Profiling the interplay and coevolution of Microcystis aeruginosa and cyanosiphophage Mic1.

The cyanosiphophage Mic1 specifically infects the bloom-forming Microcystis aeruginosa FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including gp49 that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria M. aeruginosa and cyanophages. IMPORTANCE: The highly polymorphic Microcystis aeruginosa is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in M. aeruginosa genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of M. aeruginosa and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.

Molecular mechanism of specific DNA sequence recognition by NRF1.

Nuclear respiratory factor 1 (NRF1) regulates the expression of genes that are vital for mitochondrial biogenesis, respiration, and various other cellular processes. While NRF1 has been reported to bind specifically to GC-rich promoters as a homodimer, the precise molecular mechanism governing its recognition of target gene promoters has remained elusive. To unravel the recognition mechanism, we have determined the crystal structure of the NRF1 homodimer bound to an ATGCGCATGCGCAT dsDNA. In this complex, NRF1 utilizes a flexible linker to connect its dimerization domain (DD) and DNA binding domain (DBD). This configuration allows one NRF1 monomer to adopt a U-turn conformation, facilitating the homodimer to specifically bind to the two TGCGC motifs in the GCGCATGCGC consensus sequence from opposite directions. Strikingly, while the NRF1 DBD alone could also bind to the half-site (TGCGC) DNA of the consensus sequence, the cooperativity between DD and DBD is essential for the binding of the intact GCGCATGCGC sequence and the transcriptional activity of NRF1. Taken together, our results elucidate the molecular mechanism by which NRF1 recognizes specific DNA sequences in the promoters to regulate gene expression.

Structural basis for specific DNA sequence motif recognition by the TFAP2 transcription factors.

The TFAP2 family regulates gene expression during differentiation, development, and organogenesis, and includes five homologs in humans. They all possess a highly conserved DNA binding domain (DBD) followed by a helix-span-helix (HSH) domain. The DBD-HSH tandem domain specifically binds to a GCC(N3)GGC consensus sequence, but the precise recognition mechanisms remain unclear. Here, we found that TFAP2 preferred binding to the GCC(N3)GGC sequence, and the pseudo-palindromic GCC and GGC motifs and the length of the central spacer between the two motifs determined their binding specificity. Structural studies revealed that the two flat amphipathic α-helical HSH domains of TFAP2A stacked with each other to form a dimer via hydrophobic interactions, while the stabilized loops from both DBD domains inserted into two neighboring major grooves of the DNA duplex to form base-specific interactions. This specific DNA binding mechanism controlled the length of the central spacer and determined the DNA sequence specificity of TFAP2. Mutations of the TFAP2 proteins are implicated in various diseases. We illustrated that reduction or disruption of the DNA binding ability of the TFAP2 proteins is the primary cause of TFAP2 mutation-associated diseases. Thus, our findings also offer valuable insights into the pathogenesis of disease-associated mutations in TFAP2 proteins.

Catalytic Ozonation of Polluter Benzene from -20 to >50 °C with High Conversion Efficiency and Selectivity on Mullite YMn2O5.

Catalytic decomposition of aromatic polluters at room temperature represents a green route for air purification but is currently challenged by the difficulty of generating reactive oxygen species (ROS) on catalysts. Herein, we develop a mullite catalyst YMn2O5 (YMO) with dual active sites of Mn3+ and Mn4+ and use ozone to produce a highly reactive O* upon YMO. Such a strong oxidant species on YMO shows complete removal of benzene from -20 to >50 °C with a high COx selectivity (>90%) through the generated reactive species O* on the catalyst surface (60 000 mL g-1 h-1). Although the accumulation of water and intermediates gradually lowers the reaction rate after 8 h at 25 °C, a simple treatment by ozone purging or drying in the ambient environment regenerates the catalyst. Importantly, when the temperature increases to 50 °C, the catalytic performance remains 100% conversion without any degradation for 30 h. Experiments and theoretical calculations show that such a superior performance stems from the unique coordination environment, which ensures high generation of ROS and adsorption of aromatics. Mullite's catalytic ozonation degradation of total volatile organic compounds (TVOC) is applied in a home-developed air cleaner, resulting in high efficiency of benzene removal. This work provides insights into the design of catalysts to decompose highly stable organic polluters.

