A carboxysome-based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.

Rubisco condensate formation by CcmM in ß-carboxysome biogenesis.

Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency1. The α- and ß-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-a complex of eight large (RbcL) and eight small (RbcS) subunits-and carbonic anhydrase2-4. As HCO3- can diffuse through the proteinaceous carboxysome shell but CO2 cannot5, carbonic anhydrase generates high concentrations of CO2 for carbon fixation by Rubisco6. The shell also prevents access to reducing agents, generating an oxidizing environment7-9. The formation of ß-carboxysomes involves the aggregation of Rubisco by the protein CcmM10, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain11. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS2-4. Here we have reconstituted the Rubisco-CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes12,13. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC114. Understanding carboxysome biogenesis will be important for efforts to engineer CO2-concentrating mechanisms in plants15-19.

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.

Straw application improved soil biological properties and the growth of rice plant under low water irrigation.

The application of organic amendments is one way to manage low water irrigation in paddy soils. In this 60-day greenhouse pot experiment involving paddy soil undergoing drying-rewetting cycles, we examined the effects of two organic amendments: azo-compost with a low carbon to phosphorus ratio (C:P) of 40 and rice straw with a high C:P ratio of 202. Both were applied at rates of 1.5% of soil weight (w/w). The investigation focused on changes in certain soil biochemical characteristics related to C and P in the rice rhizosphere, as well as rice plant characteristics. The irrigation regimes applied in this study included constant soil moisture in a waterlogged state (130% water holding capacity (WHC)), mild drying-rewetting (from 130 to 100% WHC), and severe drying-rewetting (from 130 to 70% WHC). The results indicated that the application of amendments was effective in severe drying-rewetting irrigation regimes on soil characteristics. Drying-rewetting decreased soil respiration rate (by 60%), microbial biomass carbon (by 70%), C:P ratio (by 12%), soil organic P (by 16%), shoot P concentration (by 7%), and rice shoot biomass (by 30%). However, organic amendments increased soil respiration rate (by 8 times), soil microbial biomass C (51%), total C (TC) (53%), dissolved organic carbon (3 times), soil available P (AP) (100%), soil organic P (63%), microbial biomass P (4.5 times), and shoot P concentration (21%). The highest significant correlation was observed between dissolved organic carbon and total C (r= 0.89**). Organic amendments also increased P uptake by the rice plant in the order: azo-compost > rice straw > control treatments, respectively, and eliminated the undesirable effect of mild drying-rewetting irrigation regime on rice plant biomass. Overall, using suitable organic amendments proves promising for enhancing soil properties and rice growth under drying-rewetting conditions, highlighting the interdependence of P and C biochemical changes in the rhizosphere during the rice vegetative stage.

Rubisco proton production can drive the elevation of CO2 within condensates and carboxysomes.

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3-, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3- accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3- accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.

Effects on soil carbon storage from municipal biosolids application to agricultural fields.

This study investigated the influence of biosolid applications on soil carbon storage and evaluated nutrient management strategies affecting soil carbon dynamics. The research assessed alterations in soil pH, soil carbon stock, and soil nitrogen content within short-term and long-term biosolids-amended soils in Bible Hill, Nova Scotia, Canada, extending to a depth of 0-60 cm. The findings indicated an increase in soil pH with alkaline treatment biosolids (ATB) applications across both study sites, with a legacy effect on soil pH noted in the long-term biosolids-amended soil following a single ATB application over 13 years. Both sites demonstrated significant increases in soil total carbon (STC) and soil organic carbon (SOC) within the 0-30 cm soil depth after biosolid application, and soil inorganic carbon (SIC) accounted for approximately 5-10% of STC, specifically in the surface soil layer (0-15 cm). In the long-term study site, annual 14, 28 and 42 Mg ATB ha-1 treatments resulted in a substantial rise in soil carbon stock (59.5, 60.1 and 68.0 Mg C ha-1), marking a 25% increase compared to control soil. The SOC content in biosolids-amended soil showed a declining trend with increasing soil depth at both study sites. Notably, the carbon stock in the short-term site was observed in composted biosolids (COMP) > ATB > liquid mesophilic anaerobically digested biosolids (LMAD) from the 0-60 cm soil depth. Approximately 79-80% of the variation in SOC response at both sites was concentrated within the top 30 cm soil. Soil total nitrogen (STN) showed no significant differences at the short-term site, and STN in biosolids-amended soil decreased with increasing soil depth at the long-term site. Biosolids-induced C retention coefficients (BCR) for ATB remained consistent at both sites, ranging from -13% to 31.4% with a mean of 11.12%. BCR values for COMP ranged from 1.9% to 34.4% with a mean of 18.73%, while those for LMAD exhibited variability, spanning from -6.2% to 106.3% with a mean of 53.9%.

