Heterocystous Anabaena Research Articles

CyAbrB Transcriptional Regulators as Safety Devices To Inhibit Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of these genes is cyabrB1 (also known as calA in Anabaena sp. strain PCC 7120), encoding a transcriptional regulator. In the present study, we investigated the function of calA/cyabrB1 in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method that we recently utilized for the photosynthetic production of a useful chemical in this strain. Conditional knockdown of calA/cyabrB1 in the presence of nitrate resulted in the formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were upregulated by calA/cyabrB1 knockdown in the presence of combined nitrogen sources. These genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by calA/cyabrB1 knockdown. hetP and hepA were repressed by direct binding of CalA/cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the overexpression of calA/cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of calB/cyabrB2 (a paralogue gene of calA/cyabrB1), in addition to knockdown of calA/cyabrB1, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and CalA/cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen stepdown. We concluded that cyAbrB proteins are essential safety devices that inhibit heterocyst differentiation.IMPORTANCE Spore formation in Bacillus subtilis and Streptomyces has been extensively studied as models of prokaryotic nonterminal cell differentiation. In these organisms, many cells/hyphae differentiate simultaneously, which is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. strain PCC 7120 is unique because it is terminal, and only 5 to 10% of vegetative cells differentiate into heterocysts. In this study, we identified CalA/cyAbrB1 as a repressor of two genes that are essential for heterocyst formation independently of HetR, a master activator for heterocyst differentiation. This finding is reasonable for unique cell differentiation of Anabaena because CalA/cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is overexpressed could differentiate into heterocysts.

Structural and enzymatic analyses of a glucosyltransferase Alr3699/HepE involved in Anabaena heterocyst envelop polysaccharide biosynthesis.

Formation of the heterocyst envelope polysaccharide (HEP) is a key process for cyanobacterial heterocyst differentiation. The maturation of HEP in Anabaena sp. strain PCC 7120 is controlled by a gene cluster termed HEP island in addition to an operon alr3698-alr3699, which encodes two putative proteins termed Alr3698/HepD and Alr3699/HepE. Here we report the crystal structures of HepE in the apo-form and three complex forms that bind to UDP-glucose (UDPG), UDP&glucose, and UDP, respectively. The overall structure of HepE displays a typical GT-B fold of glycosyltransferases, comprising two separate β/α/β Rossmann-fold domains that form an inter-domain substrate-binding crevice. Structural analyses combined with enzymatic assays indicate that HepE is a glucosyltransferase using UDPG as a sugar donor. Further site-directed mutageneses enable us to assign the key residues that stabilize the sugar donor and putative acceptor. Based on the comparative structural analyses, we propose a putative catalytic cycle of HepE, which undergoes "open-closed-open" conformational changes upon binding to the substrates and release of products. These findings provide structural and catalytic insights into the first enzyme involved in the HEP biosynthesis pathway.

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of 'new' (recently differentiated) and 'old' (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.

Novel heterocyst-specific flavodiiron proteins in Anabaena sp. PCC 7120

Flavodiiron proteins present in many prokaryotic and some eukaryotic organisms have a capacity to protect cells against nitrosative or oxidative stress. In Anabaena sp. PCC 7120, Flv1 and Flv3 proteins are encoded by families of two genes. We demonstrate here that flv1A and flv3A genes are up-regulated in vegetative cells in low CO2 and high light conditions. In contrast, flv1B and flv3B genes are expressed in N2-fixing conditions and corresponding proteins are located exclusively in heterocysts. It is suggested that Flv1B and Flv3B protect enzymes of N2-fixation in heterocysts of Anabaena 7120 by reducing molecular oxygen directly to water.

