* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Archives of Microbiology
Oncogenomics wikipedia , lookup
Gene desert wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Essential gene wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genomic library wikipedia , lookup
Transposable element wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Gene expression programming wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Human genome wikipedia , lookup
Primary transcript wikipedia , lookup
Point mutation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genome evolution wikipedia , lookup
Genome (book) wikipedia , lookup
Ridge (biology) wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Genomic imprinting wikipedia , lookup
History of genetic engineering wikipedia , lookup
Minimal genome wikipedia , lookup
Genome editing wikipedia , lookup
Helitron (biology) wikipedia , lookup
Pathogenomics wikipedia , lookup
Designer baby wikipedia , lookup
Microevolution wikipedia , lookup
Metagenomics wikipedia , lookup
Microsatellite wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Arch Microbiol (2006) 186:367–376 DOI 10.1007/s00203-006-0150-4 O RI G I NAL PAPE R The rice Weld cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase Gudrun Boison · Caroline Steingen · Lucas J. Stal · Hermann Bothe Received: 25 November 2005 / Revised: 29 May 2006 / Accepted: 10 July 2006 / Published online: 19 August 2006 © Springer-Verlag 2006 Abstract Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice Welds, expressed vanadium-containing nitrogenase when under molybdenum deWciency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-speciWc genes vnfDG are fused and clustered in a phylogenetic tree next to the corresponding genes of Methanosarcina. The expression of vnfH in cells cultured in Mo-free medium and of nifH in Mo-grown cells was shown for the Wrst time by sequencing cDNA derived from cultures of A. azotica and Anabaena sp. CH1. The vnfH sequences clustered with that of Anabaena variabilis. The vnf genes were strongly transcribed only in cultures grown either in Mo-free medium, or in W-containing medium, but also weakly in Mo-containing medium. NifH was transcribed in all media. On-line measurements of acetylene reduction by Mo-free A. azotica cultures demonstrated that the V-nitrogenase was active. Ethane was formed continuously at a rate of 2.1% of that of ethylene. Acetylene reduction of cultures grown either with or without Mo had a high temperature optimum of 42.5°C. The uptake hydrogenase gene G. Boison (&) · L. J. Stal Department of Marine Microbiology, Centre for Estuarine and Marine Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 140, 4400 AC Yerseke, The Netherlands e-mail: [email protected] C. Steingen · H. Bothe Institute of Botany, University of Cologne, Gyrhofstr. 15, 50931 Köln, Germany hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis. Keywords Alternative nitrogenase · Vanadium nitrogenase · Cyanobacteria · Tungsten · vnfDG · vnfH · Uptake hydrogenase · Gene expression Abbreviations ARA Acetylene reduction assay RT Reverse transcription Introduction Most described nitrogenases contain an FeMo-cofactor as a prosthetic group. As Wrst observed in Azotobacter, some diazotrophic microorganisms Wx N2 using alternative nitrogenases, in addition to the Mo-containing enzyme (Mo-nitrogenase). These organisms express a V-dependent enzyme (V-nitrogenase) with an FeVcofactor under Mo-deWcient growth conditions. In some strains, an Fe-only nitrogenase is active when both Mo and V are unavailable (Loveless and Bishop 1999). The three types of nitrogenase are encoded by separate gene clusters: nifHDK coding for the Mo-, vnfH/vnfDGK for the V-, and anfHDGK for the Fe-only nitrogenase (Bishop and Joerger 1990). The alternative nitrogenases are distinguished from the Moenzyme by an additional subunit encoded by vnfG or anfG, which may be involved in the insertion of the FeV- or Fe-only cofactors into the apo-proteins (Chatterjee et al. 1997). Furthermore, alternative nitrogenases can be detected by their ability to reduce C2H2 partly beyond C2H4 to C2H6 (Dilworth et al. 1987). 123 368 Alternative nitrogenases are generally repressed by Mo, but expressed in the presence of tungsten even in Mo-medium (Raina et al. 1992; Thiel et al. 2002b). Remarkably, V-nitrogenases evolve three times more molecular hydrogen and have a higher energy demand than the Mo-enzyme (Eady 2003). At present, the biological relevance of alternative nitrogenases for the organism and its habitat is unknown. Besides Azotobacter, vnf and anf genes have been reported in only a few strains of taxonomically unrelated bacteria: Rhodospirillum, Azomonas, Heliobacterium (Loveless and Bishop 1999), Clostridium (Zinoni et al. 1993), Rhodobacter (Schuddekopf et al. 1993), Rhodopseudomonas (Larimer et al. 2004), Methanosarcina (Chien et al. 2000), and Anabaena variabilis (Thiel 1993). A. variabilis is the only cyanobacterium for which the presence of a V-nitrogenase has unambiguously been shown (Kentemich et al. 1988; Thiel 1993). Circumstantial evidence for a V-nitrogenase has been presented for Anabaena azollae (Ni et al. 1990; Thiel 1993). While