Marine Cyanobacterium Synechococcus Research Articles

Interactive effects of CO2, temperature, and nitrate limitation on the growth and physiology of strain CCMP 1334 of the marine cyanobacterium Synechococcus (Cyanophyceae).

The marine cyanobacterium Synecococcus sp. (CCMP 1334) was grown in a continuous culture system on a 12:12 h light:dark cycle at all combinations of low and high pCO2 (400 and 1000 ppmv, respectively), nutrient availability (nitrate-limited and nutrient-replete conditions), and temperatures of 21, 24, 28, 32, and 35°C. The maximum nutrient-replete growth rate was ~1.15 day-1 at 32-35°C. Median nutrient-replete growth rates were higher at 1000 ppmv than at 400 ppmv pCO2 at all temperatures. Carbon:nitrogen ratios were independent of pCO2 at a fixed relative growth rate (i.e., growth rate ÷ nutrient-replete growth rate) but decreased with increasing temperature. Carbon:chlorophyll a ratios were decreased monotonically with increasing temperature and were higher under nitrate-limited than nutrient-replete conditions. Ratios of phycoerythrin to chlorophyll a were independent of growth conditions. Productivity indices were independent of temperature and nutrient limitation but were consistently higher at 1000 ppmv than 400 ppmv pCO2. Both growth rates and dark respiration rates were positively correlated with temperature, and the associated Q10 values were 2.2 and 2.3, respectively. A model of phytoplankton growth in which cellular carbon is allocated to structure, storage, or the light or dark reactions of photosynthesis accounted for the general patterns of cell composition and growth rate. This strain of Synechococcus appears well suited to changes in environmental conditions that are expected as the climate warms in response to anthropogenic emissions of CO2.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses

BackgroundMarine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO2, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses.ResultsIn this work, we cultured the marine cyanobacterium Synechococcus sp. PCC 7002 in a 3-L externally illuminated bioreactor with working volume of 2 L with a biomass productivity of ~ 0.8 g L−1 day−1. Hydrolysis of the biomass with acids released proteins and hydrolyzed glycogen while hydrolysis of the biomass with base released only proteins but did not hydrolyze glycogen. Among the different acids tested, treatment with HNO3 led to the highest release of proteins and glucose. Cyanobacterial biomass hydrolysate (CBH) prepared in HNO3 was used as a medium to produce cellulase enzyme by the Penicillium funiculosum OAO3 strain while CBH prepared in HCl and treated with charcoal was used as a medium for citric acid by Aspergillus tubingensis. Approximately 50% higher titers of both products were obtained compared to traditional media.ConclusionsThese results show that the hydrolysate of marine cyanobacteria is an effective source of nutrients/proteins for fungal bioprocesses.

Coordinated transcriptional response to environmental stress by a Synechococcus virus.

Viruses are a major control on populations of microbes. Often, their virulence is examined in controlled laboratory conditions. Yet, in nature, environmental conditions lead to changes in host physiology and fitness that may impart both costs and benefits on viral success. Phosphorus (P) is a major abiotic control on the marine cyanobacterium Synechococcus. Some viruses infecting Synechococcus have acquired, from their host, a gene encoding a P substrate binding protein (PstS), thought to improve virus replication under phosphate starvation. Yet, pstS is uncommon among cyanobacterial viruses. Thus, we asked how infections with viruses lacking PstS are affected by P scarcity. We show that the production of infectious virus particles of such viruses is reduced in low P conditions. However, this reduction in progeny is not caused by impaired phage genome replication, thought to be a major sink for cellular phosphate. Instead, transcriptomic analysis showed that under low P conditions, a PstS-lacking cyanophage increased the expression of a specific gene set that included mazG, hli2, and gp43 encoding a pyrophosphatase, a high-light inducible protein and DNA polymerase, respectively. Moreover, several of the upregulated genes were controlled by the host's phoBR two-component system. We hypothesize that recycling and polymerization of nucleotides liberates free phosphate and thus allows viral morphogenesis, albeit at lower rates than when phosphate is replete or when phages encode pstS. Altogether, our data show how phage genomes, lacking obvious P-stress-related genes, have evolved to exploit their host's environmental sensing mechanisms to coordinate their own gene expression in response to resource limitation.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700 to 800 nm) than the wavelengths typically used for oxygenic photosynthesis (400 to 700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-electron microscopy to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Dual thermal ecotypes coexist within a nearly genetically identical population of the unicellular marine cyanobacterium Synechococcus.