Transcription Activation by NtcA in the Absence of Consensus NtcA-Binding Sites in an Anabaena Heterocyst Differentiation Gene Promoter

Heterocyst differentiation is orchestrated by the N control transcriptional regulator NtcA and the differentiation-specific factor HetR. In Anabaena sp. strain PCC 7120, the devBCA operon is expressed from two different promoters activated upon N stepdown. The distal devB promoter (transcription start point [TSP] located at position -704) represents a canonical class II NtcA-activated promoter, including a consensus NtcA-binding site centered 39.5 nucleotides upstream from the TSP. Transcription activation from a second TSP (-454) requires NtcA and is impaired in hetR mutants. In a wild-type background, three different DNA fragments, including both or each individual promoter, directed gfp expression localized mainly to proheterocysts and heterocysts. Expression was undetectable in an ntcA background and, for the fragment including the proximal promoter alone, also in a hetR background. In spite of the absence of consensus NtcA-binding sequences between the two TSPs, NtcA was shown to interact with this DNA region, and NtcA and its effector, 2-oxoglutarate, were necessary and sufficient for in vitro transcription from the -454 TSP. No HetR binding to the DNA or in vitro transcription from the proximal devB TSP promoted by HetR alone were detected. However, a moderate positive effect of HetR on NtcA binding to the DNA between the two devB TSPs was observed. The proximal devB promoter appears to represent a suboptimal NtcA-activated promoter for which HetR may act as a coactivator, with the physiological effect of restricting gene activation to conditions of prevalence of high NtcA and HetR levels, such as those taking place during heterocyst differentiation.

Factors influencing microbial mat composition, distribution and dinitrogen fixation in three western Indian Ocean coral reefs

Cyanobacteria have been found to be important for primary production and nitrogen supply on coral reefs. Here, the distribution of cyanobacteria that dominate microbial mats (identified by using microscopic and molecular tools), their abundance, and specific contributions and timing of N2 fixation, were studied in two coral reef systems of the western Indian Ocean: Mayotte (Comoros) and Tulear (Toliara, Madagascar). The results were compared with previously published data from the reefs of La Réunion Island (Mascarenes). Variations in nutrient levels, temperature and light penetration (Secchi) were also measured and compared. The reefs in Mayotte are situated in oligotrophic ocean waters and support coral reefs in good condition. The reef of Tulear was overgrown by algae. The shallow lagoon and reef in La Réunion showed signs of eutrophication and coral degradation. All three reefs maintained a diverse population of benthic cyanobacterial mats. A total of 13 different cyanobacterial morphotypes were encountered, belonging mainly to the genera Anabaena, Hydrocoleum, Leptolyngbya and Lyngbya (as identified by morphotypes and 16 S RNA analyses), but with variable abundances. Twelve of the 13 dominant cyanobacteria observed in the three sites fixed dinitrogen with daily rates of up to 42 nmol N2 µg−1 Chl a. Maximum values were achieved by the heterocystous Anabaena sp. during daylight in Mayotte, which exhibited strongly light‐stimulated nitrogenase activity. Mats dominated by non‐heterocystous cyanobacteria fixed both during daylight and the night, with daily rates up to 38 nmol N2 µg−1 Chl a (by Lyngbya majuscula, Mayotte). Hydrocoleum‐dominated mats fixed N2 at rates between 0 (H. glutinosum, Tulear) and 23.4 nmol N2 µg−1 Chl a (H. coccineum, Mayotte). Cyanobacteria are normal constituents of the benthic microflora in tropical regions, but their abundance, diversity and species composition changes with environmental conditions and so do the rates of N2 fixation on the lagoon floors.

10.1007/s11183-008-2001-4

Most plants are constructed from repeating modular units such as phytomers, merophytes, and cell packets. Even an organism as simple as the filamentous cyanobacterium Anabaena shows recurrent patterns of differentiated cellular structures, notably with respect to its heterocysts. These examples reflect the inherent rhythms established within developmental processes of living organisms. In the present article, attention is paid to repetitious production of idioblasts—isolated cells, or clusters of cells, with an identity different to that of neighbouring cells from which they are derived. In higher plant root tissues, idioblasts are contained within cell packets that grow up from mother cells during the course of a number of cycles of cell production. The heterocysts of Anabaena are also discussed; they, too, are a type of idioblast. The idioblasts of root tissues originate as small cells which result from unequal cell divisions. Such divisions are usually the final ones within a cell packet which has already undergone a number of division cycles and are characteristically located at one or both ends of a packet. The packet end walls are suggested to have a role in regulating division asymmetry. Idioblastic systems discussed are root cortical trichosclereids and diaphragm cells; in their earliest stage, the cells from which lateral root primordia arise are also considered as clusters of idioblasts because they, too, are the products of asymmetric divisions of pericyclic mother cells. The division patterns of all these idioblastic systems were modelled in a consistent way using L-systems, with the assumption that the age of a cell-packet end wall plays a special role in cell determination.