vnfDGK genes are clearly diVerent and separated phylogenetically from nifDK, vnfH and nifH genes cannot be distinguished by phylogenetic analyses because of the limited number of known vnfH sequences obtained so far (Zehr et al. 2003; Raymond et al. 2004). This study addresses the questions whether the vnf system is more widespread among cyanobacteria than known hitherto and whether nifH and vnfH sequences can be distinguished by phylogenetic analyses. Ecophysiological comparisons of V-nitrogenase-positive cyanobacteria could shed light on the function of this enzyme in nature. In this study, cyanobacteria maintained in culture collections were screened for the presence and expression of vnf genes. Arch Microbiol (2006) 186:367–376 tively, vnf transcription was induced without vanadate in Mo-medium supplemented with 10 M Na2WO4 £ 2 H2O (MoW-medium). The cultures were concentrated by centrifugation (3,500g, 5 min at room temperature), washed with the corresponding medium and diluted three times (1:10) with fresh medium in 25 cm2 plastic tissue culture Xasks (TPP, Switzerland) to minimize Mo-contamination from glassware. Isolation of genomic DNA, Southern blots, and dotblot hybridization Genomic DNA was extracted from cyanobacterial cultures by vortexing cells with glass beads in phenol (Tamagnini et al. 1997). For Southern blots, 2 g of genomic DNA was restricted with EcoRI, separated on an 0.7% agarose gel, and transferred to a positively charged nylon membrane (Roche) by capillary transfer with 0.4 N NaOH. For dot-blots, 0.5 g of genomic DNA was denatured in 0.4 M NaOH/5 mM EDTA at 95°C for 5 min and placed on ice. The denatured DNA (total volume 8.5 l) was spotted onto a positively charged nylon membrane (Roche), which had previously been equilibrated with 0.4 M NaOH. Dot-blot and Southern blot membranes were washed in 2£ SSC, and the DNA was Wxed by UV cross-linking. Hybridization was performed at 60°C in 5£ SSC/0.5% blocking reagent (Roche)/0.1% N-lauroylsarcosine/0.02% SDS. The blot was washed in 2£ SSC/0.1% SDS (2£, 5 min, 60°C) and stained using NBT/X-phosphate (Roche) according to the “DIG application manual for Wlter hybridization” (Roche). Hybridization probes (vnfG from A. azotica, hupL and hoxH from A. variabilis) were labeled with digoxigenin-dUTP (Roche) by PCR using the primers given in Table 1 together with Taq polymerase (Fermentas) in 35 cycles as follows: 92°C for 40 s, 50°C for 40 s, and 72°C for 40 s. Materials and methods PCR and DNA sequencing Strains and growth conditions Anabaena azotica FACHB-118, formerly A. azotica HB686 (X. Wu, Wuhan, China, personal communication), was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan. Anabaena sp. CH1, maintained as CCY 9910 in the Culture Collection Yerseke, was kindly supplied by Prof. P. Böger, Konstanz, Germany. The cyanobacteria were cultivated in BG11 medium (Rippka et al. 1979) without combined nitrogen at 25°C (Mo-medium). To express V-nitrogenase activity, molybdate in the trace element solution was substituted by 10 M V2O5 (V-medium). Alterna- 123 Hot-start PCR was performed using genomic DNA or cDNA as templates with HotStar Taq DNA polymerase (Qiagen), which required activation (15 min, 95°C) prior to cycling. All programs were terminated with an end-elongation for 7 min at 72°C. The primers used are given in Table 1. The program for ampliWcation of the 16S rRNA was described elsewhere (Boison et al. 2004). The thermal cycle for ampliWcation of nifH using degenerated primers was as follows: 94°C for 40 s, 52°C for 40 s, and 72°C for 80 s for 35 cycles. A gradient PCR with annealing temperatures between 50 and 65°C was used to determine the highest possible annealing temperature for ampliWcation of nifH and Arch Microbiol (2006) 186:367–376 Table 1 Oligonucleotide primers used in this study The name of the target gene is followed by the position of the primers in nucleotide numbers referring to the GenBank accession number given Primer 369 Sequence in 5⬘!3⬘ orientation Source 16S rRNA (N359–N1404 of E. coli K12) CYA359 GGGGAATYTTCCGCAATGGG Nübel et al. (1997) 1387R GGGCGGWGTGTACAAGGC Marchesi et al. (1998) nifH (N276–N634 of V01215) nif-f TGYGAYCCNAARGCNGA Zehr and McReynolds (1989) nif-r ADNGCCATCATYTCNCC Zehr and McReynolds (1989) nifH (N296–N425 of V01215), sequence speciWc for A. azotica AaznifH-f CACCCGTTTGATGCTCCATGCC This work AaznifH-r TTCTACGCAACGAACACCACGG This work nifH (N297–N411 of V01215), sequence speciWc for A. variabilis and Anabaena sp. CH1 Av99niH-f ACCCGTTTGATGCTCCACGC This work Av99nifH-r CGCCACGGAAACCGGTCAACAT This work vnfH (N296–N409 of V01215), sequence speciWc for A. azotica AazvnfH-f TACTCGTTTGATCCTCCACTGT This work AazvnfH-r TCACGAAAACCATTGATAACG This work vnfH (N297–N416 of V01215), sequence speciWc for A. variabilis and Anabaena sp. CH1 Av99vnfH-f ACCCGCTTGATTCTCCACAC This work Av99vnfH-r TTTGATATCGCGGAAGCCTT This work vnfDG (N1481–N2137 of L20472) D6fm GAAGACTTYGARAAGGTCAT This work, shortened D6f, Loveless and Bishop (1999) Ga2r TGGTKCARTTCRCSGTT This work vnfG (N346–N475 of AY422692) GAaf1 TTCAAGAGCGTTGTTTGT This work GAar1 TTGCCAGTCAGTGTTTCC This work hupL (N18605–N18912 of AF368526) LF1 GAYCCYTGGTATATYAARCC