Application of artificial intelligence in modeling of nitrate removal process using zero-valent iron nanoparticles-loaded carboxymethyl cellulose.

This study explores nitrate reduction in aqueous solutions using carboxymethyl cellulose loaded with zero-valent iron nanoparticles (Fe0-CMC). The structures of this nano-composite were characterized using various techniques. Based on the characterization results, the specific surface area of Fe0-CMC measured by the Brunauer-Emmett-Teller analysis were 39.6 m2/g. In addition, Scanning Electron Microscopy images displayed that spherical nano zero-valent iron particles (nZVI) with an average particle diameter of 80 nm are surrounded by carboxymethyl cellulose and no noticeable aggregates were detected. Batch experiments assessed Fe0-CMC's effectiveness in nitrate removal under diverse conditions including different adsorbent dosages (Cs, 2-10 mg/L), contact time (t, 10-1440 min), initial pH (pHi, 2-10), temperature (T, 10-55 °C), and initial concentration of nitrate (C0, 10-500 mg/L). Results indicated decreased removal with higher initial pHi and C0, while increased Cs and T enhanced removal. The study of nitrate removal mechanism by Fe0-CMC revealed that the redox reaction between immobilized nZVI on the CMC surface and nitrate ions was responsible for nitrate removal, and the main product of this reaction was ammonium, which was subsequently completely removed by the synthesized nanocomposite. In addition, a stable deviation quantum particle swarm optimization algorithm (SD-QPSO) and a least square error method were employed to train the ANFIS parameters. To demonstrate model performance, a quadratic polynomial function was proposed to display the performance of the SD-QPSO algorithm in which the constant parameters were optimized through the SD-QPSO algorithm. Sensitivity analysis was conducted on the proposed quadratic polynomial function by adding a constant deviation and removing each input using two different strategies. According to the sensitivity analysis, the predicted removal efficiency was most sensitive to changes in pHi, followed by Cs, T, C0, and t. The obtained results underscore the potential of the ANFIS model (R2 = 0.99803, RMSE = 0.9888), and polynomial function (R2 = 0.998256, RMSE = 1.7532) as accurate and efficient alternatives to time-consuming laboratory measurements for assessing nitrate removal efficiency. These models can offer rapid insights and predictions regarding the impact of various factors on the process, saving both time and resources.

Towards engineering a hybrid carboxysome.

Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO2 environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields. To date, two carboxysome types have been characterized, the α-type that has fewer shell components and the ß-type that houses a faster Rubisco. While research is underway to construct a native carboxysome in planta, work investigating the internal arrangement of carboxysomes has identified conserved Rubisco amino acid residues between the two carboxysome types which could be engineered to produce a new, hybrid carboxysome. In theory, this hybrid carboxysome would benefit from the simpler α-carboxysome shell architecture while simultaneously exploiting the higher Rubisco turnover rates in ß-carboxysomes. Here, we demonstrate in an Escherichia coli expression system, that the Thermosynechococcus elongatus Form IB Rubisco can be imperfectly incorporated into simplified Cyanobium α-carboxysome-like structures. While encapsulation of non-native cargo can be achieved, T. elongatus Form IB Rubisco does not interact with the Cyanobium carbonic anhydrase, a core requirement for proper carboxysome functionality. Together, these results suggest a way forward to hybrid carboxysome formation.