Dinitrogen-fixing cyanobacteria in microbial mats of two shallow coral reef ecosystems.

Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using 15N2 and 13C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C2H2 reduced µg−1 Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N2 µg−1 Chl day−1 (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C µg−1 Chl day−1 (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.

Rhythmic plant morphogenesis: Recurrent patterns of idioblast cell production

Most plants are constructed from repeating modular units such as phytomers, merophytes, and cell packets. Even an organism as simple as the filamentous cyanobacterium Anabaena shows recurrent patterns of differentiated cellular structures, notably with respect to its heterocysts. These examples reflect the inherent rhythms established within developmental processes of living organisms. In the present article, attention is paid to repetitious production of idioblasts—isolated cells, or clusters of cells, with an identity different to that of neighbouring cells from which they are derived. In higher plant root tissues, idioblasts are contained within cell packets that grow up from mother cells during the course of a number of cycles of cell production. The heterocysts of Anabaena are also discussed; they, too, are a type of idioblast. The idioblasts of root tissues originate as small cells which result from unequal cell divisions. Such divisions are usually the final ones within a cell packet which has already undergone a number of division cycles and are characteristically located at one or both ends of a packet. The packet end walls are suggested to have a role in regulating division asymmetry. Idioblastic systems discussed are root cortical trichosclereids and diaphragm cells; in their earliest stage, the cells from which lateral root primordia arise are also considered as clusters of idioblasts because they, too, are the products of asymmetric divisions of pericyclic mother cells. The division patterns of all these idioblastic systems were modelled in a consistent way using L-systems, with the assumption that the age of a cell-packet end wall plays a special role in cell determination.

Light‐dependent oxygen consumption in nitrogen‐fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O2 through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O2, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O2. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer 18O2 and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O2 consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O2 concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N2‐fixing Trichodesmium colonies, Mehler activity can consume ∼75% of gross O2 production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ∼10% of gross O2 production. Moreover, evidence for the coupling between N2 fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O2 consumption by 3‐fold. Our results suggest that a major role for PSI in N2‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O2 through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.

Nitrogen‐fixation strategies and Fe requirements in cyanobacteria

Diazotrophic (nitrogen‐fixing) cyanobacteria are important contributors of new nitrogen to oligotrophic environments and greatly influence oceanic productivity. We investigated how iron availability influences the physiology of cyanobacterial diazotrophs with different strategies for segregating nitrogen fixation and photosynthesis. We examined growth, photosynthesis, nitrogen fixation, and Fe requirements of the filamentous nonheterocystous Trichodesmium, the filamentous heterocystous Anabaena, and the unicellular Cyanothece under a range of Fe concentrations. Under similar Fe concentrations the three species differed in N2‐fixation rates, photosynthetic activity, the relative abundance of the photosynthetic units PSI : PSII, elemental stoichiometry, and Fe use efficiency. Complex colonial forms such as Trichodesmium and Anabaena are more likely to be Fe limited in their natural environments and are more efficient at utilizing Fe than unicellular diazotrophs such as Cyanothece. The varied physiological responses to Fe availability of the three cyanobacteria reflect their nitrogen fixation strategies, cell size, unicellular or colonial organization, and may explain, at least in part, the ecological distribution of these photosynthetic bacteria.