[Emission Characteristics and Risk Assessment of Gas Pollutants in Typical Pesticide Manufacturing Enterprises].

In order to explore the characteristics of exhaust gas emissions, environmental impact, and human health risks in the pesticide manufacturing industry, two typical pesticide manufacturing enterprises were selected as the research objects, and samples were collected and analyzed for all exhaust pipes of each enterprise. The following results were noted:there were certain differences in the pollutants produced by different enterprises due to different products and production links. The main pollutants in enterprise A were ammonia and VOCs. The concentration of ammonia in enterprise A ranged from 0 to 847.83 mg·m-3, and the concentration of VOCs ranged from 4.21 to 91.68 mg·m-3. The main pollutants in enterprise B were VOCs, and the concentration of VOCs ranged from 3.37 to 197.30 mg·m-3. The ozone formation potential (OFP) ranged from 1.96 to 107.24 mg·m-3. Substances that required further attention in terms of ozone formation potential:enterprise A mainly included ethanol, methanol, toluene, xylene, and other substances; enterprise B mainly included 1, 1-dichloroethylene, 1, 2-dichloroethane, toluene, methylal, and other substances. The secondary organic aerosol formation potential (SOAFP) ranged from 0.94 to 74.72 mg·m-3. The main contributors to the secondary organic aerosol formation potential were aromatic hydrocarbons and oxygen-containing organics. In addition, ammonia also required additional attention. The odorous substances in pesticide enterprises were more complex, and there were differences in the exhaust pipes of different enterprises and different production links of the same enterprise. There were certain health risks in the gas pollutants of pesticide enterprises. The main carcinogens were 1, 2-dichloroethane, trichloroethylene, tetrachloroethylene, methyl chloride, and benzene. In addition, pyridine and hexachloroethane had certain non-carcinogenic risks in pesticide production enterprises.

Induced pluripotent stem cell-derived extracellular vesicles overexpressing SFPQ protect retinal Müller cells against hypoxia-induced injury.

Splicing factor proline/glutamine-rich (SFPQ) is expressed in induced pluripotent stem cells (iPSCs), which are reported to orchestrate hypoxic injury responses and release extracellular vesicles (EVs). Therefore, this study sought to explore the role of iPSC-derived EVs carrying SFPQ in hypoxia-induced injury to retinal Müller cells. We induced oxygen-glucose deprivation/reoxygenation (OGD/R) in Müller cells. SFPQ was overexpressed or knocked down in iPSCs, from which EVs were extracted. Müller cells were co-cultured with EVs, and the results indicated that SFPQ protein was transferred into retinal Müller cells by iPSC-derived EVs. We identified an interaction of SFPQ with HDAC1 in retinal Müller cells. Specifically, SFPQ recruited HDAC1 to downregulate HIF-2α by regulating its acetylation. The in vitro studies suggested that iPSC-derived EVs, SFPQ or HDAC1 overexpression, or HIF-2α silencing diminished cell injury and apoptosis but elevated proliferation in retinal Müller cells. The in vivo studies indicated that iPSC-derived EVs containing SFPQ curtailed apoptosis of retinal Müller cells, thus alleviating retinal ischemia/reperfusion (I/R) injury of rat model. Taken together, iPSC-derived EVs containing SFPQ upregulated HDAC1 to attenuate OGD/R-induced Müller cell injury via downregulation of HIF-2α.

Phylogenomics of five Pseudanabaena cyanophages and evolutionary traces of horizontal gene transfer.

