Gudrun Boison

HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).

Transcriptional analysis of hydrogenase genes in the Cyanobacteria Anacystis nidulans and Anabaena variabilis monitored by RT-PCR.

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.

Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans

Among cyanobacteria, the heterocystous, N2‐fixing Anabaena variabilis and the unicellular Anacystis nidulans have recently been shown to possess an NAD+‐dependent, bidirectional hydrogenase. A 5.0 kb DNA segment of the A. nidulans genome is now identified to harbor the structural genes hoxUYH coding for three subunits of the bidirectional hydrogenase. The gene arrangement in A. nidulans and in A. variabilis is remarkably dissimilar. In A. nidulans, but not in A. variabilis, the four accessory genes hoxW, hypA, hypB and hypF could be identified downstream of hoxH. An insertional homozygous mutant in hoxH from A. nidulans was completely inactive in performing Na2S2O4‐dependent H2 evolution but could utilize the gas with almost 50% of the activity of the wild type. These findings with the first defined hydrogenase mutant in any photosynthetic, O2‐evolving microorganism indicate that the unicellular cyanobacterium A. nidulans possesses both an uptake and a bidirectional hydrogenase. The physiological role(s) of the two hydrogenases in unicellular non‐N2‐fixing cyanobacteria is not yet understood.

Bacterial Life and Dinitrogen Fixation at a Gypsum Rock

ABSTRACT The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C2H2 reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C2H2 reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O2 tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O2 tension that allow N2 fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N2 under microaerobic conditions only.

Quantitative analysis of expression of two circadian clock-controlled gene clusters coding for the bidirectional hydrogenase in the cyanobacterium Synechococcus sp. PCC7942.

Hydrogen metabolism is of central interest in cyanobacterial research because of its potential applications. The gene expression and physiological role of the cyanobacterial bidirectional NAD(P)+‐reducing hydrogenase are poorly understood. Transcription rates of hoxEF and hoxUYH encoding this enzyme have been studied in Synechococcus sp. PCC7942. PhoxU activity was about three times higher than that of PhoxE. Circadian phasing of both promoters was found to be synchronous and influenced expression levels by at least one order of magnitude. This is the first demonstration of circadian control of gene expression for any hydrogenase. For the majority of PhoxU‐driven messages, transcription presumably terminates between hoxU and hoxH. Being part of a polycistronic hoxUYHW… operon, hoxW, encoding a protease involved in C‐terminal processing of the hydrogenase large‐subunit HoxH, is mainly expressed by its own promoter, PhoxW. The complex transcript formation may be a key feature for controlling bidirectional hydrogenase expression in vivo.

The rice field cyanobacteria Anabaena azotica and Anabaena sp. CH1 express vanadium-dependent nitrogenase

Anabaena azotica FACHB-118 and Anabaena sp. CH1, heterocystous cyanobacteria isolated from Chinese and Taiwanese rice fields, expressed vanadium-containing nitrogenase when under molybdenum deficiency. This is the second direct observation of an alternative nitrogenase in cyanobacteria. The vanadium nitrogenase-specific 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 first 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 hupL was expressed in Mo-free medium concomitantly with vnfDG in A. azotica, Anabaena sp. CH1, and A. variabilis.

Maximizing hydrogen production by cyanobacteria.

When incubated anaerobically, in the light, in the presence of C2H2 and high concentrations of H2, both Mo-grown Anabaena variabilis and either Mo- or V-grown Anabaena azotica produce large amounts of H2 in addition to the H2 initially added. In contrast, C2H2- reduction is diminished under these conditions. The additional H2-production mainly originates from nitrogenase with the V-enzyme being more effective than the Mo-protein. This enhanced H2-production in the presence of added H2 and C2H2 should be of interest in approaches to commercially exploit solar energy conversion by cyanobacterial photosynthesis for the generation of molecular hydrogen as a clean energy source

Hydrogenases in Cyanobacteria

Cyanobacteria can express at least two different hydrogenases catalyzing the consumption of molecular H2 [1,2]. A so called uptake hydrogenase is component of the thylakoid membrane [3]. It is particularly active in heterocysts of filamentous forms [4], but also occurs in the unicellular Anacystis nidulans(SynechococcusPCC 6301) [5-7]. Electron acceptor for H2-utilization catalyzed by the uptake hydrogenase is either cytochrome bor plastoquinone. The electrons are then allocated via the cytochrome bccomplex to either photosystem I or to the respiratory terminal oxidase [1,7]. H2 uptake of the thylak-oid membranes is activated by the thioredoxin system [2]. The enzyme is encoded by the hupLSgenes. In AnabaenaPCC 7120 [8], but not in NostocPCC 73102 [9], hupLis subject to gene rearrangement in parallel with heterocysts differentiation. The hupLSgenes have not been detected in unicellular cyanobacteria and are not present on the completely sequenced chromosome of SynechocystisPCC 6803 [10].