Filamentous Cyanobacterium Anabaena Sp Research Articles

HetF defines a transition point from commitment to morphogenesis during heterocyst differentiation in the cyanobacterium Anabaena sp. PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 is able to form heterocysts for nitrogen fixation. Heterocyst differentiation is initiated by combined-nitrogen deprivation, followed by the commitment step during which the developmental process becomes irreversible. Mature heterocysts are terminally differentiated cells unable to divide, and cell division is required for heterocyst differentiation. Previously, we have shown that the HetF protease regulates cell division and heterocyst differentiation by cleaving PatU3, which is an inhibitor for both events. When hetF is required during the developmental program remains unknown. Here, by controlling the timing of hetF expression during heterocyst differentiation, we provide evidence that hetF is required just before the beginning of heterocyst morphogenesis. Consistent with this finding, transcriptome data show that most of the genes known to be involved in the early step (such as hetR and ntcA) or the commitment step (such as hetP and hetZ) of heterocyst development could be expressed in the ΔhetF mutant. In contrast, most of the genes involved in heterocyst morphogenesis and nitrogen fixation remain repressed in the mutant. These results indicated that in the absence of hetF, heterocyst differentiation is able to be initiated and proceeds to the stage just before heterocyst envelope formation.

Heterologous Production of the C33-C45 Polyketide Fragment of Anticancer Apratoxins in a Cyanobacterial Host.

A polyketide synthase subcluster of cytotoxic apratoxin A was isolated from a Moorena bouillonii environmental DNA library and engineered with a thioesterase II domain for heterologous expression in the filamentous cyanobacterium Anabaena sp. PCC7120. Further engineering with a rhamnose-inducible promoter led to the production of (2R,3R,5R,7R)-3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid, a stereogenically rich chiral building block that is important to the efficient synthesis of apratoxin analogues, representing the first synthetic biology attempt for this type of polyketide fragment.

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

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

Identification and toxicity towards aquatic primary producers of the smallest fractions released from hydrolytic degradation of polycaprolactone microplastics.

Bioplastics are thought as a safe substitute of non-biodegradable polymers. However, once released in the environment, biodegradation may be very slow, and they also suffer abiotic fragmentation processes, which may give rise to different fractions of polymer sizes. We present novel data on abiotic hydrolytic degradation of polycaprolactone (PCL), tracking the presence of by-products during 132 days by combining different physicochemical techniques. During the study a considerable amount of two small size plastic fractions were found (up to∼6mg of PCL by-product/g of PCL beads after 132 days of degradation); and classified as submicron-plastics (sMPs) from 1μm to 100nm and nanoplastics (NPs, <100nm) as well as oligomers. The potential toxicity of the smallest fractions, PCL by-products < 100nm (PCL-NPs+PCL oligomers) and the PCL oligomers single fraction, was tested on two ecologically relevant aquatic primary producers: the heterocystous filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and the unicellular cyanobacterium Synechococcus sp. PCC 7942. Upon exposure to both, single and combined fractions, Reactive Oxygen Species (ROS) overproduction, intracellular pH and metabolic activity alterations were observed in both organisms, whilst membrane potential and morphological damages were only observed upon PCL-NPs+PCL oligomers exposure. Notably both PCL by-products fractions inhibited nitrogen fixation in Anabaena, which may be clearly detrimental for the aquatic trophic chain. As conclusion, fragmentation of bioplastics may render a continuous production of secondary nanoplastics as well as oligomers that might be toxic to the surrounding biota; both PCL-NPs and PCL oligomers, but largely the nanoparticulate fraction, were harmful for the two aquatic primary producers. Efforts should be made to thoroughly understand the fragmentation of bioplastics and the toxicity of the smallest fractions resulting from that degradation.