BACKGROUND: Along with the fast development and urbanization in developing countries, the waterbodies aside the growing cities become heavily polluted and highly eutrophic, thus leading to the seasonal outbreak of cyanobacterial bloom. Systematic isolation and characterization of freshwater cyanophages might provide a biological solution to control the awful blooms. However, genomic sequences and related investigations on the freshwater cyanophages remain very limited to date. RESULTS: Following our recently reported five cyanophages Pam1~Pam5 from Lake Chaohu in China, here we isolated another five cyanophages, termed Pan1~Pan5, which infect the cyanobacterium Pseudanabaena sp. Chao 1811. Whole-genome sequencing showed that they all contain a double-stranded DNA genome of 37.2 to 72.0 kb in length, with less than half of the putative open reading frames annotated with known functions. Remarkably, the siphophage Pan1 encodes an auxiliary metabolic gene phoH and constitutes, together with the host, a complete queuosine modification pathway. Proteomic analyses revealed that although Pan1~Pan5 are distinct from each other in evolution, Pan1 and Pan3 are somewhat similar to our previously identified cyanophages Pam3 and Pam1 at the genomic level, respectively. Moreover, phylogenetic analyses suggested that Pan1 resembles the α-proteobacterial phage vB_DshS-R5C, revealing direct evidence for phage-mediated horizontal gene transfer between cyanobacteria and α-proteobacteria. CONCLUSION: In addition to the previous reports of Pam1~Pam5, the present findings on Pan1~Pan5 largely enrich the library of reference freshwater cyanophages. The abundant genomic information provides a pool to identify novel genes and proteins of unknown function. Moreover, we found for the first time the evolutionary traces in the cyanophage that horizontal gene transfer might occur at the level of not only inter-species, but even inter-phylum. It indicates that the bacteriophage or cyanophage could be developed as a powerful tool for gene manipulation among various species or phyla.

Fine structure and assembly pattern of a minimal myophage Pam3.

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

Fast-Charging Zn-Air Batteries with Long Lifetime Enabled by Reconstructed Amorphous Multi-Metallic Sulfide.

Developing fast-charging Zn-air batteries is crucial for widening their application but remains challenging owing to the limitation of sluggish oxygen evolution reaction (OER) kinetics and insufficient active sites of electrocatalysts. To solve this issue, a reconstructed amorphous FeCoNiSx electrocatalyst with high density of efficient active sites, yielding low OER overpotentials of 202, 255, and 323 mV at 10, 100, and 500 mA cm-2 , respectively, is developed for fast-charging Zn-air batteries with low charging voltages at 100-400 mA cm-2 . Furthermore, the fabricated 3241.8 mAh (20 mA cm-2 , 25 °C) quasi-solid Zn-air battery shows long lifetime of 500 h at -10 and 25 °C as well as 150 h at 40 °C under charging 100 mA cm-2 . The detailed characterizations combine with density functional theory calculations indicate that the defect-rich crystalline/amorphous ternary metal (oxy)hydroxide forms by the reconstruction of amorphous multi-metallic sulfide, where the electron coupling effect among multi-active sites and migration of intermediate O* from Ni site to the Fe site breaks the scaling relationship to lead to a low theoretical OER overpotential of 170 mV, accounting for the outstanding fast-charging property. This work not only provides insights into designing advanced OER catalysts by the self-reconstruction of the pre-catalyst but also pioneers a pathway for practical fast-charging Zn-air batteries.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining.

BACKGROUND: As important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications. RESULTS: Here, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu. CONCLUSIONS: We propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth. Video abstract.

Ozone Decomposition below Room Temperature Using Mn-based Mullite YMn2O5.