This work LR1 TCRCCAGTYTTMGCATCATG This work hoxH (N2385–N2608 of X97797) HAnv3 GTAAGTGAGGCACCTMGTGGK Boison et al. (2000) HAn2 CAGGAAAGGCAGGGRTCAAAR Boison et al. (2000) vnfH using sequence and strain-speciWc primers: 94°C for 30 s, 50–65°C for 30 s, and 72°C for 1 min for 35 cycles. The annealing temperatures used in further PCR reactions with this program were 65°C for the nifH-speciWc primers, 53°C for the vnfH-speciWc primers of A. azotica, and 59°C for the vnfH-speciWc primers of Anabaena sp. CH1 and A. variabilis. For the latter primers, the program was changed to 38 cycles and denaturing and annealing times of 1 min each. A 40-cycle program was used to amplify vnfDG: 94°C for 60 s, 46°C for 60 s, and 72°C for 80 s. AmpliWcation of vnfG was performed by nested PCR using a 1-l product of a vnfDG-PCR as template according to the following protocol: 40 cycles of 94°C for 60 s, 48°C for 60 s, and 72°C for 30 s. The thermal cycle for ampliWcation of hupL and hoxH was as follows: 94°C for 40 s, 52°C for 40 s, and 72°C for 40 s for 35 cycles. PCR products of the right size were cloned with the pGEM-T Easy vector system (Promega) in Escherichia coli XL1-blue (Stratagene). White colonies were controlled by PCR using some cells as template and the general primers directed against the Sp6- and T7-promoter sites from the cloning vector. This PCR was run with 35 cycles as follows: 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s. For every product, three clones of the right size were sequenced on both strands in an ABI 3130 Genetic analyzer (Applied Biosystems). Nucleotide sequence accession numbers New sequences were deposited in GenBank under the accession numbers: A. azotica (vnfDG = AY422692, nifH = DQ294218, vnfH = DQ294219, 16S rRNA = AY422691), Anabaena sp. CH1 (vnfDG = DQ294215, nifH = DQ294216, vnfH = DQ294217, 16S rRNA = DQ294214). Computing programs for sequence analysis Sequence data were compared with the NCBI data bank entries using the BLAST program (Altschul et al. 1997). Sequences were aligned with the ClustalX software (Thompson et al. 1997), and corrected manually. The aligned sequences, editing out the primers, were used for phylogenetic analysis using the MEGA3 software package (Kumar et al. 2004). Neighbor-joining trees were calculated with Poisson correction and bootstrap values from 1,000 replicate trees. 123 370 Arch Microbiol (2006) 186:367–376 Isolation of total RNA, and reverse transcription Total RNA was extracted from cultures with warm acidic phenol followed by puriWcation with the RNeasy Kit (Qiagen) and reversely transcribed with random primers and Superscript II (Invitrogen) as described (Boison et al. 2000). PCR was performed with cDNA, a sample where Superscript has been omitted (negative control) and genomic DNA (positive control) as described above. Acetylene reduction assay Nitrogenase activity was determined by acetylene reduction assay (ARA) with an on-line system (Staal et al. 2001). The cells were concentrated on a glass Wber Wlter (47 mm, Whatman GF/F) and incubated in a temperature-controlled chamber under continuous gassing with 70% N2, 20% O2 (each containing 0.4% CO2), and 10% C2H2. For anoxic measurements, the gas Xow controller was adjusted to 90% N2 and 0% O2. Injections were made automatically every 5 min. C2H4 and C2H6 were separated and quantiWed on a gas chromatograph (Chrompack CP 9001, Varian, Netherlands) equipped with a capillary column (CP-PoraPLOT U, 27.5 m, 0.53 mm, 20 m, Varian, Netherlands) and a Xame ionization detector. The temperatures were 90, 60, and 120°C for the injector, oven, and detector, respectively. The reaction chamber was illuminated with a slide projector. The light intensity was changed using a set of neutral Wlters as slides in the projector to give an exponentially increasing light intensity. The incident light in the reaction chamber was measured using a Licor Li 250 light meter. The assay temperature was changed for temperature-dependent measurements at a random order from 15 to 47°C. Three to Wve injections were made at each temperature until the values were stable. All ARA experiments were repeated three times with cultures pre-incubated at 25°C in a 12 h/12 h light/dark regime. Chlorophyll a determination Chlorophyll a was extracted from the Wlters used for the ARA measurements by 90% acetone and soniWcation for 1 h. After removing the debris by centrifugation, absorption of the supernatant was measured at 664 nm and the chlorophyll a concentration was calculated using an absorption coeYcient of 87.7 ml mg¡1 cm¡1. Results and discussion Characterization of vnfDG genes in A. azotica and Anabaena sp. CH1 Heterocystous cyanobacteria from the culture collections in Köln (Germany) and Yerseke (CCY; The Netherlands) were screened for the presence of the V-nitrogenase-speciWc gene vnfDG by PCR. Sequences from PCR products obtained from A. azotica FACHB118 and Anabaena sp. CH1 were identiWed as cyanobacterial vnfDG by BLAST comparison. The sequences from A. azotica and Anabaena variabilis shared identities of 93% on DNA and 98% on amino acid levels, whereas identity values between the Anabaena sp. CH1 and A. variabilis sequences amounted to only 