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CURRENT MICROBIOLOGY Vol. 40 (2000), pp. 315–321 DOI: 10.1007/s002849910063 An International Journal R Springer-Verlag New York Inc. 2000 Transcriptional Analysis of Hydrogenase Genes in the Cyanobacteria Anacystis nidulans and Anabaena variabilis Monitored by RT-PCR Gudrun Boison,* Hermann Bothe, Oliver Schmitz Botanisches Institut, Universität zu Köln, Gyrhofstr. 15, D-50923 Köln, Germany Received: 22 October 1999 / Accepted: 22 November 1999 Abstract. Diverse cyanobacteria express an uptake hydrogenase, encoded by the genes hupSL, and a bidirectional, NAD(P) ⫹-reducing hydrogenase with the genes hox(E)FUYH. In the unicellular Anacystis nidulans, the hox genes are organized on two separate loci, whereas they are contiguous in one cluster, though interspersed with two unidentified reading frames, ORF 3 and 8, in the heterocystous Anabaena variabilis. The hox gene clusters of these two cyanobacteria have now been transcriptionally analyzed by RT-PCR. A polycistronic transcript was identified in both cyanobacteria. In A. nidulans, one message for each locus has been detected, the dicistronic hoxEF unit, and the polycistronic hoxUYHWhypAB one. In A. variabilis, the transcript consists of the hox genes hoxFUYH as well as the unidentified ORFs. Previous enzyme determinations on the occurrence of the uptake hydrogenase in vegetative cells and thus outside of heterocysts gave ambiguous results. Therefore, transcription of both hup and hox genes has been analyzed in both heterocysts and vegetative cells of A. variabilis. A hupL transcript is detectable in heterocysts and also, though less extensive but clearly discernible, in vegetative cells of NH4⫹-grown A. variabilis. In cyanobacteria, two different NiFe-hydrogenases have been characterized physiologically, biochemically [19, 29], and recently on the molecular level by identifying and sequencing the genes [1, 3, 4, 6, 15, 17, 26, 28, 34]. One of these enzymes, the so-called uptake hydrogenase, catalyzes only H2-oxidation in vitro. It has been described to be confined mainly [10] or exclusively [30] to heterocysts, where it utilizes the H2 formed in parallel with N2-reduction catalyzed by nitrogenase. This hydrogenase transfers the electrons from H2 to either photosystem I or to the respiratory electron transport chain to O2 [9]. Thus, the uptake hydrogenase seemingly has a beneficial role for the cells by providing energy and reductant for N2-fixation and other metabolic processes. The genes of this dimeric hydrogenase are hupL and hupS coding for the large and small subunit, respectively. They have been described for Anabaena sp. PCC 7120 [6], Nostoc sp. PCC 73102 [28], and A. variabilis [17]. The hupL gene undergoes a programmed DNA rearrangement during heterocyst differentiation in A. PCC 7120 [6, 24], but not * Present address: Folkehelsa, National Institute of Public Health, Postboks 4404 Torshov, N-0403 Oslo, Norway. Correspondence to: G. Boison at Oslo in N. PCC 73102 [28] and A. variabilis [2; G. Boison, unpublished]. The bidirectional hydrogenase catalyzes in vitro not only H2-uptake, but also the Na2S2O4/methylviologendependent evolution of the gas. The enzyme is active in heterocysts as well as in vegetative cells and in cells of unicellular cyanobacteria [19, 29]. It occurs in most species so far examined, but is missing in the filamentous strain N. PCC 73102 [5, 39]. The bidirectional hydrogenase genes have been characterized for A. variabilis [34], A. nidulans [3, 4], and Synechocystis sp. PCC 6803 [1, 26]. They are highly homologous to those coding for the NAD⫹-reducing hydrogenase of Alcaligenes eutrophus [11] and the NADP⫹-dependent hydrogenase of