Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413

Abstract

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...

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

“…strain PCC 7120 demonstrated that hupL transcription coincides with the formation of heterocysts. Subsequent studies, in other filamentous strains, have confirmed the induction of an hupL transcript under nitrogen-fixing conditions only (Axelsson et al, 1999;Happe et al, 2000; Hansel et al, 2001). One exception is Anabaena variabilis ATCC 29413, where a low level of hupL expression has been detected in vegetative cells grown with the addition of ammonia (Boison et al, 2000).…”

Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy

“…100 µmol H 2 /mg chlorophyll a/h, with light conversion efficiencies of up to 3.9 % (proportion of absorbed light energy converted to H 2 ) (Dutta et al 2005;Yoon et al 2006;. Rates were increased 3-7 fold in Anabaena mutants deficient in uptake hydrogenase activity (Borodin et al 2000;Happe et al 2000;Masukawa et al 2002;Yoshino et al 2007) and this strategy was applied in outdoor culture. However, the maximum light conversion efficiency was only 0.1 %, which has implications for the large scale application of this approach (Lindblad et al 2002;Tsygankov et al 2002).…”

Genetic Engineering of Cyanobacteria to Enhance Biohydrogen Production from Sunlight and Water

“…Elimination of Hup activity resulted in a 4-to 7-fold increase in the rates of H 2 production in an Ar atmosphere compared with wild-type cells, while the effects of inactivation of Hox activity on H 2 production were not evident under the conditions tested. Hup-disrupted mutants also were shown to be effective in enhancing H 2 production by several other Anabaena and Nostoc strains of cyanobacteria (Happe et al 2000;Lindberg et al 2002;Schütz et al 2004;Carrasco et al 2005;Yoshino et al 2007). A promising approach to further improve photobiological H 2 production in the presence of O 2 is to initially select parental strains with high nitrogenase activity and inactivate their Hup activities.…”