Phosphorus deficiency alleviates iron limitation in Synechocystis cyanobacteria through direct PhoB-mediated gene regulation.

Iron and phosphorus are essential nutrients that exist at low concentrations in surface waters and may be co-limiting resources for phytoplankton growth. Here, we show that phosphorus deficiency increases the growth of iron-limited cyanobacteria (Synechocystis sp. PCC 6803) through a PhoB-mediated regulatory network. We find that PhoB, in addition to its well-recognized role in controlling phosphate homeostasis, also regulates key metabolic processes crucial for iron-limited cyanobacteria, including ROS detoxification and iron uptake. Transcript abundances of PhoB-targeted genes are enriched in samples from phosphorus-depleted seawater, and a conserved PhoB-binding site is widely present in the promoters of the target genes, suggesting that the PhoB-mediated regulation may be highly conserved. Our findings provide molecular insights into the responses of cyanobacteria to simultaneous iron/phosphorus nutrient limitation.

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Efficient Semi-Artificial Photosynthesis of Ethylene by a Self-Assembled InP-Cyanobacterial Biohybrid System.

Biomanufacturing of ethylene is particularly important for modern society. Cyanobacterial cells are able to photosynthesize various valuable chemicals. A promising platform for next-generation biomanufacturing, the semiconductor-cyanobacterial hybrid systems are capable of enhancing the solar-to-chemical conversion efficiency. Herein, the native ethylene-producing capability of a filamentous cyanobacterium Nostoc sphaeroides is confirmed experimentally. The self-assembly characteristic of N. sphaeroides is exploited to facilitate its interaction with InP nanomaterial, and the resulting biohybrid system gave rise to further elevated photosynthetic ethylene production. Based on chlorophyll fluorescence measurement and metabolic analysis, the InP nanomaterial-augmented photosystem I activity and enhanced ethylene production metabolism of biohybrid cells are confirmed, the mechanism underlying the material-cell energy transduction as well as nanomaterial-modulated photosynthetic light and dark reactions are established. This work not only demonstrates the potential application of semiconductor-N. sphaeroides biohybrid system as a good platform for sustainable ethylene production but also provides an important reference for future studies to construct and optimize nano-cell biohybrid systems for efficient solar-driven valuable chemical production.

Functional specialization of expanded orange carotenoid protein paralogs in subaerial Nostoc species.

Orange carotenoid protein (OCP) is a photoactive protein that participates in the photoprotection of cyanobacteria. There are 2 full-length OCP proteins, 4 N-terminal paralogs (helical carotenoid protein [HCP]), and 1 C-terminal domain-like carotenoid protein (CCP) found in Nostoc flagelliforme, a desert cyanobacterium. All HCPs (HCP1 to 3 and HCP6) from N. flagelliforme demonstrated their excellent singlet oxygen quenching activities, in which HCP2 was the strongest singlet oxygen quencher compared with others. Two OCPs, OCPx1 and OCPx2, were not involved in singlet oxygen scavenging; instead, they functioned as phycobilisome fluorescence quenchers. The fast-acting OCPx1 showed more effective photoactivation and stronger phycobilisome fluorescence quenching compared with OCPx2, which behaved differently from all reported OCP paralogs. The resolved crystal structure and mutant analysis revealed that Trp111 and Met125 play essential roles in OCPx2, which is dominant and long acting. The resolved crystal structure of OCPx2 is maintained in a monomer state and showed more flexible regulation in energy quenching activities compared with the packed oligomer of OCPx1. The recombinant apo-CCP obtained the carotenoid pigment from holo-HCPs and holo-OCPx1 of N. flagelliforme. No such carotenoid transferring processes were observed between apo-CCP and holo-OCPx2. The close phylogenetic relationship of OCP paralogs from subaerial Nostoc species indicates an adaptive evolution toward development of photoprotection: protecting cellular metabolism against singlet oxygen damage using HCPs and against excess energy captured by active phycobilisomes using 2 different working modes of OCPx.

