Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002.

Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.

Production and utilization of pseudocobalamin in marine Synechococcus cultures and communities.

Cobalamin influences marine microbial communities because an exogenous source is required by most eukaryotic phytoplankton, and demand can exceed supply. Pseudocobalamin is a cobalamin analogue produced and used by most cyanobacteria but is not directly available to eukaryotic phytoplankton. Some microbes can remodel pseudocobalamin into cobalamin, but a scarcity of pseudocobalamin measurements impedes our ability to evaluate its importance for marine cobalamin production. Here, we perform simultaneous measurements of pseudocobalamin and methionine synthase (MetH), the key protein that uses it as a co-factor, in Synechococcus cultures and communities. In Synechococcus sp. WH8102, pseudocobalamin quota decreases in low temperature (17°C) and low nitrogen to phosphorus ratio, while MetH did not. Pseudocobalamin and MetH quotas were influenced by culture methods and growth phase. Despite the variability present in cultures, we found a comparably consistent quota of 300 ± 100 pseudocobalamin molecules per cyanobacterial cell in the Northwest Atlantic Ocean, suggesting that cyanobacterial cell counts may be sufficient to estimate pseudocobalamin inventories in this region. This work offers insights into cellular pseudocobalamin metabolism, environmental and physiological conditions that may influence it, and provides environmental measurements to further our understanding of when and how pseudocobalamin can influence marine microbial communities.

Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens.

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

Genome-Scale Metabolic Model Reconstruction and Investigation into the Fluxome of the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901.

The ability to convert atmospheric CO2 and light into biomass and value-added chemicals makes cyanobacteria a promising resource microbial host for biotechnological applications. A newly discovered fastest-growing cyanobacterial strain, Synechococcus sp. PCC 11901, has been reported to have the highest biomass accumulation rate, making it a preferred target host for producing renewable fuels, value-added biochemicals, and natural products. System-level knowledge of an organism is imperative to understand the metabolic potential of the strain, which can be attained by developing genome-scale metabolic models (GEMs). We present the first genome-scale metabolic model of Synechococcus sp. PCC 11901 (iRS840), which contains 840 genes, 1001 reactions, and 944 metabolites. The model has been optimized and validated under different trophic modes, i.e., autotrophic and mixotrophic, by conducting an in vivo growth experiment. The robustness of the metabolic network was evaluated by changing the biomass coefficient of the model, which showed a higher sensitivity toward pigments under the photoautotrophic condition, whereas under the heterotrophic condition, amino acids were found to be more influential. Furthermore, it was discovered that PCC 11901 synthesizes succinyl-CoA via succinic semialdehyde due to its imperfect TCA cycle. Subsequent flux balance analysis (FBA) revealed a quantum yield of 0.16 in silico, which is higher compared to that of PCC 6803. Under mixotrophic conditions (with glycerol and carbon dioxide), the flux through the Calvin cycle increased compared to autotrophic conditions. This model will be useful for gaining insights into the metabolic potential of PCC 11901 and developing effective metabolic engineering strategies for product development.

EXCRETE workflow enables deep proteomics of the microbial extracellular environment.

Extracellular proteins play a significant role in shaping microbial communities which, in turn, can impact ecosystem function, human health, and biotechnological processes. Yet, for many ubiquitous microbes, there is limited knowledge regarding the identity and function of secreted proteins. Here, we introduce EXCRETE (enhanced exoproteome characterization by mass spectrometry), a workflow that enables comprehensive description of microbial exoproteomes from minimal starting material. Using cyanobacteria as a case study, we benchmark EXCRETE and show a significant increase over current methods in the identification of extracellular proteins. Subsequently, we show that EXCRETE can be miniaturized and adapted to a 96-well high-throughput format. Application of EXCRETE to cyanobacteria from different habitats (Synechocystis sp. PCC 6803, Synechococcus sp. PCC 11901, and Nostoc punctiforme PCC 73102), and in different cultivation conditions, identified up to 85% of all potentially secreted proteins. Finally, functional analysis reveals that cell envelope maintenance and nutrient acquisition are central functions of the predicted cyanobacterial secretome. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and suggest that multiple functions of the secretome are conserved across freshwater, marine, and terrestrial species.

Machine learning reveals the transcriptional regulatory network and circadian dynamics of Synechococcus elongatus PCC 7942.