A super-low-temperature ozone decomposition is realized without energy consumption on a ternary oxide catalyst mullite YMn2O5 for the first time. The YMn2O5 oxide catalyzed ozone decomposition from a low temperature of -40 °C with 29% conversion (reaction rate: 1534.2 µmol g-1 h-1) and quickly reached 100% (5459.5 µmol g-1 h-1) when warmed up to -5 °C. The superior low-temperature performance over YMn2O5 could surpass that of the reported ozone decomposition catalysts. The structure and element valence characterizations confirmed that YMn2O5 remained the same after 100 h of room-temperature reaction, indicating excellent durability of the catalyst. O2-TPD (O2-temperature-programmed desorption) showed that the active sites are the Mn3+ sites bonded with singly coordinated oxygen on the surface. Combined with in situ Raman measurements and density functional theory calculations, we found that the ozone decomposition reaction on YMn2O5 showed a barrier of only 0.29 eV, following the Eley-Rideal (E-R) mechanism with a rate-limiting step of intermediate O22- desorption. The low barrier minimizes the accumulation of intermediate products and realizes the fast O3 decomposition even at super-low temperatures. Fundamentally, the moderate Mn-O bonding strength in the low-symmetry ternary oxides is crucial to produce singly coordinated active species on the surface responsible for the efficient ozone degradation at low temperatures.

Design Rules of a Sulfur Redox Electrocatalyst for Lithium-Sulfur Batteries.

Seeking an electrochemical catalyst to accelerate the liquid-to-solid conversion of soluble lithium polysulfides to insoluble products is crucial to inhibit the shuttle effect in lithium-sulfur (Li-S) batteries and thus increase their practical energy density. Mn-based mullite (SmMn2 O5 ) is used as a model catalyst for the sulfur redox reaction to show how the design rules involving lattice matching and 3d-orbital selection improve catalyst performance. Theoretical simulation shows that the positions of Mn and O active sites on the (001) surface are a good match with those of Li and S atoms in polysulfides, resulting in their tight anchoring to each other. Fundamentally, dz2 and dx2 -y2 around the Fermi level are found to be crucial for strongly coupling with the p-orbitals of the polysulfides and thus decreasing the redox overpotential. Following the theoretical calculation, SmMn2 O5 catalyst is synthesized and used as an interlayer in a Li-S battery. The resulted battery has a high cycling stability over 1500 cycles at 0.5 C and more promisingly a high areal capacity of 7.5 mAh cm-2 is achieved with a sulfur loading of ≈5.6 mg cm-2 under the condition of a low electrolyte/sulfur (E/S) value ≈4.6 µL mg-1 .

Characterizing Key Volatile Pollutants Emitted from Adhesives by Chemical Compositions, Odor Contributions and Health Risks.

As one of the major sources of volatile pollutants in indoor air, gaseous emissions from adhesives during interior decoration have attracted increasing concern. Identifying major volatile pollutants and the risk in adhesive gaseous emissions is of great significance, but remains rarely reported. In the present research, we assessed the major volatile pollutants emitted from white emulsion adhesive and silicone adhesive samples (n = 30) from three aspects: chemical composition, odor and health risk contributions. The results showed that a total of 21 volatile pollutants were detected. Significantly, xylene was the most concentrated compound from white emulsion adhesives, accounting for 45.51% of the total concentrations. Butanone oxime was the most concentrated compound in silicone adhesives, accounting for 69.86% of the total concentrations. The trends in odor concentration (evaluated by the odor activity value method) over time were well correlated with the total chemical concentrations. Xylene (58.00%) and butanone oxime (76.75%) showed the highest odor contribution, respectively. Moreover, from an integrated perspective of chemical emissions, odor and health risk contributions, xylene, ethylbenzene, ethyl acetate and benzene were identified as the key volatile pollutants emitted from the white emulsion adhesives, while butanone oxime, butanone, and ethanol were the key volatile pollutants emitted from the silicone adhesives. This study not only identified the key volatile pollutants but also provided characteristics of odor and health risks of gas emitted from adhesives.

Promotion of Low-Temperature Oxidation of Propane through Introduction of Ce into Mullite Oxide YMn2 O5.