Chlorophyll f production in two new subaerial cyanobacteria of the family Oculatellaceae.

Chlorophyll (Chl) f was recently identified in a few cyanobacteria as the fifth chlorophyll of oxygenic organisms. In this study, two Leptolyngbya-like strains of CCNU0012 and CCNU0013 were isolated from a dry ditch in Chongqing city and a brick wall in Mount Emei Scenic Area in China, respectively. These two strains were described as new species: Elainella chongqingensis sp. nov. (Oculatellaceae, Synechococcales) and Pegethrix sichuanica sp. nov. (Oculatellaceae, Synechococcales) by the polyphasic approach based on morphological features, phylogenetic analysis of 16S rRNA gene and secondary structure comparison of 16S-23S internal transcribed spacer domains. Both strains produced Chl a under white light (WL) but additionally induced Chl f synthesis under far-red light (FRL). Unexpectedly, the content of Chl f in P. sichuanica was nearly half that in most Chl f-producing cyanobacteria. Red-shifted phycobiliproteins were also induced in both strains under FRL conditions. Subsequently, additional absorption peak beyond 700 nm in the FRL spectral region appeared in these two strains. This is the first report of Chl f production induced by FRL in the family Oculatellaceae. This study not only extended the diversity of Chl f-producing cyanobacteria but also provided precious samples to elucidate the essential binding sites of Chl f within cyanobacterial photosystems.

Coevolution of tandemly repeated hlips and RpaB-like transcriptional factor confers desiccation tolerance to subaerial Nostoc species.

Desert-inhabiting cyanobacteria can tolerate extreme desiccation and quickly revive after rehydration. The regulatory mechanisms that enable their vegetative cells to resurrect upon rehydration are poorly understood. In this study, we identified a single gene family of high light-inducible proteins (Hlips) with dramatic expansion in the Nostoc flagelliforme genome and found an intriguingly special convergence formed through four tandem gene duplication. The emerged four independent hlip genes form a gene cluster (hlips-cluster) and respond to dehydration positively. The gene mutants in N. flagelliforme were successfully generated by using gene-editing technology. Phenotypic analysis showed that the desiccation tolerance of hlips-cluster-deleted mutant decreased significantly due to impaired photosystem II repair, whereas heterologous expression of hlips-cluster from N. flagelliforme enhanced desiccation tolerance in Nostoc sp. PCC 7120. Furthermore, a transcription factor Hrf1 (hlips-cluster repressor factor 1) was identified and shown to coordinately regulate the expression of hlips-cluster and desiccation-induced psbAs. Hrf1 acts as a negative regulator for the adaptation of N. flagelliforme to the harsh desert environment. Phylogenetic analysis revealed that most species in the Nostoc genus possess both tandemly repeated Hlips and Hrf1. Our results suggest convergent evolution of desiccation tolerance through the coevolution of tandem Hlips duplication and Hrf1 in subaerial Nostoc species, providing insights into the mechanism of desiccation tolerance in photosynthetic organisms.

Kovacikia minuta sp. nov. (Leptolyngbyaceae, Cyanobacteria), a new freshwater chlorophyll f-producing cyanobacterium.

A few groups of cyanobacteria have been characterized as having far-red light photoacclimation (FaRLiP) that results from chlorophyll f (Chl f) production. In this study, using a polyphasic approach, we taxonomically transferred the Cf. Leptolyngbya sp. CCNUW1 isolated from a shaded freshwater pond, which produces Chl f under far-red light, to the genus Kovacikia and named this taxon Kovacikia minuta sp. nov. This strain was morphologically similar to Leptolyngbya-like strains. The thin filaments were purplish-brown under white light but became grass green under far-red light. The 31-gene phylogeny grouped K. minuta CCNU0001 into order Synechococcales and family Leptolyngbyaceae. Phylogenetic analysis based on 16S rRNA gene sequences further showed that K. minuta CCNU0001 was clustered into Kovacikia with similarities of 97.2-97.4% to the recently reported type species of Kovacikia muscicola HA7619-LM3. Additionally, the internal transcribed spacer region between 16S-23S rRNA genes had a unique sequence and secondary structure compared with other Kovacikia strains and phylogenetically related taxa. Draft genome sequences of K. minuta CCNU0001 (8,564,336 bp) were assembled into one circular chromosome and two circular plasmids. A FaRLiP 20-gene cluster comprised two operons with the unique organization. In sum, K. minuta was established as a new species, and it is the first species reported to produce Chl f and for which a draft genome was produced in genus Kovacikia. This study expanded our knowledge regarding the diversity of Chl f-producing cyanobacteria in far-red light-enriched environments and provides important foundational information for future investigations of FaRLiP evolution in cyanobacteria.