Synechococcus elongatus is an important cyanobacterium that serves as a versatile and robust model for studying circadian biology and photosynthetic metabolism. Its transcriptional regulatory network (TRN) is of fundamental interest, as it orchestrates the cell's adaptation to the environment, including its response to sunlight. Despite the previous characterization of constituent parts of the S. elongatus TRN, a comprehensive layout of its topology remains to be established. Here, we decomposed a compendium of 300 high-quality RNA sequencing datasets of the model strain PCC 7942 using independent component analysis. We obtained 57 independently modulated gene sets, or iModulons, that explain 67% of the variance in the transcriptional response and 1) accurately reflect the activity of known transcriptional regulations, 2) capture functional components of photosynthesis, 3) provide hypotheses for regulon structures and functional annotations of poorly characterized genes, and 4) describe the transcriptional shifts under dynamic light conditions. This transcriptome-wide analysis of S. elongatus provides a quantitative reconstruction of the TRN and presents a knowledge base that can guide future investigations. Our systems-level analysis also provides a global TRN structure for S. elongatus PCC 7942.

A serum-free culture medium production system by co-culture combining growth factor-secreting cells and L-lactate-assimilating cyanobacteria for sustainable cultured meat production.

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an L-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant L-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.

Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable ß-caryophyllene production.

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce ß-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum ß-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO2 and light through co-culture systems.

Dissolved organic phosphorus bond-class utilization by Synechococcus.

Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus-carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5'-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5'-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus-carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5'-triphosphate, and lastly adenosine 5'-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains' exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5'-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems.

Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942.

In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Šand 2.0 Šin Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.

Quantifying the biogenic silica production of picoplankton in the oligotrophic ocean: A case study in the South China Sea.

Silicon (Si) utilization is not limited to eukaryotes. Recent research has suggested that the pattern of a large contribution of picocyanobacteria to biogenic silica (bSi) stocks might be widespread in the oligotrophic open ocean. We are the first to measure the size-fractionated bSi standing stocks and production rates in the oligotrophic South China Sea (SCS), which has obvious characteristics of oligotrophic waters. The 150 m integrated bSi standing stocks in the pico-sized fractions averaged 23 % of the total; the contribution of picoplankton to the total bSi production rate was 44 %. Interestingly, our estimated contributions of Synechococcus alone to the <2 µm bSi standing stock and < 2 µm bSi production rates averaged 14 % and 66 %, respectively, indicating that the significant and persistent contribution of bSi was strongly associated with marine picocyanobacteria. Furthermore, the dynamic changes in nutrient concentrations, especially in DIN and DIP, also potentially affected the variability in picoplankton bSi stocks and production rates, while the effects of temperature and salinity were not obvious. In this study, we have provided new information on measurable bSi in the picoplankton size fraction and its production rate in the SCS. We have demonstrated that picoplankton contributes a measurable, and at times significant, proportion to both the total bSi standing stock and its production rate in the SCS. A high silicon content within picocyanobacteria has important implications for understanding both their ecology and their contribution to biogeochemistry.

Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control.

Phycobilisomes (PBSs) are light-harvesting antenna complexes in cyanobacteria that adapt to diverse light environments through the use of phycobiliproteins within the PBS structures. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942, thrive under red light because of the presence of phycocyanin (PC) and its chromophore, phycocyanobilin (PCB), in the PBS. Cyanobacteria in shorter-wavelength light environments such as green light, employ phycoerythrin paired with phycoerythrobilin (PEB) along with PC in the PBS. Synthetic biology studies have shown that PEB production can be achieved by expression of the heterologous PEB synthases 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA) and PEB:ferredoxin oxidoreductase (PebB), leading to PEB accumulation and cellular browning. This approach is genetically unstable, and the properties of the resulting PEB-bound PBS complexes remain uncharacterized. In this study, we engineered a novel strain of Synechococcus 7942 PEB1 with finely tuned control of PEB biosynthesis. PEB1 exhibited a reversible change in the color of the culture from green to brown and pink based on PebA and PebB induction levels. High induction led to complete PCB-to-PEB substitution, causing the disassembly of the PBS rod complex. In contrast, low induction levels of PebA and PebB resulted in the formation of a stable chimeric PBS complex with partial PCB-to-PEB substitution. This acclimation enabled efficient light harvesting in the green spectrum and energy transfer to the photosynthetic reaction center. These findings, which improve our understanding of PBS and highlight the structural importance of the bilin composition, provide a foundation for future studies on PBS adaptation in bioengineering, synthetic biology, and renewable energy.

Synechococcus sp. PCC7335 responses to far-red enriched spectra and anoxic/microoxic atmospheres: Potential for astrobiotechnological applications.