A high-surface-area Ce doped mullite YMn2 O5 was developed via a facile hydrothermal approach, which exhibited higher catalytic activity with a long thermal stability towards propane oxidation in regards to pristine mullite YMn2 O5 . T90 (the temperature at 90 % conversion of reactant) of propane over the mixed oxides is ∼40 °C lower than that over pristine YMn2 O5 mullite (147 m2 /g). The complete oxidation temperature occurs at as low as 225 °C (1000 ppm C3 H8 and 10 % O2 balanced with N2 , WHSV=30,000 mL/g h). Notably, the mixed oxides maintain superior catalytic stability at 250 °C for 120 h without noticeable loss in the activity. Fundamentally, the remarkable performance stems from the abundant oxygen defects caused by the lattice mismatch between CeO2 and YMn2 O5 , which is conducive to the gas phase oxygen adsorption and activation, thereby enhancing the low temperature catalytic activity of the material. In addition, the CeO2 on the catalyst's surface acts as an oxygen reservoir and provides additional adsorption sites for propane to promote the oxidation reaction. In situ DRIFTS results indicates that the dissociation of acrylate could be the key step for propane oxidation since acrylate is more difficult to decompose and desorb than formate and acetate. These findings revealed the roles of ceria on mullite oxides for propane oxidation activity.

Structure and assembly pattern of a freshwater short-tailed cyanophage Pam1.

Despite previous structural analyses of bacteriophages, quite little is known about the structures and assembly patterns of cyanophages. Using cryo-EM combined with crystallography, we solve the near-atomic-resolution structure of a freshwater short-tailed cyanophage, Pam1, which comprises a 400-Å-long tail and an icosahedral capsid of 650 Å in diameter. The outer capsid surface is reinforced by trimeric cement proteins with a ß-sandwich fold, which structurally resemble the distal motif of Pam1's tailspike, suggesting its potential role in host recognition. At the portal vertex, the dodecameric portal and connected adaptor, followed by a hexameric needle head, form a DNA ejection channel, which is sealed by a trimeric needle. Moreover, we identify a right-handed rifling pattern that might help DNA to revolve along the wall of the ejection channel. Our study reveals the precise assembly pattern of a cyanophage and lays the foundation to support its practical biotechnological and environmental applications.

Circular RNA_0037128 aggravates high glucose-induced damage in HK-2 cells via regulation of microRNA-497-5p/nuclear factor of activated T cells 5 axis.

Circular RNAs (CircRNAs) were reported to play vital roles in the progression of DN. Herein, the action of circular RNA_0037128 (circ_0037128) was investigated in DN. The level of circ_0037128, microRNA-497-5p (miR-497-5p) and nuclear factor of activated T cells 5 (NFAT5) was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The feature of circ_0037128 was tested by RNase R and Actinomycin D treatment assays. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were conducted to evaluate the proliferation ability. The relative protein expression was determined via Western blot analysis. Levels of the inflammatory cytokines, like tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), were assessed by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were determined by the matched kits. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were conducted for evaluating the correlation between miR-497-5p and circ_0037128 or NFAT5. Circ_0037128 and NFAT5 were enhanced, while miR-497-5p was weakened in kidney tissues of DN patients and high glucose (HG)-cultured HK-2 cells. Circ_0037128 inhibition bated HG-caused inhibition effect on cell proliferation and promotion effects on oxidative stress, inflammation and fibrosis in HK-2 cells. Moreover, circ_0037128 knockdown alleviated HG-caused cell damage via regulating miR-497-5p. In addition, NFAT5 overexpression could reverse the influence of miR-497-5p on HG-induced injury in HK-2 cells. Mechanically, circ_0037128 sponged miR-497-5p to modulate NFAT5. Circ_0037128 downregulation could mitigate HG-stimulated cell damage via regulating the miR-497-5p/NFAT5 axis in HK-2 cells in vitro, providing a possible therapy target for DN.

NO is involved in H2-induced adventitious rooting in cucumber by regulating the expression and interaction of plasma membrane H+-ATPase and 14-3-3.