Desulfovibrio fructosovorans [22]. Sequence comparisons and protein purifications indicated that the cyanobacterial enzyme consists, like the one of A. eutrophus, of two entities [34, 37]. The hydrogenase dimer HoxYH catalyzes the H2-oxidation, whereas the diaphorase moiety HoxFU acts in transferring electrons to NAD(P)⫹. These implications from sequence data have been ascertained by enzyme measurements in crude extracts from A. nidulans [32]. An additional ORF, termed hoxE [1], described for unicellular strains, has been suggested to 316 code for a third diaphorase subunit [4]. The diaphorase subunits Hox(E)FU have been discussed to also serve as components of the NADH:ubiquinone oxidoreductase of respiratory complex I [1, 4, 5, 33]. In A. variabilis and S. PCC 6803, different ORFs of unknown functions are interspersed within the hox cluster at different positions [4]. In contrast, the hox genes of A. nidulans are contiguous, but hoxEF and hoxUYH are located on different loci. A gene set coding for accessory hydrogenase proteins is located downstream of hoxUYH in A. nidulans. These genes are spread over the genome in S. PCC 6803 [26] and have not yet been characterized for A. variabilis. Although the hydrogenase genes from several cyanobacteria have been described up to now, transcript analyses of the hup and hox genes have been performed solely for Nostoc muscorum CCAP 1453/12 [2]. By RT-PCR, these authors demonstrated a constitutive transcription for hoxH both under N2- and non-N2-fixing conditions, whereas a hupL transcript was detected under N2-fixing conditions only. Currently, the operon structure, location of promoters, and regulation of transcription are still unknown for this and all other cyanobacteria. In particular, no information is available whether the unidentified ORFs in the hox clusters are transcribed at all. The complete transcriptional analyses of the hoxclusters of A. variabilis and A. nidulans are shown by RT-PCR in the present communication. Experiments with isolated heterocysts and vegetative cells of A. variabilis have also been performed to examine the occurrence of transcripts of the uptake and the bidirectional hydrogenase in both cell types. Materials and Methods Culture and growth of cyanobacteria. A. nidulans (⫽ Synechococcus leopoliensis sp. PCC 6301), purchased from the Algensammlung des Pflanzenphysiologischen Instituts der Universität Göttingen, Germany (SAUG 1402-1) and A. variabilis ATCC 29413 were grown in BG11 medium with or without nitrate as described [34]. To obtain heterocystfree cultures of A. variabilis, cells were grown in nitrate-free BG11 medium with 5 mM NH4Cl. The medium was buffered with 2.1 mM HEPES and adjusted to pH 8.0 with NaOH prior to autoclaving. NH4⫹-grown A. variabilis filaments were free of heterocysts as examined by light microscopy. Heterocyst preparation. The preparation of heterocysts from N2-fixing cultures of A. variabilis [13] was slightly modified. An N2-fixing culture (750 ml) was harvested by centrifugation (10 min, 3000 g, 4°C). The pellet was resuspended in 5 ml cold (4°C) STET (8% sucrose, 5% Triton X-100, 50 mM EDTA pH 8.0, 50 mM Tris-Cl pH 8.0) with 1 mg/ml lysozyme and vortexed (2–3 min, 25°C). Further homogenization was achieved by sonification (Ultrasonics model W-225R, maximal output, 3 min on ice). Intact heterocysts were separated from broken vegetative cells by centrifugation (5 min, 3000 g, 4°C). The sedimented heterocysts were washed 4⫻ in SET (as STET, without Triton) followed by centrifugation (5 min, 4°C) with decreasing g-values (2000 g, 300 g, CURRENT MICROBIOLOGY Vol. 40 (2000) 2 ⫻ 200 g). The last pellet was frozen in liquid nitrogen for subsequent RNA preparation. The