The extent and ecological significance of intraspecific functional diversity within marine microbial populations is still poorly understood, and it remains unclear if such strain-level microdiversity will affect fitness and persistence in a rapidly changing ocean environment. In this study, we cultured 11 sympatric strains of the ubiquitous marine picocyanobacterium Synechococcus isolated from a Narragansett Bay (RI) phytoplankton community thermal selection experiment. Thermal performance curves revealed selection at cool and warm temperatures had subdivided the initial population into thermotypes with pronounced differences in maximum growth temperatures. Curiously, the genomes of all 11 isolates were almost identical (average nucleotide identities of >99.99%, with >99% of the genome aligning) and no differences in gene content or single nucleotide variants were associated with either cool or warm temperature phenotypes. Despite a very high level of genomic similarity, sequenced epigenomes for two strains showed differences in methylation on genes associated with photosynthesis. These corresponded to measured differences in photophysiology, suggesting a potential pathway for future mechanistic research into thermal microdiversity. Our study demonstrates that present-day marine microbial populations can harbor cryptic but environmentally relevant thermotypes which may increase their resilience to future rising temperatures.

Engineering Synechococcus sp. PCC 7002 for sustainable production of sucrose

Sucrose is a readily available substrate and feedstock for biotechnology, which is biosynthesized by plants, algae, and cyanobacteria. Engineered cyanobacteria with enhanced sucrose production may be a viable alternative to sucrose synthesis from plants. The marine cyanobacteria which can be cultivated in seawater have the potential to provide a sustainable solution. The model marine cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) has many features for a good chassis, including a fast growth rate and a wide range of stress tolerances. Syn7002 synthesizes mainly glucosylglycerol and traces of sucrose as compatible solutes under salt stress. The compatible solute glucosylglycerate was also found in small amounts in Syn7002. To enhance sucrose production in Syn7002, the sucrose biosynthesis genes and the sucrose transporter gene were overexpressed. And the competing pathway genes and the sucrose hydrolysis gene were inactivated. The sucrose production in the engineered strain can reach 2282 mg/L in 8 days under 1 M NaCl stress. With the further substitution of sucrose biosynthesis genes from Synechocystis sp. PCC 6803, the engineered strain can efficiently produce sucrose over a wide range of NaCl concentrations. The efficient sucrose production by marine cyanobacteria will promote the sustainable supply of sugar feedstocks for biotechnology and aquaculture.

Using δFIP as a potential biomarker for risk assessment of environmental pollutants in aquatic ecosystem: A case study of marine cyanobacterium Synechococcus sp. PCC7002

Increased hazardous substances application causes more environmental pollution and risks for human health. Microalgae are the important biological groups in marine ecosystem, and considered to be sensitive to environmental pollutants. Therefore, toxicity test on marine microalgae could provide the most efficient method for aquatic toxicity assessment, and could also be used as the early warning signals in aquatic ecosystem. In view of this, our study aimed at investigating the toxicity potential of two typical organic compounds, and screening out novel photosynthetic indicators for the risk assessment of environmental pollutants. In this study, benzyl alcohol and 2-phenylethanol were chosen as the target organic compounds, and preliminary toxicity mechanism of these organic compounds on marine cyanobacterium Synechococcus sp. PCC7002 was investigated with chlorophyll fluorescence technology. Results showed that PCC7002 could be affected by benzyl alcohol or 2-phenylethanol stress, and the toxicity effect was concentration-dependent. And external benzyl alcohol and 2-phenylethanol stress damaged the oxygen evolving complex, and suppressed electron transport at the donor and receptor sides of photosystem II (PSII), influencing the absorption, transfer, and application of light energy. Furthermore, potential biomarkers were screened by half maximal inhibitory concentration (IC50) on the basis of pearson correlation coefficient analysis, and fluorescence intensity difference between the I-step and P-step of OJIP curve (δFIP) seems to be the most sensitive indicator for external stress. This study would be of significant interest to the biomarker community, and pave the way for the practical resource for marine pollution monitoring and assessment.

