Heterocyst-forming Cyanobacterium Anabaena Research Articles

Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments.

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.IMPORTANCECircadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena, under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.

The organization of the phycobilisome-photosystem I supercomplex depends on the ratio between two different phycobilisome linker proteins

The phycobilisome (PBS) is an antenna protein complex in cyanobacteria, Glaucocystophytes, and red algae. In the standard PBS, the rod-core PBS, the rods are connected to the core by the rod-core linker protein CpcG. The rod-core PBS transfers the light energy mainly to photosystem (PS) II and to a lesser extent to PSI. Cyanobacteria assemble another type of PBS, the CpcL-PBS, which consists of only one rod. This rod-type PBS is connected to the thylakoid membrane by the linker protein CpcL and is a PSI-specific antenna. In the filamentous heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120, the CpcL-PBS forms a complex with the tetrameric PSI (PBS-PSI supercomplex). The CpcL-PBS and the rod part of the rod-core PBS are identical except for the linker proteins CpcL and CpcG. How cells control the accumulation of the two different types of PBS is unknown. Here, we analyzed two mutant strains which either lack the major rod-core linker CpcG4 or overexpress the rod-membrane linker CpcL. In both mutant strains, more and larger PBS-PSI supercomplexes accumulated compared to the wild type. Our results suggest that CpcL and CpcG4 compete for the same phycobiliprotein pool, and therefore the CpcL/CpcG4 ratio determines the levels of PBS-PSI supercomplexes. We propose that the CpcL-PBS and the rod-core PBS fulfill distinct functions in light harvesting.Graphical abstract

Single-Cell Measurements of Fixation and Intercellular Exchange of C and N in the Filaments of the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

ABSTRACTUnder diazotrophic conditions, the model filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [13C]bicarbonate and 15N2 and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG-SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The 13C/12C and 15N/14N ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-l-arginyl-poly(l-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, 13C/12C and 15N/14N ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N2, and the 13C/12C ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions.

A TonB-Like Protein, SjdR, Is Involved in the Structural Definition of the Intercellular Septa in the Heterocyst-Forming Cyanobacterium Anabaena.

ABSTRACTCyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR (septal junction disk regulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure.

Structural and functional insights into the tetrameric photosystem I from heterocyst-forming cyanobacteria.

Two large protein-cofactor complexes, photosystem I and photosystem II, are the central components of photosynthesis in the thylakoid membranes. Here, we report the 2.37-Å structure of a tetrameric photosystem I complex from a heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Four photosystem I monomers, organized in a dimer of dimer, form two distinct interfaces that are largely mediated by specifically orientated polar lipids, such as sulfoquinovosyl diacylglycerol. The structure depicts a more closely connected network of chlorophylls across monomer interfaces than those seen in trimeric PSI from thermophilic cyanobacteria, possibly allowing a more efficient energy transfer between monomers. Our physiological data also revealed a functional link of photosystem I oligomerization to cyclic electron flow and thylakoid membrane organization.

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.

Two DevBCA-like ABC transporters are involved in the multidrug resistance of the cyanobacterium Anabaena sp. PCC 7120.

The filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is an important model organism for studying cell differentiation, nitrogen fixation, and photosynthesis. This cyanobacterium possesses a high number of membrane transporters. Not much is known about the roles of the membrane transporters, especially the ATP-binding cassette (ABC) transporters, in the multidrug resistance of this cyanobacterium. In the present work, we performed a mutational analysis of the genes alr4280/alr4281/alr4282 and alr3647/alr3648/alr3649 that code for the components of putative ABC exporters and are homologous to the DevBCA heterocyst-specific glycolipid exporter. We show that these genes are essential for resistance to different drugs and are not essential for heterocyst development.

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine.

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal α/β propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, β-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.

Cyanobacterial Septal Junctions: Properties and Regulation.