Planktonic nitrogen fixation in Lake Malawi/Nyasa

Nitrogen (N2) fixation has been identified as possibly an important source of “new” nitrogen (N) to the epilimnion of Lake Malawi but studies in 1999–2000 and 2002 (September–December) estimated that the contribution of N2-fixation by heterocystous Anabaena filaments to the N budget of the lake’s epilimnion is only 3–4% of total N input. N2-fixation rates in Lake Malawi, as estimated by the acetylene reduction assay were higher during the stratified season (September–March) than during the rest of the year. Planktonic N2-fixation in Lake Malawi can be monitored by measuring heterocyst biovolume concentrations because a significant correlation (r2 = 0.945, P < 0.0001) was observed between the two parameters. Heterocyst density cannot be used to estimate N2-fixation because heterocyst sizes in the lake change continuously as at least two Anabaena species with different heterocyst dimensions are present. During September–October 2002, a species similar to A. maxima, contained larger heterocysts (16.34 ± 2.46 and 13.25 ± 1.89 μm in cross and apical section dimensions, respectively). In November–December of 2002, A. discoidea dominated and had smaller sized heterocysts (8.92 ± 1.13 and 7.25 ± 0.95 μm in cross and apical section dimensions). Since planktonic N2-fixation is higher near shore than offshore, its contribution to the N budget in the littoral zone where high densities of grazing benthic fish occur may be more critical to maintaining the high rates of benthic algal productivity observed.

The proteome of the heterocyst cell wall in Anabaena sp. PCC 7120

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.

Isolation and Identification of an Epibiotic Bacterium Associated with HeterocystousAnabaenaCells

Previous articleNext article No AccessResearch NoteIsolation and Identification of an Epibiotic Bacterium Associated with Heterocystous Anabaena CellsBradley S. Stevenson and John B. WaterburyBradley S. Stevenson1. Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019 Search for more articles by this author and John B. Waterbury2. Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Search for more articles by this author Received 8 November 2005; accepted 28 January 2006.PDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 210, Number 2April 2006 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.2307/4134596 Views: 94Total views on this site PDF download Crossref reports no articles citing this article.

Inorganic Nutrient Concentrations and Chlorophyll in the Euphotic Zone of Lake Tanganyika

Chlorophyll a and inorganic plant nutrient levels were measured in the euphotic zone at a pelagic station in Lake Tanganyika for 12 months. In October, chlorophyll a had a mean concentration of 5.0 μg/l, the highest value obtained during the study period. A secondary peak (mean 2.5 μg/l) was obtained in May. Upwelling in July/August may have been responsible for the high biomass in October, while secondary upwelling in April/May may have caused the secondary peak in May. Secchi disc transparency ranged between 7.71 and 16.25 m. The low transparency coincided with maximum chlorophyll a in October. An inverse relationship was found between chlorophyll and transparency. High nitrate-nitrogen was observed in July and December, when the values were 67 and 76 μg N/l, respectively. Nitrogen fixation by the heterocystous Anabaena flos-aquae may have been responsible for the high nitrate values in October/December. The mean nitrate-nitrogen concentration was 45.64 μg N/l and an inverse relationship was found between chlorophyll a and nitrate-nitrogen. Very low soluble reactive phosphorus levels (range 0.4 and 4.0 μg P/l) were found throughout the 12 month period. A mean SRP concentration of 1.86 μg P/l was found. A positive correlation was found between chlorophyll a and SRP. N:P molar ratio gave a value of 54.33, showing that in the euphotic zone of the lake, P is limiting primary production. Silica concentrations were high throughout the study period with a mean value of 1.56 mg SiO2/l.

A Two-component System Mediates Developmental Regulation of Biosynthesis of a Heterocyst Polysaccharide