Development of shuttle vectors for rapid prototyping of engineered Synechococcus sp. PCC7002.

Cyanobacteria are of particular interest for chemical production as they can assimilate CO2 and use solar energy to power chemical synthesis. However, unlike the model microorganism of Escherichia coli, the availability of genetic toolboxes for rapid proof-of-concept studies in cyanobacteria is generally lacking. In this study, we first characterized a set of promoters to efficiently drive gene expressions in the marine cyanobacterium Synechococcus sp. PCC7002. We identified that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002. Next, a set of shuttle vectors was constructed based on the endogenous pAQ1 plasmid to facilitate the rapid pathway assembly. Moreover, we used the shuttle vectors to modularly optimize the amorpha-4,11-diene synthesis in PCC7002. By modularly optimizing the metabolic pathway, we managed to redistribute the central metabolism toward the amorpha-4,11-diene production in PCC7002 with enhanced product titer. Taken together, the plasmid toolbox developed in this study will greatly accelerate the generation of genetically engineered PCC7002. KEY POINTS: • Promoter characterization revealed that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002 • A set of shuttle vectors with different antibiotic selection markers was constructed based on endogenous pAQ1 plasmid • By modularly optimizing the metabolic pathway, amorpha-4,11-diene production in PCC7002 was improved.

Farming on Mars: Treatment of basaltic regolith soil and briny water simulants sustains plant growth.

A fundamental challenge in human missions to Mars is producing consumable foods efficiently with the in situ resources such as soil, water, nutrients and solar radiation available on Mars. The low nutrient content of martian soil and high salinity of water render them unfit for direct use for propagating food crops on Mars. It is therefore essential to develop strategies to enhance nutrient content in Mars soil and to desalinate briny water for long-term missions on Mars. We report simple and efficient strategies for treating basaltic regolith simulant soil and briny water simulant for suitable resources for growing plants. We show that alfalfa plants grow well in a nutrient-limited basaltic regolith simulant soil and that the alfalfa biomass can be used as a biofertilizer to sustain growth and production of turnip, radish and lettuce in the basaltic regolith simulant soil. Moreover, we show that marine cyanobacterium Synechococcus sp. PCC 7002 effectively desalinates the briny water simulant, and that desalination can be further enhanced by filtration through basalt-type volcanic rocks. Our findings indicate that it is possible to grow food crops with alfalfa treated basaltic regolith martian soil as a substratum watered with biodesalinated water.

Sustainable citric acid production from CO2 in an engineered cyanobacterium.

Citric acid is one of the most widely used organic acids in the world, with applications ranging from acidity regulation in food and beverages to metal chelation in hydrometallurgical processes. Most of its production is currently derived from fermentative processes, using plant-derived carbon feedstocks. While these are currently dominant, there is an increasing need to develop closed-loop production systems that reduce process carbon footprint. In this work, we demonstrate for the first time that an engineered marine cyanobacterium Synechococcus sp. PCC 7002 can be used as a sustainable chassis for the photosynthetic conversion of CO2 to citric acid. Decreased citric acid cycle flux, through the use of a theophylline-responsive riboswitch, was combined with improved flux through citrate synthase and enhanced citric acid excretion, resulting in a significant improvement to citric acid production. While allowing citrate production, this strategy induces a growth defect which can be overcome by glutamate supplementation or by fine-tuning aconitase levels, resulting in an increase in production relative to WT of over 100-fold. This work represents a first step toward sustainable production of a commodity organic acid from CO2.

Interactive effects of CO2 , temperature, irradiance, and nutrient limitation on the growth and physiology of the marine cyanobacterium Synechococcus (Cyanophyceae).