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

An early modern human presence in Sumatra 73,000-63,000 years ago.

Genetic evidence for anatomically modern humans (AMH) out of Africa before 75 thousand years ago (ka) and in island southeast Asia (ISEA) before 60 ka (93-61 ka) predates accepted archaeological records of occupation in the region. Claims that AMH arrived in ISEA before 60 ka (ref. 4) have been supported only by equivocal or non-skeletal evidence. AMH evidence from this period is rare and lacks robust chronologies owing to a lack of direct dating applications, poor preservation and/or excavation strategies and questionable taxonomic identifications. Lida Ajer is a Sumatran Pleistocene cave with a rich rainforest fauna associated with fossil human teeth. The importance of the site is unclear owing to unsupported taxonomic identification of these fossils and uncertainties regarding the age of the deposit, therefore it is rarely considered in models of human dispersal. Here we reinvestigate Lida Ajer to identify the teeth confidently and establish a robust chronology using an integrated dating approach. Using enamel-dentine junction morphology, enamel thickness and comparative morphology, we show that the teeth are unequivocally AMH. Luminescence and uranium-series techniques applied to bone-bearing sediments and speleothems, and coupled uranium-series and electron spin resonance dating of mammalian teeth, place modern humans in Sumatra between 73 and 63 ka. This age is consistent with biostratigraphic estimations, palaeoclimate and sea-level reconstructions, and genetic evidence for a pre-60 ka arrival of AMH into ISEA. Lida Ajer represents, to our knowledge, the earliest evidence of rainforest occupation by AMH, and underscores the importance of reassessing the timing and environmental context of the dispersal of modern humans out of Africa.

Critical review on phytoremediation of polyfluoroalkyl substances from environmental matrices: Need for global concern.

Poly- and perfluoroalkyl substances (PFAS) are a class of emerging organic contaminants that are impervious to standard physicochemical treatments. The widespread use of PFAS poses serious environmental issues. PFAS pollution of soils and water has become a significant issue due to the harmful effects of these chemicals both on the environment and public health. Owing to their complex chemical structures and interaction with soil and water, PFAS are difficult to remove from the environment. Traditional soil remediation procedures have not been successful in reducing or removing them from the environment. Therefore, this review focuses on new phytoremediation techniques for PFAS contamination of soils and water. The bioaccumulation and dispersion of PFAS inside plant compartments has shown great potential for phytoremediation, which is a promising and unique technology that is realistic, cost-effective, and may be employed as a wide scale in situ remediation strategy.

Reviewing the role of biochar in paddy soils: An agricultural and environmental perspective.

The main challenge of the twenty-first century is to find a balance between environmental sustainability and crop productivity in a world with a rapidly growing population. Soil health is the backbone of a resilient environment and stable food production systems. In recent years, the use of biochar to bind nutrients, sorption of pollutants, and increase crop productivity has gained popularity. This article reviews key recent studies on the environmental impacts of biochar and the benefits of its unique physicochemical features in paddy soils. This review provides critical information on the role of biochar properties on environmental pollutants, carbon and nitrogen cycling, plant growth regulation, and microbial activities. Biochar improves the soil properties of paddy soils through increasing microbial activities and nutrient availability, accelerating carbon and nitrogen cycle, and reducing the availability of heavy metals and micropollutants. For example, a study showed that the application of a maximum of 40 t ha-1 of biochar from rice husks prior to cultivation (at high temperature and slow pyrolysis) increases nutrient utilization and rice grain yield by 40%. Biochar can be used to minimize the use of chemical fertilizers to ensure sustainable food production.

Micro (nano) plastics uptake, toxicity and detoxification in plants: Challenges and prospects.

