Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.Cyanobacteria are phototrophic organisms that often form water blooms in eutrophic or estuarine waters. These water blooms undergo fluctuations and may exhibit toxic states. One common genus in such water blooms, Microcystis, produces the hepatotoxin microcystin (6). There are approximately 65 known isoforms of microcystin, representing a family of cyclic heptapeptides having the common structure cyclo(D-Ala-L-X-D-MeAsp-L-Z-Adda-D-Glu-Mdha), where L-X and L-Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decandienoic acid, D-MeAsp is 3-methylaspartic acid, and Mdha is N-methyl-dehydroalanine (Fig.

An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD'-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP' -dependent protein from Desulfovibrio fructosovorarzs. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxv and for the diaphorase moiety (hoxU and hoxfl described for the A. eurroplius enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxl: respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 71 19 and Anucystis nidulan.7 was amplified by PCR. Using the labeled segment from A. 71 19 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 71 19, Anabaena cylindrica, Arzacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A. variabilis. Therefore the 8.9-kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-dependent H, evolution corroborating the molecular biological demonstration of the NAD(P)'.-dependent hydrogenase in 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).

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 NH(4)(+)-grown A. variabilis.

The bidirectional, NAD+-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803. In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E. coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKmR cassette into the gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H2-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake. A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes.

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 (C 2 H 2 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, C 2 H 2 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 O 2 tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O 2 tension that allow N 2 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 N 2 under microaerobic conditions only.

SummaryHydrogen 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)1 -reducing hydrogenase are poorly understood. Transcription rates of hoxEF and hoxUYH encoding this enzyme have been studied in Synechococcus sp. PCC7942. P hoxU activity was about three times higher than that of P hoxE . 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 P hoxU -driven messages, transcription presumably terminates between hoxU and hoxH.

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