The marine cyanobacterium Synechococcus elongatus was grown in a continuous culture system to study the interactive effects of temperature, irradiance, nutrient limitation, and the partial pressure of CO2 (pCO2) on its growth and physiological characteristics. Cells were grown on a 14:10 h light:dark cycle at all combinations of low and high irradiance (50 and 300 μmol photons ⋅ m-2 ⋅ s-1 , respectively), low and high pCO2 (400 and 1000 ppmv, respectively), nutrient limitation (nitrate-limited and nutrient-replete conditions), and temperatures of 20-45°C in 5°C increments. The maximum growth rate was ~4.5 · d-1 at 30-35°C. Under nutrient-replete conditions, growth rates at most temperatures and irradiances were about 8% slower at a pCO2 of 1000 ppmv versus 400 ppmv. The single exception was 45°C and high irradiance. Under those conditions, growth rates were ~45% higher at 1000 ppmv. Cellular carbon:nitrogen ratios were independent of temperature at a fixed relative growth rate but higher at high irradiance than at low irradiance. Initial slopes of photosynthesis-irradiance curves were higher at all temperatures under nutrient-replete versus nitrate-limited conditions; they were similar at all temperatures under high and low irradiance, except at 20°C, when they were suppressed at high irradiance. A model of phytoplankton growth in which cellular carbon was allocated to structure, storage, or the light or dark reactions of photosynthesis accounted for the general patterns of cell composition and growth rate. Allocation of carbon to the light reactions of photosynthesis was consistently higher at low versus high light and under nutrient-replete versus nitrate-limited conditions.

A metallothionein from an open ocean cyanobacterium removes zinc from the sensor protein controlling its transcription

Bacterial metallothioneins are known for a limited range of phyla including cyanobacteria. We have characterised the BmtA from the marine cyanobacterium Synechococcus sp. WH8102 (SynBmtA). This strain inhabits the open ocean, one of the most nutrient-poor environments on Earth, with very low total and free Zn2+ concentrations. Therefore, the presence of a metallothionein, usually associated with zinc and cadmium tolerance, in this strain is intriguing. Previous transcriptomics work revealed that unprecedentedly, expression of SynBmtA is activated by the Synechococcus sp. WH8102 “zinc uptake regulator” (SynZur) at elevated [Zn2+]. SynBmtA binds four Zn2+ ions, and its first 37 residues adopt the zinc-finger fold characteristic of BmtAs. In contrast, sequence similarity to other BmtAs in the C-terminal stretch is low. This is expected to affect especially the most reactive site in zinc-transfer reactions. Indeed, chelators were unable to extract Zn2+ from SynBmtA, even in the presence of denaturant. This indicates an extremely stable protein fold, with no accessibility to any bound zinc ions in the folded protein. In addition, the zinc-binding affinity of SynBmtA exceeds those of any other metallothioneins. Apo-SynBmtA is capable of removing zinc from the sensory site of SynZur, providing one possible avenue of de-activating transcription of the synbmtA gene. All of these properties are consistent with a role in safely sequestering any excess zinc, to prevent toxic effects. The fact that this strain stores zinc in a metallothionein rather than employing an efflux pump implies that zinc is a valuable resource for Synechococcus sp. WH8102 and related strains.