Structure Insights Into Photosystem I Octamer From Cyanobacteria

The diversity of photosystem oligomers is essential to understanding how photosynthetic organisms adapt to light conditions. Due to its structural and physiological significance, the assembly of the PSI supercomplex has been of great interest recently in terms of both chloroplast and cyanobacteria. In this study, two novel photosystem I supercomplexes were isolated for the first time from the low light incubated culture of filamentous cyanobacterium Anabaena sp. PCC 7120. These complexes were defined as PSI hexamers and octamers through biochemical and biophysical characterization. Their 77K emission spectra indicated that the red forms of chlorophylls seemed not to be affected during oligomerization. By cryo-EM single-particle analysis, a near-atomic (7.0 Å) resolution structure of a PSI octamer was resolved, and the molecular assemblies of a stable PSI octamer were revealed.

Grazer-induced changes in molecular signatures of cyanobacteria

Algal biomass production is an emerging renewable source of fuels, nutrients, manufacturing materials, and pharmaceuticals. Industrial-scale production is predominantly performed in open raceway ponds that are inexpensive to build, operate, and maintain compared to closed bioreactors. However, these open pond systems suffer from increased opportunities for biological contamination from predators, pathogens, and competitors, which result in reduced biomass quality and yields and often the complete destruction of the crop over a short period of time. Early detection of contaminants is a necessary step of integrated pest management for triggering and informing interventions to prevent crop losses. To develop a sensitive method of detection utilizing mass spectrometry (MS), we used three methods – imaging mass spectrometry (imaging MS), liquid chromatography MS/MS (LC-MS/MS) combined with molecular networking, and gas chromatography MS/MS (GC–MS/MS) – to observe and identify molecular signatures from a model predator-prey system of the heterolobosean amoeba HGG1 preying on the filamentous cyanobacterium Anabaena sp. PCC 7120. Imaging MS enabled the association of molecules with the crop, the predator, or the activity of protozoan grazing, while LC-MS/MS-based molecular networking identified a subset of the grazing-specific signals as the chlorophyll breakdown products pyropheophytin, pheophorbide A, and pyropheophorbide. Application of MS techniques to other amoeba-cyanobacterial predator-prey pairs allowed categorization of grazing signatures as universal or specific to the species of prey, predator, or the prey-predator pairing, indicating that MS-based techniques can distinguish crops from competitors and potentially identify predators. Finally, GCMS/MS was shown to be capable of monitoring novel volatile organic compounds (VOCs), including those predicted to be released through chlorophyll breakdown, in the headspace over algal cultures under predation. These results demonstrate that the combination of multiple MS technologies creates a predictive framework to identify and catalog relevant molecular signatures for informing crop protection strategies.

Comparative Phenotypic Analysis of Anabaena sp. PCC 7120 Mutants of Porinlike Genes.

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.

Inactivation of Three RG(S/T)GR Pentapeptide-Containing Negative Regulators of HetR Results in Lethal Differentiation of Anabaena PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 produces, during the differentiation of heterocysts, a short peptide PatS and a protein HetN, both containing an RGSGR pentapeptide essential for activity. Both act on the master regulator HetR to guide heterocyst pattern formation by controlling the binding of HetR to DNA and its turnover. A third small protein, PatX, with an RG(S/T)GR motif is present in all HetR-containing cyanobacteria. In a nitrogen-depleted medium, inactivation of patX does not produce a discernible change in phenotype, but its overexpression blocks heterocyst formation. Mutational analysis revealed that PatX is not required for normal intercellular signaling, but it nonetheless is required when PatS is absent to prevent rapid ectopic differentiation. Deprivation of all three negative regulators—PatS, PatX, and HetN—resulted in synchronous differentiation. However, in a nitrogen-containing medium, such deprivation leads to extensive fragmentation, cell lysis, and aberrant differentiation, while either PatX or PatS as the sole HetR regulator can establish and maintain a semiregular heterocyst pattern. These results suggest that tight control over HetR by PatS and PatX is needed to sustain vegetative growth and regulated development. The mutational analysis has been interpreted in light of the opposing roles of negative regulators of HetR and the positive regulator HetL.