Heterocyst-forming cyanobacteria are multicellular organisms that grow as chains of cells (filaments or trichomes) in which the cells exchange regulators and nutrients. In this article, we review the morphological, physiological and genetic data that have led to our current understanding of intercellular communication in these organisms. Intercellular molecular exchange appears to take place by simple diffusion through proteinaceous structures, known as septal junctions, which connect the adjacent cells in the filament and traverse the septal peptidoglycan through perforations known as nanopores. Proteins that are necessary to produce, and that may be components of, the septal junctions―SepJ, FraC and FraD―have been identified in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 model. Additionally, several proteins that are necessary to produce a normal number of nanopores and functional septal junctions have been identified, including AmiC-type amidases, peptidoglycan-binding proteins and some membrane transporters. Available reports and reevaluation of intercellular molecular transfer data for some mutants of Anabaena suggest that the septal junctions can be regulated, likely by a mechanism of gating.

Excision of the 59-kb fdxN DNA element is required for transcription of the nifD gene in Anabaena PCC 7120 Heterocysts

ABSTRACTIn the heterocyst-forming cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120, the nitrogen-fixation (nif) genes are expressed specifically in heterocysts during the late stages of development. We used gfp-reporter fusions to examine the regulation of the nifH and nifD genes. Plasmid-borne reporter fusions containing up to 500-bp of the nifH upstream region, which extended into the nifU gene, did not show developmentally regulated GFP fluorescence after removal of a nitrogen source from the growth medium, which indicated that sequences essential for transcriptional regulation are outside of the tested regions. Therefore, a gfp-reporter fusion was engineered into the chromosome at the 5′ end of nifD to produce strain AMC1774. This reporter construct at the native locus showed strong developmentally regulated GFP expression in differentiating heterocysts by 18 h after removal of combined nitrogen. We then screened for UV-induced mutants of AMC1774 that differentiated heterocysts but failed to show GFP-reporter fluorescence. Several dark mutants were obtained and then complemented with an expression library. One mutant was complemented with a clone that contained the xisF and alr1460 genes. The xisF gene is required for excision of a 59,428-bp DNA element present within the fdxN gene in the nifB-fdxN-nifS-nifU gene cluster, which is upstream of the nifHDK gene cluster. Analysis of the original UV-induced mutant showed that it was defective for excision of the fdxN DNA element. Together, our data shows that transcription of the nifHDK genes requires a distant promoter upstream of the fdxN DNA element. We used a gfp transcriptional reporter to demonstrate developmentally regulated promoter activity from the intergenic region upstream of nifB. We also show that the promoter for a second nifH gene, nifH2 (alr0874), lies proximal to nifH2 in its upstream intergenic region.

Specific mutations in the permease domain of septal protein SepJ differentially affect functions related to multicellularity in the filamentous cyanobacterium Anabaena.

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two interdependent cell types that exchange nutrients and regulators. Vegetative cells provide heterocysts with reduced carbon, and heterocysts provide vegetative cells with fixed nitrogen. Additionally, heterocyst differentiation from vegetative cells is regulated by inhibitors of differentiation produced by prospective heterocysts and heterocysts. Proteinaceous structures known as septal junctions join the cells in the filament. The SepJ protein is involved in formation of septal junctions in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SepJ bears extra-membrane and membrane (permease) domains and is located at the cell poles in the intercellular septa of the filament. Here we created Anabaena mutants that produce SepJ proteins altered in the permease domain. Some of these mutant SepJ proteins did not provide functions needed for Anabaena to form long filaments and (in some cases) differentiate heterocysts, identifying amino acids and amino acid stretches that are important for the structure or function of the protein. Some other mutant SepJ proteins fulfilled filamentation and heterocyst differentiation functions but failed to provide normal communication function assessed via the intercellular transfer of the fluorescent marker calcein. These mutant SepJ proteins bore mutations in amino acids located at the cytoplasmic face of the permease, which could affect access of the fluorescent marker to the septal junctions. Overall, the data are consistent with the idea that SepJ carries out multiple roles in the multicellular function of the Anabaena filament.

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.

Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density.

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N2. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13-15 and 16-18% under air and 1% CO2-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5-11 and 9.7-13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H2 productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H2 production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H2 productivity by outdoor cultures of cyanobacteria in high-light environments.