MAIN CONCLUSION: NO was involved in H2-induced adventitious rooting by regulating the protein and gene expressions of PM H+-ATPase and 14-3-3. Simultaneously, the interaction of PM H+-ATPase and 14-3-3 protein was also involved in this process. Hydrogen gas (H2) and nitric oxide (NO) have been shown to be involved in plant growth and development. The results in this study revealed that NO was involved in H2-induced adventitious root formation. Western blot (WB) analysis showed that the protein abundances of plasma membrane H+-ATPase (PM H+-ATPase) and 14-3-3 protein were increased after H2, NO, H2 plus NO treatments, whereas their protein abundances were down regulated when NO scavenger carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTI O) was added. Moreover, the mRNA abundances of the HA3 and 14-3-3(7) gene as well as the activities of PM H+-ATPase (EC 3.6.1.35) and H+ pump were in full agreement with the changes of protein abundance. Phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein were detected by co-immunoprecipitation analysis. H2 and NO significantly up regulated the phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein. Conversely, the stimulation of PM H+-ATPase phosphorylation and protein interaction were significantly diminished by cPTIO. Protein interaction activator fusicoccin (FC) and inhibitor adenosine monophosphate (AMP) of PM H+-ATPase and 14-3-3 were used in this study, and the results showed that FC significantly increased the abundances of PM H+-ATPase and 14-3-3, while AMP showed opposite trends. We further proved the critical roles of PM H+-ATPase and 14-3-3 protein interaction in NO-H2-induced adventitious root formation. Taken together, our results suggested that NO might be involved in H2-induced adventitious rooting by regulating the expression and the interaction of PM H+-ATPase and 14-3-3 protein.

Molecular basis for the assembly of RuBisCO assisted by the chaperone Raf1.

The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL-Raf1-RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL8Raf18 to the holoenzyme RbcL8RbcS8. In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1.

Genomic Analysis of Mic1 Reveals a Novel Freshwater Long-Tailed Cyanophage.

Lake Chaohu, one of the five largest freshwater lakes in China, has been suffering from severe cyanobacterial blooms in the summer for many years. Cyanophages, the viruses that specifically infect cyanobacteria, play a key role in modulating cyanobacterial population, and thus regulate the emergence and decline of cyanobacterial blooms. Here we report a long-tailed cyanophage isolated from Lake Chaohu, termed Mic1, which specifically infects the cyanobacterium Microcystis aeruginosa. Mic1 has an icosahedral head of 88 nm in diameter and a long flexible tail of 400 nm. It possesses a circular genome of 92,627 bp, which contains 98 putative open reading frames. Genome sequence analysis enabled us to define a novel terminase large subunit that consists of two types of intein, indicating that the genome packaging of Mic1 is under fine control via posttranslational maturation of the terminase. Moreover, phylogenetic analysis suggested Mic1 and mitochondria share a common evolutionary origin of DNA polymerase γ gene. All together, these findings provided a start-point for investigating the co-evolution of cyanophages and its cyanobacterial hosts.

Crystal structure of a novel fold protein Gp72 from the freshwater cyanophage Mic1.

Cyanophages, widespread in aquatic systems, are a class of viruses that specifically infect cyanobacteria. Though they play important roles in modulating the homeostasis of cyanobacterial populations, little is known about the freshwater cyanophages, especially those hypothetical proteins of unknown function. Mic1 is a freshwater siphocyanophage isolated from the Lake Chaohu. It encodes three hypothetical proteins Gp65, Gp66, and Gp72, which share an identity of 61.6% to 83%. However, we find these three homologous proteins differ from each other in oligomeric state. Moreover, we solve the crystal structure of Gp72 at 2.3 Å, which represents a novel fold in the α + ß class. Structural analyses combined with redox assays enable us to propose a model of disulfide bond mediated oligomerization for Gp72. Altogether, these findings provide structural and biochemical basis for further investigations on the freshwater cyanophage Mic1.