Acid-base properties of Synechococcus-derived organic matter

Marine cyanobacteria play an important role in trace metal cycling in the oceans. However, the influence of bacterial detritus following cell lysis – that includes both particulate organic matter (POM) and dissolved organic matter (DOM) – is less understood. Previous investigations have shown that the ratio of bacterial detritus to living cells increases significantly with ocean depth, indicating that the former plays a central role in trace metal cycling and eventual sequestration in deep marine sediments. Thus, in this study we produced detritus from the mechanical lysis of the common marine cyanobacterium Synechococcus sp. PCC 7002 (referred to as cyPOM and cyDOM), and then measured their buffering capacity through proton adsorption and release via potentiometric acid-base titrations. Both cyPOM and cyDOM were best modelled using a 2-site non-electrostatic protonation model. The cyPOM fraction possessed ligands with pKa values of 5.78 (±0.07) and 9.01 (±0.29) with corresponding site concentrations of 41.8 (±8.15) µmol g−1 and 41.2 (±5.28) µmol g−1, while cyDOM had pKa values of 4.89 (±0.22) and 6.80 (±0.22) and corresponding site concentrations of 42.8 (±14.6) µmol g−1 and 37.3 (±16.5) µmol g−1. As a comparison, intact Synechococcus cells were previously reported to be best modelled by invoking 3-sites with overall ligand densities more than double that of cyPOM and cyDOM. Trace metal (Cd, Co, Cu, Ni, and Zn) adsorption experiments indicate that cyPOM has a lower affinity towards trace metals than intact Synechococcus cells, evidenced by lower trace metals adsorption per gram of dry mass and therefore less trace metal sequestration potential. This implies that intact Synechococcus cells are chiefly responsible for trace metal sequestration, even at high cyPOM proportions (10:1 – cyPOM:Synechococcus) that are more representative of POM that reaches the seafloor. The cyDOM-trace metal association could not be determined experimentally; thus we applied existing thermodynamic data from modern marine DOM studies acquired via potentiometric titrations and metal-binding constants. Unlike cyPOM, metal speciation is almost entirely accounted for in the dissolved fraction (>99.9%). These results further the view that DOM is the most important microbial fraction in controlling metals speciation in the water column.

Growth and grazing of the chlorarachniophyte Bigelowiella natans (Chlorarachniophyceae) on the marine cyanobacterium Synechococcus

ABSTRACT Chlorarachniophytes are cosmopolitan, typically unicellular marine phototrophic protists with green plastids of secondary endosymbiotic origin. However, there are few studies of their eco-physiology. Bigelowiella natans feeds mixotrophically on diverse strains of Synechococcus. We investigated feeding by B. natans (Chlorarachniophyceae) on diverse strains of the marine cyanobacterium Synechococcus and quantified the ingestion and clearance rates of B. natans on Synechococcus strains CC9311 and CC9605 as a function of prey concentration, as well as the growth rates of B. natans feeding on these Synechococcus strains. The maximum ingestion rates of B. natans on Synechococcus strains CC9311 and CC9605 were 2.77 and 2.18 cells predator–1 h–1, respectively. The maximum clearance rates of B. natans on Synechococcus CC9311 and CC9605 were 197.2 and 331.5 nl predator–1 h–1, respectively. The maximum mixotrophic growth rates of B. natans on Synechococcus CC9311 and CC9605 were 0.38 d–1 and 0.28 d–1, respectively, while its autotrophic growth rates were 0.174 ± 0.019 d–1. The results of the present study suggest that B. natans is an effective protistan grazer of Synechococcus and that it obtains a significant ecological advantage from a mixotrophic strategy in marine planktonic food webs.

Amino Acid Analog Induces Stress Response in Marine Synechococcus.

Characterizing the cell-level metabolic trade-offs that phytoplankton exhibit in response to changing environmental conditions is important for predicting the impact of these changes on marine food web dynamics and biogeochemical cycling. The time-selective proteome-labeling approach, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to provide insight into differential allocation of resources at the cellular level, especially when coupled with proteomics. However, the application of this technique in marine phytoplankton remains limited. We demonstrate that the marine cyanobacteria Synechococcus sp. and two groups of eukaryotic algae take up the modified amino acid l-homopropargylglycine (HPG), suggesting that BONCAT can be used to detect translationally active phytoplankton. However, the impact of HPG addition on growth dynamics varied between groups of phytoplankton. In addition, proteomic analysis of Synechococcus cells grown with HPG revealed a physiological shift in nitrogen metabolism, general protein stress, and energy production, indicating a potential limitation for the use of BONCAT in understanding the cell-level response of Synechococcus sp. to environmental change. Variability in HPG sensitivity between algal groups and the impact of HPG on Synechococcus physiology indicates that particular considerations should be taken when applying this technique to other marine taxa or mixed marine microbial communities. IMPORTANCE Phytoplankton form the base of the marine food web and substantially impact global energy and nutrient flow. Marine picocyanobacteria of the genus Synechococcus comprise a large portion of phytoplankton biomass in the ocean and therefore are important model organisms. The technical challenges of environmental proteomics in mixed microbial communities have limited our ability to detect the cell-level adaptations of phytoplankton communities to a changing environment. The proteome labeling technique, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to address some of these challenges by simplifying proteomic analyses. This study explores the ability of marine phytoplankton to take up the modified amino acid, l-homopropargylglycine (HPG), required for BONCAT, and investigates the proteomic response of Synechococcus to HPG. We not only demonstrate that cyanobacteria can take up HPG but also highlight the physiological impact of HPG on Synechococcus, which has implications for future applications of this technique in the marine environment.