heterocyst fraction was controlled microscopically and revealed no contaminating vegetative cells. Isolation of total RNA. Total RNA from both A. variabilis and A. nidulans was isolated by a combination of different standard techniques with the RNeasy Kit supplied by Qiagen (D-Hilden). In the case of A. variabilis, either 50 ml of an N2-fixing or NH4⫹-grown culture or the heterocyst preparation from 750 ml of an N2-fixing culture were harvested and frozen in liquid nitrogen. Cells were crushed with a pestle and mortar in liquid N2 in the presence of 1 ml RLT buffer from the RNeasy Kit, supplemented with 10 µl -mercaptoethanol, were then heated (2 min, 50°C), extracted once with acidic phenol/chloroform/ isoamylalcohol (25/24/1, vol/vol/vol) and twice with chloroform/ isoamylalcohol, followed by centrifugation in a microfuge (5 min, 18,000 g, 4°C). The supernatant was purified with the columns of the RNeasy Kit according to the manufacturer’s protocol. The RNA was eluted from the column twice with 30 µl H2O, and the two fractions were combined. To remove contaminating DNA, the eluate was precipitated with 3 vol of 4 M Na-acetate pH 7.0 (1 h, ⫺20°C). The RNA sedimented in a microfuge (20 min, 5000 g, 0°C), and the pellet was resuspended in 50 µl DEPC-H2O. The amount and purity of the RNA was determined from the optical density at 260 nm and 280 nm. Each preparation was monitored for contaminating DNA on an agarose gel and also by PCR. Total RNA from A. nidulans was prepared in a different way (K.-P. Michel, D-Bielefeld, personal communication) from 20 ml cells, which were harvested by centrifugation (10 min, 4000 g, 4°C), washed in 1 ml Bgl1 medium, centrifuged, and resuspended in 100 µl TE buffer. After addition of 350 µl RLT buffer from the RNeasy Kit (supplemented with -mercaptoethanol), the cells were vortexed extensively and placed on ice. Lysis was completed by incubating in 300 µl prewarmed acidic phenol (10 min, 65°C) and vortexing. After addition of 300 µl chloroform/isoamylalcohol and vortexing, the preparation was centrifuged (5 min, 18,000 g, 4°C), and extracted 1–2⫻ by chloroform/ isoamylalcohol. The supernatant was then transferred to the RNeasy Kit column and treated as in the case of A. variabilis RNA. Reverse transcription (RT) and PCR. Prior to RT, 2 µg RNA was digested with 2 U DNase (deoxyribonuclease I, amplification grade, Gibco/BRL) in a volume of 20 µl (15 min, 25°C) with the buffer supplied by the manufacturer. The digestion was stopped by adding 2 µl 25 mM EDTA and incubating (10 min, 65°C). The sample was split into two aliquots. One was filled up to 20 µl with DEPC-H2O and used as a control. The other was used for reverse transcription with Superscript II (RNase H⫺ reverse transcriptase, Gibco/BRL) according to the manufacturer’s protocol, with the following exceptions: Sequence-specific primers were used (2 pmol, Table 1), the amount of reaction buffer was reduced to 2.6 µl, the assay was incubated (2 min, 47°C), 200 U Superscript II was added, and the incubation was continued (50 min, 47°C). No RNase H digestion was performed. The PCR was performed either with 2 µl (up to 5 µl) of the cDNA obtained by the transcription reaction, 2 µl (up to 5 µl) of the control tube, or 2–5 ng genomic DNA in a 50-µl assay (100 pmol of each primer, 2 mM MgSO4, 2 U Taq polymerase from Promega) running 35 cycles after 4 min at 95°C (program: 1 min 92°C, 1 min 50/56°C for A. variabilis/A. nidulans, respectively, 1.5 min 72°C) and a final elongation step of 7 min at 72°C. The PCR products obtained were separated on 1–1.5% horizontal agarose gels with 10 µl of each reaction. Results Transcriptional analysis of the hox cluster of A. nidulans. Total RNA of