Functional Dissection of Genes Encoding DNA Polymerases Based on Conditional Mutants in the Heterocyst-Forming Cyanobacterium Anabaena PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 develops N2-fixing heterocyst cells under condition of combined-nitrogen deprivation and constitutes an excellent model for studying cell differentiation. The mechanism of heterocyst development has been extensively investigated and a network of regulating factors has been identified. A few studies have showed that the process of heterocyst differentiation relates with cell cycle events, but further investigation is still required to understand this relationship. In a previous study, we created a conditional mutant of PolI encoding gene, polA, by using a CRISPR/Cpf1 gene-editing technique. Here, we were able to create another conditional mutant of a PolIII encoding gene dnaENI using a similar strategy and subsequently confirmed the essential roles of both polA and dnaENI in DNA replication. Further investigation on the phenotype of the mutants showed that lack of PolI caused defects in chromosome segregation and cell division, while lack of DnaENI (PolIII) prevented bulk DNA synthesis, causing significant loss of DNA content. Our findings also suggested the possible existence of a SOS-response like mechanism operating in Anabaena PCC 7120. Moreover, we found that heterocyst development was differently affected in the two conditional mutants, with double heterocysts/proheterocysts found in PolI conditional mutant. We further showed that formation of such double heterocysts/proheterocysts are likely caused by the difficulty in nucleoids segregation, resulting delayed, or non-complete closure of the septum between the two daughter cells. This study uncovers a link between DNA replication process and heterocyst differentiation, paving the way for further studies on the relationship between cell cycle and cell development.

Enhanced H2 production with efficient N2-fixation by fructose mixotrophically grown Anabaena sp. PCC 7120 strain disrupted in uptake hydrogenase

To explore the potential of a model filamentous cyanobacterium Anabaena sp. PCC 7120 for solar based biohydrogen production, an inactivated uptake hydrogenase ΔHupL strain was used in combination with fructose induced N2-fixing ability of the cells. The results revealed that fructose supports N2-fixing and H2-metabolism in ΔHupL strain. Cells showed an increase in growth rate, pigment content, and the formation of mature heterocysts within 6 h after growing under light-fructose mixotrophic condition. Upon supplementation of optimum fructose concentration at 60 mM, the cells grown for 24 h showed a 3.1-fold increase of nitrogenase activity in conjunction with an increase of nifD expression, with increased electron flow from utilization of fructose through the oxidative pentose phosphate pathway. Nitrogenase rather than bidirectional hydrogenase was likely responsible for the fructose mediated increase of H2-production. The maximum H2-production rate of ΔHupL strain under fructose mixotrophic growth was 101.33 μmolH2 mg chl a−1 h−1, accounting for a 4.7-fold increase compared to wild type under the same condition. Overall results indicated that fructose supplementation enabled Anabaena sp. PCC 7120 strain ΔHupL to increase N2-fixation leading to an enhanced H2-production.

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.

Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP-galactose 4-epimerase HgdA.

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaenasp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here, we studied the function of the putative nucleoside‐diphosphate‐sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site‐directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdAmutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP‐tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP‐galactose 4‐epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst‐specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC.

The Absence of Thioredoxin m1 and Thioredoxin C in Anabaena sp. PCC 7120 Leads to Oxidative Stress.

Thioredoxin (Trx) family proteins perform redox regulation in cells, and they are involved in several other biological processes (e.g. oxidative stress tolerance). In the filamentous cyanobacterium Anabaena sp. PCC7120 (A. 7120), eight Trx isoforms have been identified via genomic analysis. Among these Trx isoforms, the absence of Trx-m1 and TrxC appears to result in oxidative stress in A. 7120 together with alterations of the thylakoid membrane structure and phycobiliprotein composition. To analyze the physiological changes in these Trx disruptants thoroughly, quantitative proteomics was applied. Certainly, the mutants exhibited similar alterations in the proteome including decreased relative abundance of phycobiliproteins and an increased level of proteins involved in amino acid and carbohydrate metabolism. Nevertheless, the results also indicated that the mutants exhibited changes in the relative abundance of different sets of proteins participating in reactive oxygen species detoxification, such as Fe-SOD in Δtrx-m1 and PrxQ in ΔtrxC, suggesting distinct functions of Trx-m1 and TrxC.

Soft X-Ray Imaging of Cellular Carbon and Nitrogen Distributions in Heterocystous Cyanobacteria.