77% for the nucleotides and 73% for the amino acids. VnfD and vnfG, which are separate genes in other bacteria, are fused into a single entity in A. azotica and Anabaena sp. CH1, as was Wrst observed in A. variabilis (Thiel 1993). Surprisingly, this sequence in Anabaena sp. CH1 is 21 nucleotides longer than those from the other Anabaena strains. This additional stretch is located at the 3⬘ end of the vnfD part of the fused gene (Fig. 1). It is neither known whether the functional and regulatory properties An. azotica AIYSPLMQLAAFDVRDDAPKAP------AKTKEIEH-LNEKVTNITTYIQERCLW An. variabilis AIYSPLMQLAAIDVRDDAPKAP------AKTKEIEH-LNEKVTNITTYIQERCLW An. CH1 AIYSPLMQLAGIDVRDDEPKKDNSESLKQQSEEVTAYIQERTEEITKFIQERCLW M. acetivorans AIYSPMWKLAGKDPRETDSPMWSLT-EKDSGVVQESM-NEKIEEITALIQERCLW M. barkeri GIYSPMWSLAGKDPR----------------VVQELM--KKLEEVTALIQKQCLW R. palustris AVHNPLLKLAATDIRGETSTR---------LLEAAEMSAEQIDQLYNYCQERYLW Az. chroococcum AVHNPLRHLAAVDIRDSSQTTP-------VIVRGAAMSQSHLDDLFDYTEERCLW Az. paspali AVHNPLRHLAAVDIRDKSQTTP-------VIVRGAAMSQSHLDDLFAYVEERCLW Az. salinestris AVHNPLRHLAAVDIRDKSQTTP-------IIVRGAAMSQSRLDDLFAYVEERCLW Az. vinelandii AVHNPLRHLAAVDIRDKSQTTP-------VIVRGAAMSQSHLDDLFAYVEERCLW * ** * * ** Fig. 1 Alignment of the C-terminal end of VnfD and N-terminal end of VnfG sequences. The arrow indicates the start methionine of VnfG. In Anabaena spp., VnfD and VnfG are fused into one 123 entity. Identical amino acids are marked by an asterisk. An Anabaena, M Methanosarcina, R Rhodopseudomonas, Az Azotobacter Arch Microbiol (2006) 186:367–376 371 of the VnfDG products are altered by this gene fusion, nor whether the unique addition of 21 nucleotides in the vnfDG fusion area of Anabaena sp. CH1 is of functional signiWcance. In a neighbor-joining analysis of all available deduced VnfDG sequences, those from cyanobacteria clustered next to those from Methanosarcina spp. and were clearly separated from AnfDAnfG (Fig. 2). The close similarity of the cyanobacterial vnfDG to vnfDvnfG of Methanosarcina spp. might strengthen the idea of an archaeal origin of conventional and alternative nitrogenases (Raymond et al. 2004). Since alternative nitrogenases occur in bacteria of totally unrelated taxonomic aYnities and because the composition of the structural genes nifHDK and vnfHDGK is largely diVerent, a selection pressure must have existed for the acquisition of the V-nitrogenase genes by the organisms by lateral gene transfer. The evolution of the cyanobacterial V-nitrogenase gene vnfDG may have occurred in a common ancestor, since these genes are contiguous in the three Anabaena spp. but not in other bacteria. PCR experiments with vnfDG-primers and nested PCR with vnfG-primers were negative for Fischerella sp. SAG 1427-1, Chlorogloeopsis sp. ATCC 27193, Calothrix spp. (ATCC 27905, CCY 0013, and CCY 0202), and Anabaena spp. (CH2 and CCY 0015). A dot-blot hybridization using a vnfDG probe of A. azotica gave unambiguous positive signals only with A. variabilis, A. azotica, and Anabaena sp. CH1 (not documented). IdentiWcation of nifH and vnfH sequences An investigation of nitrogenase and hydrogenase genes in A. azotica carried out by Southern blotting yielded two signals with a probe for nifH, possibly due to the presence of nifH and vnfH, and one signal each using a probe for the uptake hydrogenase gene hupL and the bidirectional hydrogenase gene hoxH. These results were corroborated by PCR using the degenerate primers LF1/LR1 for hupL and HAnv3/HAn2 for hoxH. In order to identify nifH and vnfH sequences in A. azotica and Anabaena sp. CH1, reverse transcription (RT)-PCR products obtained with the universal nifHprimers from cultures of these strains grown in Mo- and V-medium were cloned and three clones were sequenced from both media and strains. Two clearly diVerent genes were expressed in the cultures grown in Mo- or V-medium and the three clones from each medium were identical in both cases. In a phylogenetic analysis of deduced NifH sequences, the genes expressed in Mo-medium clustered with the majority of heterocystous NifH sequences next to NifH1 from A. variabilis (Fig. 3), which is known to be active in Monitrogenase. The other gene, expressed in V-medium, occupied a separate cluster comprising the sequence from A. variabilis tentatively identiWed as vnfH (Thiel et al. 2002a). Thus, the diVerent genes from A. azotica and Anabaena sp. CH1 were termed nifH and vnfH. X15077 Azotobacter chroococcum 95 71 AF152913 uncultured bacterium AF152914 uncultured bacterium 100 AF058782 Azotobacter salinestris 84 88 98 100 99 100 99 100 84 100 100 68 AF058781 Azotobacter paspali M32371 Azotobacter vinelandii AF152909 uncultured bacterium AF152910 uncultured bacterium VnfDVnfG AF152908 uncultured bacterium AF152912 uncultured bacterium AF152911 uncultured bacterium NC005296 Rhodopseudomonas palustris NC003552 Methanosarcina acetivorans C2A AF254784 Methanosarcina barkeri Anabaena sp. CH1 AY422692 Anabaena azotica AY422707 uncultured bacterium BSC VnfDG CAA52044 Anabaena variabilis M23528 Azotobacter vinelandii AnfDAnfG 10 % substitutions Fig. 2 Neighbor-joining tree of deduced amino acid sequences of part of V-nitrogenase subunits VnfDG (corresponding to