A. nidulans prepared from exponentially growing cultures was digested by DNase to 317 G. Boison et al.: Hydrogenase Genes in Cyanobacteria Table 1. Primers used for reverse transcription and subsequent PCR. The positions of the primers refer to the nucleotide numbers of the corresponding EMBL entry: AN ⫽ Anacystis nidulans: Y13471 (hoxEF), X97797 (hoxU–hypF); AV ⫽ Anabaena variabilis: X79285, 7120 ⫽ Anabaena PCC 7120: U08013.1. Reverse primers are marked by an asterisk. IS F/U: intergenic spacer between hoxF and hoxU Primer Gene/ specificity EAn1 EAn2 FAn1 FAnv3 FAn4 FAv1 FAv2 FAv4 Av6 UAn1 UAn2 UAn3 UAv2 UAv3 8Av1 8Av2 YAnv1 YAnv2 YAv3 HAn2 HAnv3 HAv1 W1 W2 A1 A2 B4 B5 hypF1 hypF2 H4A [39] H6B [39] hoxE-AN hoxE-AN hoxF-AN hoxF-AN hoxF-AN hoxF-AV hoxF-AV hoxF-AV IS F/U-AV hoxU-AN hoxU-AN hoxU-AN hoxU-AV hoxU-AV ORF8-AV ORF8-AV hoxY-AN hoxY-AN hoxY-AV hoxH-AN hoxH-AN hoxH-AV hoxW-AN hoxW-AN hypA-AN hypA-AN hypB-AN hypB-AN hypF-AN hypF-AN hupL-7120 hupL-7120 Sequence 58 = 38 Position ctacttctgaaacgacaccc cttggacttgttgccagacc atttggggcgtttggctaac tttcaagccggtgggatagc tggtagtcatggacgag gatcgctgcctatgctgtgg accaagaccacacaagctgg tcagtctgcacctaatccgg gcgccagtttccccttcaag gatgtctgtcgtcaccttac gtgcttcggccaagaacgc agacgacagcgtcctac ggtaaatgcgtcaatgcttg aacacggatgcagcgagtac tgtcgatgtgcttatctccg tgccaatcttgaacaacgcg gmtgttcgggytgccayatg armggcggcacaattccggg aaggacatatgacagccagaac caggaaaggcagggrtcaaar gtaagtgaggcacctmgtggk atactaatcttggcgtgacc cactccaccatccgtgcaatcg gtaatcggctatggcaatgc cgcagcaaatcgtcagtctg ggaacaggcagctaacagcg tgccagcgatcgcttcc atcgtgggtgatctggc cgctcagattgaagtgg gtgcgattgcgatcgtaggg gaagtcggccccctagcccgc gtggacagtacacaccagacaagagtcaaa 78–97 *529–548 584–603 *1085–1104 1853–1869 831–850 *1661–1680 1680–1699 *2373–2392 0–19 *673–691 *130–146 3298–3317 *3164–3183 3602–3621 *4096–4115 742–761 *1083–1102 *4253–4274 *2588–2608 2385–2405 *5560–5579 *2899–2920 2717–2736 3221–3240 *3401–3420 *3427–3443 3736–3752 4436–4452 *4740–4759 2189–2209 *2642–2671 remove any residual DNA. All preparations were controlled for contaminating DNA by PCR with appropriate primers (not documented, but see Fig. 3). The DNA-free RNA aliquots were used for several RT assays with an RT primer for the 38 end of each gene of the hox cluster (Fig. 1, Table 1). The cDNA fractions obtained were used in PCR reactions. By applying primer pairs for each gene lying upstream of the primer that was used for the reverse transcription, co-transcription of genes could be demonstrated (Fig. 1). The data obtained indicated that the potential diaphorase gene hoxE [4] is co-transcribed with hoxF. PCR amplificates have been obtained both for hoxE and hoxF with the same cDNA generated with primer FAnv3. No amplificate of either hoxE or hoxF was obtained from cDNA generated by primer UAn2, thus ascertaining that hoxEF are transcribed separately from the residual hydrogenase genes. By using either RT primer B4, A2, W1, HAn2, YAnv2, or UAn2 (Table 1) followed by PCR on all cDNAs generated, transcription of all genes lying upstream of the corresponding RT primer was demonstrated (Fig. 1). This implies that the genes hoxUYHWhypAB are transcribed as a unit. Transcription of hypF has also been shown. However, no PCR products of the expected size were obtained with the primer pairs A1/B4 and B5/hypF2 with cDNA generated with RT primer hypF2. This might indicate that hypF is transcribed separately from hoxUYHWhypAB. Transcriptional characterization of the hox cluster from A. variabilis. In the same way as for A. nidulans, DNA-free total RNA of A. variabilis was transcribed with reverse primers for the genes hoxF, hoxU, ORF8, hoxY, or hoxH (Fig. 2). Subsequent PCRs with corresponding primers (Table 1) resulted in amplificates