Plastic pollution has emerged as a global challenge affecting ecosystem health and biodiversity conservation. Terrestrial environments exhibit significantly higher plastic concentrations compared to aquatic systems. Micro/nano plastics (MNPs) have the potential to disrupt soil biology, alter soil properties, and influence soil-borne pathogens and roundworms. However, limited research has explored the presence and impact of MNPs on aquaculture systems. MNPs have been found to inhibit plant and seedling growth and affect gene expression, leading to cytogenotoxicity through increased oxygen radical production. The article discusses the potential phytotoxicity process caused by large-scale microplastics, particularly those unable to penetrate cell pores. It also examines the available data, albeit limited, to assess the potential risks to human health through plant uptake.

Effect of systemic inflammation biomarkers on overall survival after lung cancer radiotherapy: a single-center large-cohort study.

INTRODUCTION: Recent studies suggest that immune-related cells can be recruited for anti-tumor functions as well as tumor progression and the interplay between systemic inflammation and local immune response may play a major role in the development and progression of various cancers including lung cancer. Inflammatory markers, such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII) can be used as surrogate biomarkers of host immune status. In this work, associations between neutrophils, lymphocytes, platelets, NLR, PLR, SII and overall survival (OS) are investigated in two cohorts of non-small cell lung cancer (NSCLC) patients treated with fractionated radiotherapy (RT) and stereotactic body radiation therapy (SBRT) and a cohort of small cell lung cancer (SCLC) patients treated with fractionated RT. MATERIAL AND METHODS: Data from 2513 lung cancer patients were retrospectively analyzed. Baseline NLR, PLR, and SII (NLR × platelet count) were calculated from full blood test prior to RT initiation. Cox proportional hazards regression analyses were used to evaluate the association between systemic inflammation markers and known clinical factors with OS. RESULTS: The two-year OS was 42%, 63%, and 62% in the NSCLC fractionated RT, SBRT, and SCLC cohort. NLR (per 1 unit: hazard ratio [HR]: 1.04, p < 0.05) and SII (per 100 × 109/L: HR: 1.01, p < 0.05) remained the strongest independent factors of OS in multivariable Cox analyses, correcting for clinical factors in early-stage and locally advanced NSCLC and SCLC patients treated with RT. DISCUSSION: This single-center large-cohort study suggests that baseline NLR and SII are independent prognostic biomarkers associated with OS in locally advanced and early-stage NSCLC patients treated with either curative-intent fractionated RT or SBRT and SCLC patients treated with curative-intent fractionated RT. External validation is warranted to evaluate the utility of these biomarkers for patients' stratification and adapting new treatment approaches.

Structural Basis for the Allosteric Regulation of the SbtA Bicarbonate Transporter by the PII-like Protein, SbtB, from Cyanobium sp. PCC7001.

Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP, and ATP with micromolar-range affinities. X-ray crystal structures of apo and nucleotide-bound SbtB7001 revealed that while AMP, ADP, and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop, and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased 10-fold in the presence of Ca2+, and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.

Measuring CO2 and HCO3- permeabilities of isolated chloroplasts using a MIMS-18O approach.

To support photosynthetic CO2 fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO2 or bicarbonate. However, the mechanisms allowing the rapid passage of this gas and this charged molecule through the bounding membranes of the chloroplast envelope are not yet completely elucidated. We describe here a method allowing us to measure the permeability of these two molecules through the chloroplast envelope using a membrane inlet mass spectrometer and 18O-labelled inorganic carbon. We established that the internal stromal carbonic anhydrase activity is not limiting for this technique, and precisely measured the chloroplast surface area and permeability values for CO2 and bicarbonate. This was performed on chloroplasts from several plant species, with values ranging from 2.3 × 10-4 m s-1 to 8 × 10-4 m s-1 permeability for CO2 and 1 × 10-8 m s-1 for bicarbonate. We were able to apply our method to chloroplasts from an Arabidopsis aquaporin mutant, and this showed that CO2 permeability was reduced 50% in the mutant compared with the wild-type reference.

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

MALDI-TOF MS meets WGS in a VRE outbreak investigation.

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.