amino acid residues 373–578 of A. variabilis, accession no. CAA52044), rooted to AnfDAnfG of Fe-nitrogenase of A. vinelandii. The sequences of the separate subunits VnfD/AnfD and VnfG/AnfG from bacteria other than cyanobacteria were concatenated (VnfDVnfG/AnfDAnfG) for the analysis. VnfDG is one entity in cyanobacteria. Numbers at the branches indicate the percentage of occurrence of the respective node in a bootstrap analysis of 1,000 re-samplings; only values above 50% are shown 123 372 Arch Microbiol (2006) 186:367–376 Anabaena sp. CH1 VnfH * 99 DQ315787 Anabaena variabilis VnfH AAC36070 unidentified marine eubacterial clone BT1118 47 U49515 Fischerella sp. UTEX1931 Anabaena azotica VnfH * 74 AAQ64036 uncultured bacterium cluster M 73 ZP00111243 Nostoc punctiforme (extra copy) U73129 Calothrix sp. ATCC27901 69 52 AJ716389 uncultured bacterial clone MING-50A V01482 Nostoc sp. PCC7120 NifH1 AAS22069 Anabaena aphanizomenoides A49831 Anabaena oscillarioides 49 AAA22011 Anabaena oscillarioides AAT48543 Cylindrospermopsis raciborskii AAC64640 Fischerella UTEX1903 44 AAB37315 Chlorogloepsis sp. ATCC27193 AAA22014 Anabaena sp. L-31 Anabaena azotica NifH1 ** 43 43 40 62 AAA87251 Anabaena azollae U89346 Anabaena variabilis NifH1 Anabaena sp. CH1 NifH1 ** 42 58 69 AAB37316 Chlorogloeopsis sp. CCAP1411/1 AAP48972 Tolypothrix sp. ZP_00162494 Anabaena variabilis (extra copy) 42 42 AF012326 Nostoc sp. PCC7120 (extra copy) AAS22068 Anabaenopsis sp. NRE1 86 ZP_00112319 Nostoc punctiforme NifH1 L23514 Nostoc commune UTEX584 AAS75595 Nodularia sphaerocarpa 53 AAB37308 Scytonema sp. 54 77 51 ZP_00109382 Nostoc punctiforme (extra copy) U04054 Nostoc muscorum NifH2 Trichodesmium, Symploca Gloeothece, Synechococcus, Crocosphaera X07866 Rhodobacter capsulatus 5 % substitutions Fig. 3 Neighbor-joining tree of deduced amino acid sequences of part of nitrogenase reductase NifH (corresponding to amino acid residues 48–152 of Nostoc sp. PCC7120, accession no. AAA22008) rooted to the sequence of Rhodobacter capsulatus. Bootstrap analysis is as in Fig. 2; only values above 40% are given. Gray boxes—sequences of the genomes of A. variabilis, Nostoc sp. PCC7120, and N. punctiforme PCC73102, which are described as VnfH or NifH1 of Mo-nitrogenase, and cDNA sequences of A. azotica and Anabaena sp. CH1 expressed in V-medium (asterisk) and Mo-medium (double asterisks). Copies of nifH known from the genome sequences as not belonging to a nifHDK cluster are marked as extra copies. The condensed NifH2 cluster comprises the same sequences as in Boison et al. (2004). The other condensed clusters contain—L15554 Gloeothece sp., U22146 Synechococcus sp. RF-1, AAP48976 Crocosphaera watsonii, AAB81940 Symploca atlantica PCC8002, AAP48970 Oscillatoria sancta, M29709 Trichodesmium thiebautii, AAB70120 Trichodesmium erythraeum IMS101 Since only a few vnfH sequences have been deposited in the databanks, a clear separation between a vnfH and nifH cluster in phylogenetic studies has not emerged (Zehr et al. 2003; Raymond et al. 2004). The situation is further complicated by the occurrence of multiple copies of nifH in several heterocystous cyanobacteria. Hence, it is not known whether the sequences from Fischerella sp. UTEX 1931 and Calothrix sp. ATCC 27901 and a few environmental sequences that cluster with the vnfH sequence of A. variabilis (Fig. 3) belong to a V-nitrogenase system. No vnfDG genes were detected in other strains of Fischerella or Calothrix in the present study, but it cannot be ruled out that such genes exist but are too divergent to be detected. However, it is known that the nifH copy from Nostoc punctiforme clustering with the vnfH sequence does not belong to a vnf system (Thiel et al. 2002a). By the sequencing of cDNA of V- and Mo-grown cultures 123 Arch Microbiol (2006) 186:367–376 in the present study, it appears to be possible for the Wrst time to distinguish between vnfH and nifH. This might give a new start for re-investigating the still unresolved nifH/vnfH phylogeny. 373 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 + - M A Transcription of vnf genes and hupL in Anabaena spp. Primers were designed to distinguish between nifH and vnfH sequences of the Anabaena strains and tested for their speciWcity on cloned nifH and vnfH products. These primers are sequence- and strain-speciWc and ampliWed under high stringency only the desired target. The expression of nitrogenase and hydrogenase genes was then investigated in A. azotica, Anabaena sp. CH1, and A. variabilis cultures grown in either Mo-, MoW-, or V-medium by RT-PCR (Fig. 4). The diVerent products of each strain were ampliWed from the same cDNA pool. The universal nifH-primers, which do not distinguish between nifH and vnfH, were used as positive control, and products were obtained with these primers from the cDNA of all three strains grown in all media (Fig. 4). The nifH-speciWc primers revealed that nifH was transcribed in all three strains in Mo-grown cultures and also in MoW- and V-grown cultures. This eVect was described earlier for A. variabilis (Thiel et al. 1997). However, vnfH and vnfDG was strongly expressed in the Anabaena strains only in MoW- and V-medium (Fig. 4). Expression in