for hoxF with cDNA derived from RT primer FAv2. Similarly, amplificates consisting of part of hoxF and part of the intergenic spacer between hoxF and hoxU were obtained with cDNA derived from both RT primers UAv3 and 8Av2. With the latter RT primer, transcription of ORF8 was also shown in a PCR with primers 8Av1 and 8Av2. In addition, a combined amplificate for hoxU(part)/ORF8/hoxY(part) with cDNA derived from RT primer YAv3 as well as a combined amplificate for hoxY(part)/ORF3/hoxH(part) with cDNA derived from reverse primer HAv1 was demonstrated (Fig. 2). Thus, ORF8 and ORF3 are co-transcribed with the adjacent hox genes. In view of all these data, co-transcription of hoxFhoxUORF8hoxYORF3hoxH is expected. Transcriptional analysis of hox and hup genes in heterocysts and vegetative cells of A. variabilis. N2-fixing A. variabilis cultures from the exponential growth phase were harvested and split into two fractions. One was used for immediate preparation of total RNA; the other part was subjected to heterocyst isolation followed by RNA preparation. Total RNA of vegetative cells was obtained from NH4⫹-grown cultures, which were free of heterocysts, as indicated by microscopic examination. The transcription of hox genes of both the diaphorase entity (HoxFU) and the hydrogenase dimer (HoxYH) was detected in all three RNA preparations (N2-grown filaments, heterocysts, NH4⫹-grown filaments) by the RT-PCR protocol described above (Fig. 3). With RT primer H6B for the 38 part of hupL, coding for the large subunit of the uptake hydrogenase [6], a strong signal became visible with the primer pair H4A/H6B for the heterocyst fraction and for RNA isolated from 318 CURRENT MICROBIOLOGY Vol. 40 (2000) Fig. 1. Transcriptional analysis of the hox loci of Anacystis nidulans by RT-PCR. Primers used in the reverse transcriptions RT1–9 and subsequent PCRs are given as arrows under the gene clusters (see Table 1). For each RT-reaction, the reverse primer (gray arrow) as well as the PCR-amplificates obtained with that cDNA are indicated. The maximum lengths of the determined transcripts are given as a curved line. Fig. 2. Transcriptional analysis of the hox locus of Anabaena variabilis by RT-PCR. Primers used in the reverse transcriptions RT1–5 and subsequent PCRs are given as arrows under the gene cluster (see Table 1). For each RT-reaction, the reverse primer, as well as the PCR-amplificates obtained with that cDNA is indicated. The maximum length of the determined transcript is given as a curved line. N2-grown cells (Fig. 3). Such findings agree well with physiological data, which indicated the occurrence of the uptake hydrogenase in heterocysts [10, 19, 30]. Remarkably, there was a distinct, though weaker, signal also for the RNA isolated from NH4⫹-grown cultures (Fig. 3). Thus, at least a low basic level of transcription of hupL is detectable in NH4⫹-grown filaments of A. variabilis. G. Boison et al.: Hydrogenase Genes in Cyanobacteria Fig. 3. Demonstration of transcription of hoxYH, hoxF, and hupL in N2-grown filaments (lanes 2 and 3), heterocysts (lanes 4 and 5), and vegetative cells (lane 6 and 7) of Anabaena variabilis. RNA isolated from N2-grown filaments (lane 3), heterocysts (lane 5), and ammoniagrown filaments (lane 7) was used in RT-reactions with the reverse primers HAv1 (hoxH), FAv2 (hoxF) and H6B (hupL) (see Table 1). Subsequent PCRs were performed with the primer pairs YAv2/HAv1 (hoxY-ORF3-hoxH), FAv1/FAv2 (hoxF) and H4A/H6B (hupL) with genomic DNA of N2-grown cultures (lane 1), DNase-treated RNA, not transcribed into cDNA, as control (lanes 2, 4, and 6), and reversetranscribed RNA (lanes 3, 5, and 7). The amplificates obtained from the cDNA have the same sizes as