Functional Significance of NADPH-Thioredoxin Reductase C in the Antioxidant Defense System of Cyanobacterium Anabaena sp. PCC 7120.

The redox regulation system is widely accepted as a crucial mechanism for controlling the activities of various metabolic enzymes. In addition to thioredoxin reductase/thioredoxin cascades, NADPH-thioredoxin reductase C (NTRC), a hybrid protein formed by an NADPH-thioredoxin reductase domain and a thioredoxin (Trx) domain, is present in chloroplasts and in most cyanobacteria species. Although several target proteins and physiological functions of NTRC in chloroplasts have been characterized, little is known about NTRC functions in cyanobacteria. Therefore, we investigated the molecular basis and physiological significance of NTRC-dependent redox regulation in the filamentous heterocyst-forming nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120). Initially, we identified six candidate NTRC targets in Anabaena 7120 using NTRC affinity chromatography. Subsequently, we compared the efficiency of reducing-equivalent transfer from NTRC and Trx-m1 to the NTRC target protein 2-Cys peroxiredoxin. Biochemical analyses revealed that compared with Trx-m1, NTRC more efficiently transfers reducing equivalents to 2-Cys peroxiredoxin. Subsequently, we constructed and analyzed an ntrC knockout strain in Anabaena 7120. The mutant showed impaired growth under oxidative stress conditions and lower concentrations of reduced 2-Cys peroxiredoxin in cells. Taken together, the present in vitro and in vivo results indicate that NTRC is a significant electron donor for 2-Cys peroxiredoxin and plays a pivotal role in antioxidant defense systems in Anabaena 7120 cells.

Expanding the Role of FurA as Essential Global Regulator in Cyanobacteria.

In the nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA plays a global regulatory role. Failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we developed a selectively regulated furA expression system by the replacement of furA promoter in the Anabaena sp. chromosomes with the Co2+/Zn2+ inducible coaT promoter from Synechocystis sp. PCC 6803. By removing Co2+ and Zn2+ from the medium and shutting off furA expression, we showed that FurA was absolutely required for cyanobacterial growth. RNA-seq based comparative transcriptome analyses of the furA-turning off strain and its parental wild-type in conjunction with subsequent electrophoretic mobility shift assays and semi-quantitative RT-PCR were carried out in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approaches led us to identify 15 novel direct iron-dependent transcriptional targets belonging to different functional categories including detoxification and defences against oxidative stress, phycobilisome degradation, chlorophyll catabolism and programmed cell death, light sensing and response, heterocyst differentiation, exopolysaccharide biosynthesis, among others. Our analyses evidence novel interactions in the complex regulatory network orchestrated by FurA in cyanobacteria.

Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.

Intercellular diffusion of a fluorescent sucrose analog via the septal junctions in a filamentous cyanobacterium.

ABSTRACTMany filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJ ΔfraC ΔfraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose.

All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with kcat = 48.2 s−1 at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

Sustained photobiological hydrogen production in the presence of N2 by nitrogenase mutants of the heterocyst-forming cyanobacterium Anabaena

Heterocyst-forming cells of the cyanobacterium Anabaena sp. strain PCC 7120 ΔHup, lacking an uptake hydrogenase, photobiologically produce H2 by nitrogenase. Under N2-rich atmosphere, the nitrogenase activity declines in a rather short time due to the sufficiency of combined nitrogen. From the parental ΔHup strain, site-directed double-crossover variants, dc-Q193S and dc-R284H, were created with amino acid substitutions presumed to be located in the vicinity of the FeMo-cofactor of nitrogenase. Unlike the case for the ΔHup strain, H2 production activities of the variants were not decreased by the presence of high concentrations of N2 and they continuously produced H2 over 21 days with occasional headspace gas replacement. This property of N2 insensitivity is a potentially useful strategy for reducing the cost of the culture gas in future practical applications of sustainable biofuel production. This Anabaena strain has only the Mo-containing nitrogenase which reduces acetylene to ethylene, but the dc-Q193S variant also produced ethane at low but measurable rates along with greater rates of ethylene production.