the presence of Mo and W might be due to the fact that the high aYnity Mo-transporter is competitively inhibited by W (Thiel et al. 2002b; Zahalak et al. 2004). Surprisingly, the vnf genes were weakly transcribed also in the Mo-medium. Expression of vnf genes in the presence of Mo is a new Wnding and might be explained by the higher sensitivity of RT-PCR compared to -galactosidase assays and Northern blots, which were used in earlier investigations (Thiel et al. 1997). Whether this basic transcription also reXects expression and activity of vanadium nitrogenase cannot be concluded from these results. The uptake hydrogenase gene hupL, which is expressed under nitrogen-starvation only, was transcribed equally in Mo-grown and Mo-deWcient cultures (not documented), suggesting that this enzyme recycles H2 evolved by Mo- or by V-nitrogenase. Activity of the V-nitrogenase from A. azotica measured by ARA V-grown cultures of both A. azotica and Anabaena sp. CH1 reduced C2H2 partly beyond C2H4 to C2H6, which is a characteristic of alternative nitrogenases (Dilworth et al. 1987). Under all conditions tested, the C2H6 formed amounted to 2.1 § 0.5% of the measured C2H4, B C D Fig. 4 Transcription of nifH and vnf genes in Anabaena spp. Total RNA, isolated from A. azotica (Aa), Anabaena sp. CH1 (CH1), and A. variabilis (Av) cultures grown in Mo-, MoW-, or V-medium was reverse-transcribed with hexanucleotides. PCR from the cDNA pools and from negative controls to which no reverse transcriptase was added (nc) was performed with degenerated primers nif-f/-r directed against nifH/vnfH (A), with speciWc primers AaznifH-f/-r and Av99nifH-f/-r directed against nifH (B), with speciWc primers AazvnfH-f/-r and Av99vnfH-f/-r directed against vnfH (C), and with degenerated primers D6fm/Gar2 directed against vnfDG (D). Lane 1/2 cDNA/nc of Aa-Mo; lane 3/4 cDNA/nc of Aa-MoW; lane 5/6 cDNA/nc of Aa-V; lane 7/8 cDNA/nc of CH1-Mo; lane 9/10 cDNA/nc of CH1-V; lane 11/12 cDNA/nc of Av-Mo; lane 13/14 cDNA/nc of Av-V; (+) positive control with genomic DNA in PCR; (¡) negative control with no template added in PCR; M DNA ladder, low range (Fermentas). A fragment size of 500 bp is indicated by a line which is as high as in V-grown cultures of A. variabilis (Kentemich et al. 1988; Zahalak et al. 2004) or Azotobacter spp. (Dilworth et al. 1987). Cultures grown in Mo-medium did not form detectable amounts of C2H6, except above 40°C when some C2H6 was formed, as observed earlier with Azotobacter chroococcum (Dilworth et al. 1993). Further studies were pursued only with A. azotica, because the geographical origin and the characteristics of this strain are well known (Li 1981). Thus, physiological experiments with this cyanobacterium were carried out under conditions reXecting the growth 123 Conclusions In this paper, the occurrence of a functional V-nitrogenase in A. azotica and Anabaena sp. CH1, both isolates from rice Welds (Ley et al. 1959; Chen 1984), is conclusively presented from physiological and genetic data. This is the second unambiguous example of V-nitrogenase in cyanobacteria, previously shown to be in A. variabilis (Kentemich et al. 1988; Thiel 1993). 123 50 2.5 40 2.0 30 1.5 20 1.0 10 0.5 0 C2H6 [µmol h-1 mg Chla-1] demands experienced in its natural habitat. A. azotica was isolated from rice Welds in the Chinese province Hubei (Ley et al. 1959), where it is used as a bio-fertilizer in the late-rice crop (Li 1981). The area is characterized by hot summers with average temperatures of about 30°C (Domroes and Peng 1988), and temperatures in the open water body of the rice Welds of up to 40°C (Halwart and Gupta 2004). One of the aspects of this study was, therefore, to test whether high temperatures may favor V-nitrogenase activity in this special cyanobacterium. Measurements of light response curves, at 25 and 35°C in a range of 0–980 mol photons m¡2 s¡1, were Wrst performed under a Xow of 20% O2 and then repeated without O2 using the same cells. The ratio of the amounts of C2H4 and C2H6 formed in V-grown cultures was constant over the whole light range both under aerobic and anaerobic conditions (not documented). Light saturation of C2H2-reduction was reached at around 200 mol photons m¡2 s¡1 with both Mo- and V-grown cultures under all conditions. Saturating light intensities of 245 mol photons m¡2 s¡1 were then chosen for assaying the temperature dependence of C2H2-reduction. Over the whole temperature range, C2H4-production was 1.5-fold higher in Mothan in V-grown cultures and the ratio of the amounts of C2H6 and C2H4 in V-grown cells was constant over the measured temperature range (Fig. 5). The maximum of C2H4-formation for both media was at 42.5°C. This rather high temperature optimum reXects the natural habitat of A. azotica. However, neither the light response curve nor the temperature dependence revealed diVerences in the optimal conditions for C2H2reduction of Mo- and V-grown cultures of A. azotica. Since Mo-nitrogenases are more eYcient, the use of this enzyme might be preferred in A. azotica in rice Welds and will be independent of the temperature, unless its expression is hampered by the presence of W. Tungsten is an abundant mineral in the