Soft x-ray microscopy (SXM) is a minimally invasive technique for single-cell high-resolution imaging as well as the visualization of intracellular distributions of light elements such as carbon, nitrogen, and oxygen. We used SXM to observe photosynthesis and nitrogen fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120, which can form heterocysts during nitrogen starvation. Statistical and spectroscopic analyses from SXM images around the K-absorption edge of nitrogen revealed a significant difference in the carbon-to-nitrogen (C/N) ratio between vegetative cells and heterocysts. Application of this analysis to soft x-ray images of Anabaena sp. PCC 7120 revealed inhomogenous C/N ratios in the cells. Furthermore, soft x-ray tomography of Anabaena sp. PCC 7120 revealed differing cellular C/N ratios, indicating different carbon and nitrogen distributions between vegetative cells and heterocysts in three dimensions.

ATP-binding cassette transporters of the multicellular cyanobacterium Anabaena sp. PCC 7120: a wide variety for a complex lifestyle.

Two hundred genes or 3% of the known or putative protein-coding genes of the filamentous freshwater cyanobacterium Anabaena sp. PCC 7120 encode domains of ATP-binding cassette (ABC) transporters. Detailed characterization of some of these transporters (14-15 importers and 5 exporters) has revealed their crucial roles in the complex lifestyle of this multicellular photoautotroph, which is able to differentiate specialized cells for nitrogen fixation. This review summarizes the characteristics of the ABC transporters of Anabaena sp. PCC 7120 known to date.

Determination of Intracellular Osmolytes in Cyanobacterial Cells

Most of the cyanobacteria accumulate osmolytes including sucrose, glucosylglycerol, in their cells in response to salt stress. Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular sucrose and glucosylglycerol. We have confirmed this protocol was applicable to at least four kinds of cyanobacteria, filamentous cyanobacterium Anabaena sp. PCC 7120, unicellular cyanobacterium Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 and halotolerant unicellular cyanobacterium Synechococcus sp. PCC 7002.

Spatial separation of photosynthesis and ethanol production by cell type-specific metabolic engineering of filamentous cyanobacteria

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO2 and H2O using light energy. Although cyanobacteria capable of producing diverse chemicals have been generated by metabolic engineering, anaerobic non-photosynthetic culture conditions are often necessary for their production. In this study, we conducted cell type-specific metabolic engineering of the filamentous cyanobacterium Anabaena sp. PCC 7120, which forms a terminally differentiated cell called a heterocyst with a semi-regular spacing of 10-15 cells. Because heterocysts are specialized cells for nitrogen fixation, the intracellular oxygen level of heterocysts is maintained very low even when adjacent cells perform oxygenic photosynthesis. Pyruvate decarboxylase of Zymomonas mobilis and alcohol dehydrogenase of Synechocystis sp. PCC 6803 were exclusively expressed in heterocysts. Ethanol production was concomitant with nitrogen fixation in genetically engineered Anabaena sp. PCC 7120. Engineering of carbon metabolism in heterocysts improved ethanol production, and strain ET14, with an extra copy of the invB gene expressed from a heterocyst-specific promoter, produced 130.9mgL-1 of ethanol after 9days. ET14 produced 1681.9mgL-1 of ethanol by increasing the CO2 supply. Ethanol production per heterocyst cell was approximately threefold higher than that per cell of unicellular cyanobacterium. This study demonstrates the potential of heterocysts for anaerobic production of biofuels and commodity chemicals under oxygenic photosynthetic conditions.

Determination of carbon-to-nitrogen ratio in the filamentous and heterocystous cyanobacterium Anabaena sp. PCC 7120 with single-cell soft X-ray imaging

Vegetative cells and heterocysts in the filamentous cyanobacterium Anabaena sp. PCC 7120 were observed by soft X-ray microscopy. Carbon-to-nitrogen (C/N) ratio of each cell was estimated by the difference of the absorbance of the images below and above the nitrogen K-edge absorption. It was revealed that the C/N ratios in vegetative cells and heterocysts are 4.54 and 2.46, respectively.

In silico characterization and transcriptomic analysis of nif family genes from Anabaena sp. PCC7120.

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.