those obtained from the genomic DNA, which have the expected sizes calculated from the sequences. The control lanes show no amplificates. The 100-bp ladder from GIBCO/ BRL was used as size marker. Discussion Up to now, two reports only about transcription of hydrogenase genes in cyanobacteria have been published. Axelsson et al. [2] investigated the transcription of hoxH and hupL in Nostoc sp. PCC 73102. Gubili and Borthakur [16] compared the differential expression of the accessory gene hupB of the uptake hydrogenase of A. PCC 7120, under N2- and non-N2-fixing culture conditions. In the present investigation, the complete transcriptional analysis of the hox genes in A. nidulans and A. variabilis has been performed, and the presence of hox and hup transcripts in N2-fixing and NH4⫹-grown A. variabilis has been demonstrated. Thus, this is the first comprehensive transcript analysis of hydrogenase genes in cyanobacte- 319 ria. All investigations thus far mentioned have been performed by RT-PCR. This fairly new technique has been introduced as a method for transcription analysis in cyanobacteria only recently [see e.g. 12]. Transcript analyses by conventional Northern blot analysis, where transcript levels can more easily be quantified, have never been published for cyanobacterial hydrogenases. Northern blot experiments with the hox cluster of A. nidulans gave no clear-cut results (G. Boison, unpublished), probably owing to a very low abundance of these transcripts. This assumption is based on the low enzyme activity (⬍10 nmol min⫺1 mg protein⫺1 [29]), which is at least two orders of magnitude lower than that of the NAD⫹-reducing hydrogenase of Alcaligenes eutrophus [35]. Promoter activities measured by the -galactosidase assay of hox::lacZ fusions of A. nidulans (G. Boison, unpublished) and A. eutrophus [36] also reveal a much lower transcriptional activity of the cyanobacterial hox genes. Additionally, short half-lives of prokaryotic mRNA [31] render transcriptional analysis difficult, especially for large operons. Therefore, the more sensitive RT-PCR offers better perspectives in analyzing genes, which are transcribed at low levels only, as in the case of hydrogenase genes. The gene cluster arrangement of the bidirectional hydrogenase has been described to be quite dissimilar in three cyanobacterial species [4]. The differences refer mainly to the existence of different unidentified ORFs, which are interspersed in the cluster of A. variabilis and S. sp. PCC 6803, but are missing in A. nidulans. The present communication shows that the unidentified ORFs 3 and 8 of A. variabilis are co-transcribed with the hydrogenase genes hoxF, hoxU, hoxY, and hoxH. Thus, these ORFs might not be silent genes or even non-coding DNA regions, but encode proteins of unknown functions. Sequence alignments of ORF3 to the database showed us homology to an unknown ORF of Prochlorothrix hollandica being located in the same position, between hoxY and hoxH, in the hox cluster of this isolate. Outside of hydrogenases, ORF3 has been reported to show homologies to CP12 of higher plant chloroplasts [41], a protein that is involved in oligomerization of glyceraldehyde-3phosphate dehydrogenase and phosphoribulokinase. Further homologies of ORF3 exist to the unidentified ORF4 of A. PCC 7120, located downstream of the fdxH gene [23]. The real functions of the putative proteins encoded by ORF3 as well as by ORF8 remain to be elucidated. Interestingly, the present RT-PCR analysis of the A. variabilis hox cluster gave also clear evidence for transcription of the rather long intergenic stretch between hoxF and hoxU [34]. The analysis of the A. variabilis hox gene cluster by RT-PCR demonstrated one