Expansion of bilin-based red light sensors in the subaerial desert cyanobacterium Nostoc flagelliforme.

Light is the crucial environmental signal for desiccation-tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin-binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF-containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin-producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near-UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first-ever reported GAF-only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual-cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn.

Iron transport in cyanobacteria - from molecules to communities.

Iron is an essential micronutrient for the ecologically important photoautotrophic cyanobacteria which are found across diverse aquatic environments. Low concentrations and poor bioavailability of certain iron species exert a strong control on cyanobacterial growth, affecting ecosystem structure and biogeochemical cycling. Here, we review the iron-acquisition pathways cyanobacteria utilize for overcoming these challenges. As the molecular details of cyanobacterial iron transport are being uncovered, an overall scheme of how cyanobacteria handle and exploit this scarce and redox-active micronutrient is emerging. Importantly, the range of biological solutions used by cyanobacteria to increase iron fluxes goes beyond transport and includes behavioral traits of colonial cyanobacteria and intricate cyanobacteria-bacteria interactions.

New types of ATP-grasp ligase are associated with the novel pathway for complicated mycosporine-like amino acid production in desiccation-tolerant cyanobacteria.

Mycosporine-like amino acids (MAAs) were widespread in diverse organisms to attenuate UV radiation. We recently characterized the large, complicated MAA mycosporine-2-(4-deoxygadusolyl-ornithine) in desert cyanobacterium Nostoc flagelliforme. Synthesis of this MAA requires the five-gene cluster mysABDC2C3. Here, bioinformatic analysis indicated that mysC duplication within five-gene mys clusters is strictly limited to drought-tolerant cyanobacteria. Phylogenic analysis distinguished these duplicated MysCs into two clades that separated from canonical MysCs. Heterologous expression of N. flagelliforme mys genes in Escherichia coli showed that MysAB produces 4-deoxygadusol. The ATP-grasp ligase of MysC3 catalyses the linkage of the δ- or ε-amino group of ornithine/lysine to 4-deoxygadusol, yielding mycosporine-ornithine or mycosporine-lysine respectively. The ATP-grasp ligase of MysC2 strictly condenses the α-amino group of mycosporine-ornithine to another 4-deoxygadusol. MysD (D-Ala-D-Ala ligase) functions following MysC2 to catalyse the formation of mycosporine-2-(4-deoxygadusolyl-ornithine). High arginine content likely provides a greater pool of ornithine over other amino acids during rehydration of desiccated N. flagelliforme. Duplication of ATP-grasp ligases is specific for the use of substrates that have two amino groups (such as ornithine) for the production of complicated MAAs with multiple chromophores. This five-enzyme biosynthesis pathway for complicated MAAs is a novel adaptation of cyanobacteria for UV tolerance in drought environments.

Reading and surviving the harsh conditions in desert biological soil crust: the cyanobacterial viewpoint.

