A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5 start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N 2 -fixing conditions, the mutant strain exhibited significantly increased rates in H 2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H 2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.The only microorganisms with an O 2 -producing photosynthesis that have a hydrogen metabolism are cyanobacteria and green algae (23,25). In cyanobacteria, up to three enzymes can be involved in hydrogen metabolism: the nitrogenase which produces H 2 during nitrogen fixation (24), the membranebound hydrogenase which reoxidizes the H 2 (10), and the bidirectional hydrogenase catalyzing both oxidation of molecular hydrogen and reduction of protons (38). In cyanobacteria, the genetics of bidirectional hydrogenases are especially well characterized (3, 7). But, the H 2 production in filamentous cyanobacteria during the reduction of nitrogen to NH 3 is mainly catalyzed by the nitrogenase in the heterocysts. The Anabaena cells can oxidize the hydrogen with the uptake hydrogenase via the oxyhydrogen (Knallgas) reaction. It was suggested that the organism gets additional ATP while the Knallgas reaction can protect the O 2 -sensitive nitrogenase by removing the oxygen in the heterocysts (30).Hydrogenases have been described for a large number of microorganisms and studied intensively in diverse phylogenetic groups of bacteria (22,34,54). The uptake hydrogenases are membrane-bound enzymes which consist of two subunits with [Fe-S] clusters as prosthetic groups. The large subunit, HupL, carries additionally a Ni atom in the active center. In most of the bacterial families, the hupSL genes are clustered in an operon in which hupS is located upstream of the hupL gene (20,51,53). Recently, some hydrogenase sequences from filamentous cyanobacteria were published (10, 31, 38). The uptake hydrogenase i...
In heterocysts of the filamentous cyanobacterium Anabaena 7120 a specific [2Fe-2S] ferredoxin is synthesized, serving as immediate electron donor to nitrogenase. The structural gene for this heterocyst ferredoxin, fdxH, was isolated from a recombinant lambda library, using an oligonucleotide probe derived from a unique segment of the N-terminal amino acid sequence of the purified protein. The sequence of the entire fdxH coding region was determined including 3' and 5' flanking sequences. Assuming proteolytic cleavage of the first methionine residue, the molecular weight of Anabaena 7120 heterocyst ferredoxin is 10,806. Compared with the ferredoxin from vegetative cells, 47 out of 98 amino acid residues are different, including conversions within a highly conserved region responsible for binding of the iron-sulfur cluster. Northern hybridization with a 0.64 kb EcoRI DNA fragment containing the entire fdxH gene indicated two major transcripts of 0.59 and 1.85 kb, which are expressed at a late stage of heterocyst differentiation. By S1 nuclease digestion and primer extension a possible start site of transcription was mapped, 132 bp upstream of fdxH; however, neither a typical Escherichia coli nor nif-type promoter sequence was apparent. Southern hybridization detected only one copy of the fdxH gene in the Anabaena 7120 genome. FdxH is located approximately 7 kb downstream from the nifHDK gene cluster.
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