Some cyanobacteria couple oxygenic photosynthesis in vegetative cells with O2-sensitive N2 fixation in differentiated cells called heterocysts. Heterocyst differentiation involves extensive biochemical and structural changes that collectively permit heterocysts to assimilate N2 aerobically and supply the products of N2 fixation to vegetative cells. HepK and DevR are required for the development of functional heterocysts in Anabaena and Nostoc, respectively. We show that HepK is an autokinase and that Anabaena DevRA is its cognate response regulator, together comprising part or all of a two-component system that mediates developmental regulation of biosynthesis of a heterocyst envelope polysaccharide. Recombinant N-hexahistidine-tagged HepK (H6HepK), the cytoplasmic portion H6'HepK of H6HepK, H6DevR, and H6DevRA were overexpressed in Escherichia coli and purified to homogeneity. H6'HepK, but not H6HepK, autophosphorylates with [gamma-32P]ATP. ADP, specifically, elicits dephosphorylation of phosphorylated H6'HepK. The phosphoryl group of H6'HepK is transferred rapidly and efficiently to both H6DevR and H6DevRA but not to His-tagged OmpR, whose cognate sensor kinase is EnvZ. Sequence comparisons, the results of site-specific mutagenesis, and tests of chemical stability support identification of HepK-His348 and DevR-Asp53 as the phosphorylated residues. The mutation HepK-H348A abolishes both in vitro autokinase activity and in vivo functionality of HepK. Heterocysts of both hepK Anabaena and devRA Anabaena lack an envelope polysaccharide layer and are nonfunctional. Consistent with the normal site of deposition of that polysaccharide, a hepK::gfp transcriptional fusion is expressed principally in proheterocysts. HepK/DevRA is the first two-component system identified that regulates the biosynthesis of a polysaccharide as part of a patterned differentiation process.

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.

Molecular evidence for the aerobic expression of nifJ, encoding pyruvate:ferredoxin oxidoreductase, in cyanobacteria

Pyruvate:ferredoxin (flavodoxin) oxidoreductase (PFO, EC 1.2.7.1) catalyses the oxidative cleavage of pyruvate and coenzyme A to acetylcoenzyme A and CO 2 with the simultaneous reduction of ferredoxin or flavodoxin. PFO occurs in anaerobes and in some aerobic archaea and bacteria. For cyanobacteria, activity measurements indicated the occurrence of PFO in heterocystous forms. The completely sequenced genomes of the unicellular Synechocystis sp. PCC 6803 and the heterocystous Anabaena sp. PCC 7120 and Nostoc punctiforme revealed the existence of one PFO (encoded by nifJ) in Synechocystis 6803 and N. punctiforme but two different PFOs, encoded by nifJ1 and nifJ2, in Anabaena. Sequence comparison now indicates that all cyanobacterial PFOs are more closely related to those of anaerobes than to those of aerobes. Reverse transcription-polymerase chain reaction (RT-PCR) experiments show that nifJ is transcribed in the presence of saturating iron concentrations in aerobically grown cells of the unicellular Synechococcus sp. PCC 6301 and Synechocystis 6803. Both nifJ genes are transcribed in aerobically grown Anabaena 7120. These findings are corroborated by luciferase reporter gene analysis of nifJ in Synechococcus sp. PCC 7942. The occurrence of PFO in these cyanobacteria is enigmatic.

Molecular evidence for the aerobic expression of nifJ, encoding pyruvate:ferredoxin oxidoreductase, in cyanobacteria.

Pyruvate:ferredoxin (flavodoxin) oxidoreductase (PFO, EC 1.2.7.1) catalyses the oxidative cleavage of pyruvate and coenzyme A to acetylcoenzyme A and CO2 with the simultaneous reduction of ferredoxin or flavodoxin. PFO occurs in anaerobes and in some aerobic archaea and bacteria. For cyanobacteria, activity measurements indicated the occurrence of PFO in heterocystous forms. The completely sequenced genomes of the unicellular Synechocystis sp. PCC 6803 and the heterocystous Anabaena sp. PCC 7120 and Nostoc punctiforme revealed the existence of one PFO (encoded by nifJ) in Synechocystis 6803 and N. punctiforme but two different PFOs, encoded by nifJ1 and nifJ2, in Anabaena. Sequence comparison now indicates that all cyanobacterial PFOs are more closely related to those of anaerobes than to those of aerobes. Reverse transcription-polymerase chain reaction (RT-PCR) experiments show that nifJ is transcribed in the presence of saturating iron concentrations in aerobically grown cells of the unicellular Synechococcus sp. PCC 6301 and Synechocystis 6803. Both nifJ genes are transcribed in aerobically grown Anabaena 7120. These findings are corroborated by luciferase reporter gene analysis of nifJ in Synechococcus sp. PCC 7942. The occurrence of PFO in these cyanobacteria is enigmatic.

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

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.