Recently, cyanobacteria have gained attention in space exploration to support long-term crewed missions via Bioregenerative Life Support Systems. In this frame, cyanobacteria would provide biomass and profitable biomolecules through oxygenic photosynthesis, uptaking CO2, and releasing breathable O2. Their growth potential and organic matter production will depend on their ability to photoacclimate to different light intensities and spectra, maximizing incident light harvesting. Studying cyanobacteria responses to different light regimes will also benefit the broader field of astrobiology, providing data on the possibility of oxygenic photosynthetic life on planets orbiting stars with emission spectra different than the Sun. Here, we tested the acclimation and productivity of Synechococcus sp. PCC7335 (hereafter PCC7335), capable of Far-Red Light Photoacclimation (FaRLiP) and type III chromatic acclimation (CA3), in an anoxic, CO2-enriched atmosphere and under a spectrum simulating the low energetic light regime of an M-dwarf star, also comparable to a subsuperficial environment. When exposed to the light spectrum, with few photons in the visible (VIS) and rich in far-red (FR), PCC7335 did not activate FaRLiP but acclimated only via CA3, achieving a biomass productivity higher than expected, considering the low VIS light availability, and a higher production of phycocyanin, a valuable pigment, with respect to solar light. Its growth or physiological responses of PCC7335 were not affected by the anoxic atmosphere. In these conditions, PCC7335 efficiently produced O2 and scavenged CO2. Results highlight the photosynthetic plasticity of PCC7335, its suitability for astrobiotechnological applications, and the importance to investigate biodiversity of oxygenic photosynthesis for searching life beyond Earth.

Elucidating the role of primary and secondary sphere Zn2+ ligands in the cyanobacterial CO2 uptake complex NDH-14: The essentiality of arginine in zinc coordination and catalysis.

Inorganic carbon uptake in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO2 hydration. This active site of CO2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-14 CO2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which closely interact with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO2-hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO2-hydration activity from proton pumping. The results are discussed in terms of possible alternative CO2 hydration mechanisms.

Cryo-electron tomography reveals the packaging pattern of RuBisCOs in Synechococcus ß-carboxysome.

Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO2-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact ß-carboxysomes from Synechococcus elongatus PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the ß-carboxysome. These results provide new insights into the biogenesis of ß-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.

Contaminant removal performance and lipid productivity of a cyanobacteria-bacteria consortium containing exogenous phytohormones during the treatment of antibiotic-polluted wastewater.

In this study, a cyanobacteria-bacteria consortium containing native wastewater bacteria and immobilized Synechococcus sp. was constructed. The cyanobacterial cellular responses (including growth, biomass and lipid productivity) and contaminant removal ability (for TN, TP, COD and antibiotics) in the consortium were evaluated during the advanced treatment of wastewater containing 10-50 µg/L of mixed antibiotics (amoxicillin, tetracycline, erythromycin, sulfadiazine and ciprofloxacin) with the addition of a certain phytohormone (indole-3-acetic acid, gibberellin A3 or 6-benzylaminopurine) at trace level within a period of four days. Each phytohormone promoted the growth of Synechococcus sp. and increased the tolerance of Synechococcus sp. to mixed antibiotics. Indole-3-acetic acid coupled to moderate antibiotic stress could elevate lipid productivity and lipid content of Synechococcus sp. to 33.50 mg/L/day and 43.75%, respectively. Phytohormones increased the pollutant removal performance of the cyanobacteria-bacteria consortium through the stimulation of cyanobacterial growth and the regulation of cyanobacteria-bacteria interaction, which increased the abundances of microalgae-associated bacteria including Flavobacterium, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Bosea, Sphingomonas and Emticicia. Up to 80.83%, 98.06%, 83.26%, 99.84%, 99.50%, 89.41%, 65.61% and 60.65% of TN, TP, COD, amoxicillin, tetracycline, erythromycin, sulfadiazine and ciprofloxacin were removed by the consortium with the addition of phytohormones. In general, indole-3-acetic acid was the optimal phytohormone for enhancing lipid production and contaminant removal performance of the cyanobacteria-bacteria consortium.

Expanding the synthetic biology repertoire of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. The promoters PcpcB, PpsbA1, and P11770 exhibited consistently high fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for identifying RBSs, using an operon construct comprising fluorescent protein genes eyfp and mTurq under PcpcB (PcpcB:eyfp:RBS:mTurq:TrrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942.

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria.

Direct interaction between marine cyanobacteria mediated by nanotubes.

Microbial associations and interactions drive and regulate nutrient fluxes in the ocean. However, physical contact between cells of marine cyanobacteria has not been studied thus far. Here, we show a mechanism of direct interaction between the marine cyanobacteria Prochlorococcus and Synechococcus, the intercellular membrane nanotubes. We present evidence of inter- and intra-genus exchange of cytoplasmic material between neighboring and distant cells of cyanobacteria mediated by nanotubes. We visualized and measured these structures in xenic and axenic cultures and in natural samples. We show that nanotubes are produced between living cells, suggesting that this is a relevant system of exchange material in vivo. The discovery of nanotubes acting as exchange bridges in the most abundant photosynthetic organisms in the ocean may have important implications for their interactions with other organisms and their population dynamics.