Biological soil crusts (BSCs) are found in drylands, cover ∼12% of the Earth's surface in arid and semi-arid lands and their destruction is considered an important promoter of desertification. These crusts are formed by the adhesion of soil particles to polysaccharides excreted mostly by filamentous cyanobacteria, which are the pioneers and main primary producers in BSCs. Desert BSCs survive in one of the harshest environments on Earth, and are exposed to daily fluctuations of extreme conditions. The cyanobacteria inhabiting these habitats must precisely read the changing conditions and predict, for example, the forthcoming desiccation. Moreover, they evolved a comprehensive regulation of multiple adaptation strategies to enhance their stress tolerance. Here, we focus on what distinguishes cyanobacteria able to revive after dehydration from those that cannot. While important progress has been made in our understanding of physiological, biochemical and omics aspects, clarification of the sensing, signal transduction and responses enabling desiccation tolerance are just emerging. We plot the trajectory of current research and open questions ranging from general strategies and regulatory adaptations in the hydration/desiccation cycle, to recent advances in our understanding of photosynthetic adaptation. The acquired knowledge provides new insights to mitigate desertification and improve plant productivity under drought conditions.

Genomic and transcriptomic insights into the habitat adaptation of the diazotrophic paddy-field cyanobacterium Nostoc sphaeroides.

Nitrogen-fixing cyanobacteria are common in paddy fields, one of the most productive wetland ecosystems. Here, we present the complete genome of Nostoc sphaeroides, a paddy-field diazotroph used for food and medicine for more than 1700 years and deciphered the transcriptional regulation during the developmental transition from hormogonia to vegetative filaments with heterocysts. The genome of N. sphaeroides consists of one circular chromosome (6.48 Mb), one of the largest ever reported megaplasmids (2.34 Mb), and seven plasmids. Multiple gene families involved in the adaption to high solar radiation and water fluctuation conditions were found expanded, while genes involved in anoxic adaptation and phosphonate utilization are located on the megaplasmid, suggesting its indispensable role in environmental adaptation. Distinct gene expression patterns were observed during the light-intensity-regulated transition from hormogonia to vegetative filaments, specifically, genes encoding proteins involved in photosynthetic light reaction, carbon fixation, nitrogen metabolism and heterocyst differentiation were significantly upregulated, whereas genes related to cell motility were down-regulated. Our results provide genomic and transcriptomic insights into the adaptation of a filamentous nitrogen-fixing cyanobacterium to the highly dynamic paddy-field habitat, suggesting N. sphaeroides as an excellent system to understand the transition from aquatic to terrestrial habitats and to support sustainable rice production.

Long-term iron deprivation and subsequent recovery uncover heterogeneity in the response of cyanobacterial populations.

Cyanobacteria are globally important primary producers and nitrogen fixers. They are frequently limited by iron bioavailability in natural environments that often fluctuate due to rapid consumption and irregular influx of external Fe. Here we identify a succession of physiological changes in Synechocystis sp. PCC 6803 occurring over 14-16 days of iron deprivation and subsequent recovery. We observe several adaptive strategies that allow cells to push their metabolic limits under the restriction of declining intracellular Fe quotas. Interestingly, cyanobacterial populations exposed to prolonged iron deprivation showed discernible heterogeneity in cellular auto-fluorescence during the recovery process. Using FACS and microscopy techniques we revealed that only cells with high auto-fluorescence were able to grow and reconstitute thylakoid membranes. We propose that ROS-mediated damage is likely to be associated with the emergence of the two subpopulations, and, indeed, a rapid increase in intracellular ROS content was observed during the first hours following iron addition to Fe-starved cultures. These results suggest that an increasing iron supply is a double-edged sword - posing both an opportunity and a risk. Therefore, phenotypic heterogeneity within populations is crucial for the survival and proliferation of organisms facing iron fluctuations within natural environments.

Identification and characterization of SaeIF1 from the eukaryotic translation factor SUI1 family in cadmium hyperaccumulator Sedum alfredii.

