig. 1), were selected for further transcriptional analyses. Non-nitrogen-¢xing cultures (cells grown with the addition of ammonia) exhibited no in vivo light-dependent H2 -uptake activity (Fig. 3, [11]). Cells of N. muscorum were further analyzed in detail since they contain both an uptake hydrogenase and a bidirectional enzyme (Fig. 1B). A transfer to media lacking combined nitrogen induced the presence of an in
Fig. 2. Comparison of the nucleotide sequence of the recombination site within hupL of Anabaena PCC 7120 [2] with the corresponding potential sequences in Nostoc PCC 73102 [3] and Nostoc muscorum.
Fig. 3. Induction of an in vivo light-dependent H2 -uptake in non-nitrogen-¢xing cells of Nostoc muscorum transferred to a medium lacking combined nitrogen. Ammonia grown cells were transferred to nitrogen-¢xing conditions (time=0) and analyzed for the appearance of an in vivo light-dependent H2 -uptake (A), and the presence of hupL (B) and hoxH (C) transcripts after 14, 24, 41, and 61 h, respectively. (A) In vivo light-dependent H2 -uptake measured using an H2 -electrode. (B,C) Presence of hupL (B, left panel) and hoxH (C, left panel) transcripts visualized using RNA prepared from cells sampled at di¡erent time intervals (0, 14, 24, 41, and 61 h) and RT-PCR as detailed in Section 2 (negative images). M, Marker, 100-bp DNA-ladder. , Primer artefacts (see [16]). Controls (right panel) include only a PCR (-RT; all RNA samples, only t=0 h shown, negative control), replacing RNA with water (H2 O, negative control), and using genomic DNA instead of RNA (DNA, positive control).
vivo light-dependent H2 -uptake activity after 24^41 h (Fig. 3A). Similarly, a hupL transcript could be detected after 24 h (Fig. 3B), and the relative quantity increased in parallel with an increase in the in vivo light-dependent H2 -uptake activity (Fig. 3A,B). This is in agreement with an earlier observation using the heterocystous strain A. PCC 7120 in which a 10.5 DNA fragment prevents the transcription of hupL in vegetative cells [2]. However, in N. muscorum hupL is contiguous in both vegetative cells and heterocysts. Recently, the cellular pools of transcripts for both hydrogenases in Alcaligenes eutro-
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phus were shown to correlate with the activities of the respective promoters [17]. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Further studies are needed in order to identify the external/internal condition(s)/signal(s) initiating the transcription of hupL in heterocystous strains with a contiguous gene in both vegetative cells and heterocysts. hoxH transcripts (structural gene encoding the large subunit of the bidirectional hydrogenase) were detected throughout the induction of the in vivo light-dependent H2 -uptake activity with no apparent increase, or decrease, during nitrogen-¢xing conditions (Fig. 3C). The physiological function(s) of the bidirectional enzyme remains to be studied. Speci¢cally, the presence during nitrogen-¢xing conditions and the cellular localization of transcript, and corresponding polypeptide/enzyme, deserve additional attention.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments We thank P. Tamagnini for excellent technical assistance and Prof. J. Golden for providing the Anabaena sp. strain PCC 7120 xisC clone. This work was ¢nancially supported by grants from the Swedish National Energy Administration, and the Swedish Natural Science Research Council (P. Lindblad).
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