polycistronic transcript for all bidirectional hydrogenase genes. The same is true for the diaphorase gene hoxU, the hydrogenase genes hoxYH, 320 and the accessory genes hoxWhypAB of A. nidulans. However, the existence of further smaller transcripts, either generated by different termination of transcription or via further promoters in the cluster, cannot be ruled out by the method chosen. The genes of the hox clusters of R. opacus [14], A. eutrophus [27], and of the hnd cluster of the NADP⫹-reducing hydrogenase of D. fructosovorans [22] are also transcribed as polycistronic mRNA. Additional smaller transcripts, which have been interpreted as degraded or processed mRNA, have been observed in these three investigations. Apart from the polycistronic message hoxUYHWhypAB, the gene hypF encoding an accessory protein involved in Ni incorporation and maturation of the large subunit of NiFe hydrogenases [8, 21], is probably transcribed separately in A. nidulans. The same has been shown for Rhodobacter capsulatus [7] and Escherichia coli [21]. In A. nidulans, the diaphorase gene hoxF is separated from the other hydrogenase genes by at least 16 kb [4]. Upstream of hoxF, the gene hoxE has been found in S. PCC 6803 [1] and A. nidulans [4]. Up to now, participation and function of HoxE in the bidirectional hydrogenase has not been demonstrated. HoxE has been suggested to function as a further subunit of the diaphorase moiety [1, 4] because of clear sequence identities of HoxF, HoxU, and HoxE to the NADH oxidizing homologous subunits of respiratory complex I of E. coli. However, a function of HoxE in the bidirectional hydrogenase has to be demonstrated by either protein purification or measuring the NAD(P) ⫹-dependent H2-uptake activity of hoxE-mutants. The present communication describes transcription of hoxE in a dicistronic mRNA together with hoxF, strengthening a function of HoxE in the hydrogenase complex. The question remains to be solved how the transcription of hoxEF and hoxUYHWhypAB is coordinated in A. nidulans. In the present communication, the transcription of the bidirectional hydrogenase genes hoxF and hoxYH has been demonstrated for both heterocysts and vegetative cells of A. variabilis. The uptake hydrogenase gene hupL is strongly expressed in heterocysts, as expected from the enzyme measurements [10, 30]. Unexpectedly, a weaker transcription of hupL was also observed in vegetative cells of A. variabilis. H2-uptake in heterocyst-free cultures has been detected in A. variabilis [40]. Additionally, immunological localizations indicated the presence of the uptake hydrogenase in N. sp. PCC 73102, a strain lacking the bidirectional enzyme [5, 39], both in heterocysts and vegetative cells [20, 38]. The RT-PCR signal of hupL could reflect a basic activity of the hupSL promoter, not necessarily leading to translation and expression of HupL in vegetative cells. Such translational control in cyanobacteria has been reported for S. PCC 6803 [18, 25]. CURRENT MICROBIOLOGY Vol. 40 (2000) ACKNOWLEDGMENT This work was kindly supported by a grant from the Deutsche Forschungsgemeinschaft. Literature Cited 1. Appel J, Schulz R (1996) Sequence analysis of an operon of a NAD(P)-reducing nickel hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803 gives additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase (complexI). Biochim Biophys Acta 1298:142–147 2. Axelsson R, Oxelfelt F, Lindblad P (1999) Transcriptional regulation of Nostoc uptake hydrogenase. FEMS Microbiol Lett 170: 77–81 3. Boison G, Schmitz O, Mikheeva L, Shestakov S, Bothe H (1996) Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans. FEBS Lett 394:153–158 4. 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