neighboring province of Hunan (Tanelli 1982) and it is known to interfere strongly with the activity of Mo-nitrogenases (Kumar and Kumar 1980). Arch Microbiol (2006) 186:367–376 C2H4 [µmol h-1 mg Chla-1] 374 0 0 15 25 35 Temp. [°C] 45 55 Fig. 5 C2H2-reduction and C2H4- and C2H6-formation in Moand V-grown cultures of A. azotica. Temperature-dependent curve under saturating light conditions of 245 mol photons m¡2 s¡1. C2H4 (Mo-culture)—solid triangle; C2H4 (V-culture)— solid square; C2H6 (V-culture)—open square. One representative measurement from three independent experiments is given. Internal errors of integration were not signiWcant. C2H6 values below 0.2 mol h¡1 mg Chla¡1 are at the detection limit of the method and may be overestimated by up to 20% Circumstantial evidence for the occurrence of V-nitrogenase in another rice Weld strain, A. azollae (Ni et al. 1990), is not supported by published sequence information. Hardly anything is known about the function, distribution, and expression of alternative nitrogenases in nature. Bacteria containing alternative nitrogenase genes have been isolated from diverse environments (Loveless et al. 1999). However, to our knowledge, the expression of anf genes in gut symbionts of termites (Noda et al. 1999) is the only example where transcription of alternative nitrogenase genes in environmental samples has been documented. Expression of alternative nitrogenases in Mo-deWcient soils or micro-zones of microbial colonies (Maynard et al. 1994), at unusually high tungsten concentrations, high alkalinity (pH 10) (Tsygankov et al. 1997) or low temperatures (Walmsley and Kennedy 1991) has been suggested in several studies. So far, all but one of the Anabaena strains able to express V-nitrogenase have been isolated from rice Welds of diVerent geographic origin. 16S rRNA analysis suggests that Anabaena sp. CH1 is closely related to A. variabilis, but that A. azotica is more closely related to Trichormus azollae and Anabaenopsis circularis (not documented). Thus, conditions that favor the use of alternative nitrogenases might exist in the anoxic lower soil levels of Xooded rice Welds . It was speculated that these enzymes may have been advantageous in ancient anoxic oceans, when Mo may have been scarce due to the formation of insoluble sulWdes under anoxic conditions (Raymond et al. 2004). Tungsten sulWde, by contrast, is more soluble (Hille 2002). This might increase Arch Microbiol (2006) 186:367–376 Mo-deWciency in anoxic environments by inhibiting Mo-uptake. Indigenous cyanobacteria like A. azotica are important biological nitrogen fertilizers in rice Welds, and the distribution and possible contribution of V-nitrogenases to total N2-Wxation in these habitats need further investigation. Acknowledgments We are indebted to Prof. M.G. Yates, Brighton, United Kingdom, for helpful comments on the English and to Prof. H. Dai, Wuhan, Peoples Republic of China, for supplying the A. azotica culture. We warmly thank M. Doeleman for the excellent technical assistance, Dr M. Staal for generous help with the on-line GC system, and Dr X. Zhai for kind help with the Chinese translations. This is NIOO publication number 3897. References Altschul SF, Madden TL, SchaVer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 Bishop PE, Joerger RD (1990) Genetics and molecular biology of alternative nitrogen Wxation systems. Annu Rev Plant Physiol Plant Mol Biol 41:109–125 Boison G, Bothe H, Schmitz O (2000) Transcriptional analysis of hydrogenase genes in the cyanobacteria Anacystis nidulans and Anabaena variabilis monitored by RT-PCR. Curr Microbiol 40:315–321 Boison G, Mergel A, Jolkver H, Bothe H (2004) Bacterial life and dinitrogen Wxation at a gypsum rock. Appl Environ Microbiol 70:7070–7077 Chatterjee R, Ludden PW, Shah VK (1997) Characterization of vnfG, the delta subunit of the vnf-encoded apodinitrogenase from Azotobacter vinelandii: implications for its role in the formation of functional dinitrogenase 2. J Biol Chem 272:3758–3765 Chen P-C (1984) Physiology of nitrogen Wxation in two new strains of Anabaena. Z Naturforsch 40c:406–408 Chien Y-T, Auerbuch V, Brabban AD, Zinder SH (2000) Analysis of genes encoding an alternative nitrogenase in the archaeon Methanosarcina barkeri 227. J Bacteriol 182:3247–3253 Dilworth MJ, Eady RR, Robson RL, Miller RW (1987) Ethane formation from acetylene as a potential test for vanadium nitrogenase in vivo. Nature 326:167–168 Dilworth MJ, Eldridge ME, Eady RR (1993) The molybdenum and vanadium nitrogenases of Azotobacter chroococcum: eVect of elevated temperature on N2 reduction. Biochem J 289:395–400 Domroes M, Peng G (1988) The climate of China. Springer, Berlin Heidelberg New York Eady RR (2003) Current status of structure function relationships of vanadium nitrogenase. Coord Chem Rev 237:23–30 Halwart M, Gupta MV (eds) (2004) Culture of Wsh in rice Welds. FAO, World Fish Center, Rome, Italy, Penang, Malaysia Hille R (2002) Molybdenum and tungsten in biology. Trends Biochem Sci 27:360–367 Kentemich T, Danneberg G, Hundeshagen B, Bothe H (1988) Evidence for the occurrence of the alternative, vanadiumcontaining nitrogenase in the cyanobacterium Anabaena variabilis. FEMS