MAIN CONCLUSION: Cadmium-sensitive yeast screening resulted in the isolation of protein translation factor SaeIF1 from the hyperaccumulator Sedum alfredii which has both general and special regulatory roles in controlling cadmium accumulation. The hyperaccumulator of Sedum alfredii has the extraordinary ability to hyperaccumulate cadmium (Cd) in shoots. To investigate its underlying molecular mechanisms of Cd hyperaccumulation, a cDNA library was generated from leaf tissues of S. alfredii. SaeIF1, belonging to the eukaryotic protein translation factor SUI1 family, was identified by screening Cd-sensitive yeast transformants with this library. The full-length cDNA of SaeIF1 has 582 bp and encodes a predicted protein with 120 amino acids. Transient expression assays showed subcellular localization of SaeIF1 in the cytoplasm. SaeIF1 was constitutively and highly expressed in roots and shoots of the hyperaccumulator of S. alfredii, while its transcript levels showed over 100-fold higher expression in the hyperaccumulator of S. alfredii relative to the tissues of a nonhyperaccumulating ecotype of S. alfredii. However, the overexpression of SaeIF1 in yeast cells increased Cd accumulation, but conferred more Cd sensitivity. Transgenic Arabidopsis thaliana expressing SaeIF1 accumulated more Cd in roots and shoots without changes in the ratio of Cd content in shoots and roots, but were more sensitive to Cd stress than wild type. Both special and general roles of SaeIF1 in Cd uptake, transportation, and detoxification are discussed, and might be responsible for the hyperaccumulation characteristics of S. alfredii.

A unique porin meditates iron-selective transport through cyanobacterial outer membranes.

Cyanobacteria are globally important primary producers and nitrogen fixers with high iron demands. Low ambient dissolved iron concentrations in many aquatic environments mean that these organisms must maintain sufficient and selective transport of iron into the cell. However, the nature of iron transport pathways through the cyanobacterial outer membrane remains obscure. Here we present multiple lines of experimental evidence that collectively support the existence of a novel class of substrate-selective iron porin, Slr1908, in the outer membrane of the cyanobacterium Synechocystis sp. PCC 6803. Elemental composition analysis and short-term iron uptake assays with mutants in Slr1908 reveal that this protein is primarily involved in inorganic iron uptake and contributes less to the accumulation of other metals. Homologues of Slr1908 are widely distributed in both freshwater and marine cyanobacteria, most notably in unicellular marine diazotrophs. Complementary experiments with a homologue of Slr1908 in Synechococcus sp. PCC 7002 restored the phenotype of Synechocystis knockdown mutants, showing that this siderophore producing species also possesses a porin with a similar function in Fe transport. The involvement of a substrate-selective porins in iron uptake may allow cyanobacteria to tightly control iron flux into the cell, particularly in environments where iron concentrations fluctuate.

Photosynthetic adaptation to light availability shapes the ecological success of bloom-forming cyanobacterium Pseudanabaena to iron limitation.

The poorly understood filamentous cyanobacterium Pseudanabaena is commonly epiphytic on Microcystis colonies and their abundances are often highly correlated during blooms. The response and adaptation of Microcystis to iron limitation have been extensively studied, but the strategies Pseudanabaena uses to respond to iron limitation are largely unknown. Here, physiological responses to iron limitation were compared between one Pseudanabaena and two Microcystis strains grown under different light intensities. The results showed that low-intensity light exacerbated, but high-intensity light alleviated, the negative effect of iron limitation on Pseudanabaena growth relative to two Microcystis strains. It was found that robust light-harvesting and photosynthetic efficiency allowed adaptation of Pseudanabaena to low light availability relative to two Microcystis strains only during iron sufficiency. The results also indicated that a larger investment in the photosynthetic antenna probably contributed to light/iron co-limitation of Pseudanabaena relative to two Microcystis strains under both light and iron limitation. Furthermore, the lower antenna pigments/chlorophyll a ratio and photosynthetic efficiency, and higher nonphotochemical quenching and saturation irradiance provided Pseudanabaena photoadaptation and photoprotection advantages over the two Microcystis strains under the high-light condition. The lower investment in antenna pigments of Pseudanabaena than the two Microcystis strains under high-light intensity is likely an efficient strategy for both saving iron quotas and decreasing photosensitivity. Therefore, when compared with Microcystis, the high plasticity of antenna pigments, along with the excellent photoadaptation and photoprotection ability of Pseudanabaena, probably ensures its ecological success under iron limitation when light is sufficient.