Microbiol Lett 51:19–24 Kumar A, Kumar HD (1980) Tungsten-induced inactivation of molybdoenzymes in Anabaena. Biochim Biophys Acta 613:244–248 375 Kumar S, Tamura K, Nei M (2004) Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163 Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, Land ML, Pelletier DA, Beatty JT, Lang AS, Tabita FR, Gibson JL, Hanson TE, Bobst C, Torres J, Peres C, Harrison FH, Gibson J, Harwood CS (2004) Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat Biotechnol 22:55–61 Ley S-H, Yeh T-C, Liu F-J, Wang L-M, Ts’ui S-K (1959) The nitrogen Wxation of some blue-green algae from Chinese rice-Welds. Acta Hydrobiol Sin 4:429–439 Li S (1981) Studies on the nitrogen-Wxing blue-green algae as biofertilizer in the late rice crop. Acta Hydrobiol Sin 7:417–423 Loveless TM, Bishop PE (1999) IdentiWcation of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs. Can J Microbiol 45:312–317 Loveless TM, Saah JR, Bishop PE (1999) Isolation of nitrogenWxing bacteria containing molybdenum-independent nitrogenases from natural environments. Appl Environ Microbiol 65:4223–4226 Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Dymock D, Wade WG (1998) Design and evaluation of useful bacterium-speciWc PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 64:795–799 Maynard RH, Premakumar R, Bishop PE (1994) Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum. J Bacteriol 176:5583–5586 Ni CV, Yakunin AF, Gogotov IN (1990) InXuence of molybdenum, vanadium, and tungsten on growth and nitrogenase synthesis of the free-living cyanobacterium Anabaena azollae. Microbiology 59:395–398 Noda S, Ohkuma M, Usami R, Horikoshi K, Kudo T (1999) Culture-independent characterization of a gene responsible for nitrogen Wxation in the symbiotic microbial community in the gut of the termite Neotermes koshunensis. Appl Environ Microbiol 65:4935–4942 Nübel U, Garcia-Pichel F, Muyzer G (1997) PCR primers to amplify 16S rRNA genes from cyanobacteria. Appl Environ Microbiol 63:3327–3332 Raina R, Bageshwar UK, Das HK (1992) Construction of a vnfHlacZ fusion and study of expression from the vnfH promoter of the vanadium-dependent nitrogen-Wxation pathway in Azotobacter vinelandii. FEMS Microbiol Lett 98:169–173 Raymond J, Siefert JL, Staples CR, Blankenship RE (2004) The natural history of nitrogen Wxation. Mol Biol Evol 21:541–554 Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61 Schuddekopf K, Hennecke S, Liese U, Kutsche M, Klipp W (1993) Characterization of anf genes speciWc for the alternative nitrogenase and identiWcation of nif genes required for both nitrogenases in Rhodobacter capsulatus. Mol Microbiol 8:673–684 Staal M, Lintel-Hekkert ST, Harren F, Stal L (2001) Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and online monitoring. Environ Microbiol 3:343–351 Tamagnini P, Troshina O, Oxelfelt F, Salema R, Lindblad P (1997) Hydrogenases in Nostoc sp. strain PCC 73102, a strain lacking a bidirectional enzyme. Appl Environ Microbiol 63:1801–1807 Tanelli G (1982) Geological setting, mineralogy and genesis of tungsten mineralization in Dayu district, Jiangxi (Peoples 123 376 Republic of China)—an outline. Mineralium Deposita 17:279–294 Thiel T (1993) Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis. J Bacteriol 175:6276–6286 Thiel T, Lyons EM, Zahalak M (1997) Regulation of alternative nitrogenase systems by environmental factors in the cyanobacterium Anabaena variabilis. In: Legocki A, Bothe H, Pühler A (eds) Biological Wxation of nitrogen for ecology and sustainable agriculture. NATO ASI Series, vol. G39. Springer, Berlin Heidelberg New York, pp. 159–162 Thiel T, Meeks JC, Elhai J, Potts M, Larimer F, Lamerdin J, Predki P, Atlas R (2002a) Nitrogen Wxation: analysis of the genome of the cyanobacterium Nostoc punctiforme. In: Finan TM, O’Brian MR, Layzell DB, Vessey JK, Newton W (eds) Nitrogen Wxation: global perspectives. CABI Publishing, New York, pp. 88–92 Thiel T, Pratte B, Zahalak M (2002b) Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413. Arch Microbiol 179:50–56 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: Xexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 123 Arch Microbiol (2006) 186:367–376 Tsygankov AS, Serebryakova LT, Sveshnikov DA, Rao KK, Gogotov IN, Hall DO (1997) Hydrogen photoproduction by three diVerent nitrogenases in whole cells of Anabaena variabilis and the dependence on pH. Int J Hydrogen Energy 22:859–867 Walmsley J, Kennedy C (1991) Temperature-dependent regulation by molybdenum and vanadium of expression of the structural genes encoding three nitrogenases in Azotobacter vinelandii. Appl Environ Microbiol 57:622–624 Zahalak M, Pratte B, Werth KJ, Thiel T (2004) Molybdate transport and its eVect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413. Mol Microbiol 51:539–549 Zehr JP, McReynolds LA (1989) Use of degenerate oligonucleotides for ampliWcation of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526 Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a crosssystem comparison. Environ Microbiol 5:539–554 Zinoni F, Robson RM, Robson RL (1993) Organization of potential alternative nitrogenase genes from Clostridium pasteurianum. Biochim Biophys Acta 1174:83–86