Distribution and functional analysis of the two types of 8-vinyl reductase involved in chlorophyll biosynthesis in marine cyanobacteria.

In the chlorophyll biosynthesis pathway, the 8-vinyl group of the chlorophyll precursor is reduced to an ethyl group by 8-vinyl reductase. Two isozymes of 8-vinyl reductase have been described in oxygenic photosynthetic organisms: one encoded by BciA and another by BciB. Only BciB contains an [Fe-S] cluster and most cyanobacteria harbor this form; whereas a few contain BciA. Given this disparity in distribution, cyanobacterial BciA has remained largely overlooked, which has limited understanding of chlorophyll biosynthesis in these microorganisms. Here, we reveal that cyanobacterial BciA encodes a functional 8-vinyl reductase, as evidenced by measuring the in vitro activity of recombinant Synechococcus and Acaryochloris BciA. Genomic comparison revealed that BciB had been replaced by BciA during evolution of the marine cyanobacterium Synechococcus, and coincided with replacement of Fe-superoxide dismutase (SOD) with Ni-SOD. These findings imply that the acquisition of BciA confers an adaptive advantage to cyanobacteria living in low-iron oceanic environments.

Uranium adsorption and oil emulsification by extracellular polysaccharide (EPS) of a halophilic unicellular marine cyanobacterium Synechococcus elongatus BDU130911

An integrated approach on uranium adsorption and emulsification of oils was evaluated using extracellular polysaccharides (EPS) of a marine unicellular cyanobacterium Synechococcus elongatus BDU130911. EPS gel of S. elongatus BDU130911 was able to adsorb uranium at the rate of 75% per gram dry weight. Uranium follows a monolayer adsorption onto EPS gel surface as revealed by Langmuir isotherm. Energy-dispersive X-ray fluorescence (EDXRF) analysis and powder X-ray diffraction analysis further confirms the uranium adsorption to the EPS gel of S. elongatus BDU130911. The culture filtrate of S. elongatus BDU130911 exhibited biosurfactant property and formed a stable emulsion with petrol (EI24 51.2%) due to the presence of uronic acid and rhamnose sugar. These findings suggest that the EPS of S. elongatus BDU130911 biomass has the potential to be used as a green adsorbing agent for the effective extraction of radionucleotide and petroleum hydrocarbons.

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.

Cadmium adsorption to clay-microbe aggregates: Implications for marine heavy metals cycling

Interactions between microorganisms and clay minerals influence the transport and cycling of metal contaminants in both marine and terrestrial environments. The present study was conducted to quantify the adsorption of dissolved cadmium, Cd(II), under seawater-like conditions to the marine cyanobacterium Synechococcus sp. PCC 7002, three common clay minerals (kaolinite, montmorillonite and illite), as well as cell-clay aggregates. We show here that the Synechococcus-only experiments removed the most Cd above pH 5.5, followed in decreasing order by aggregates of 50% cells:50% individual clays, aggregates of cells and all 3 clays, and individual clays. Electron microscope imaging showed that clays associated in a tangential edge-on orientation to the cells in Synechococcus-clay mineral aggregates. A non-electrostatic surface complexation modeling approach was used to fit Cd adsorption onto Synechococcus cells and individual clay minerals. The resulting Cd binding constants were then used in consort with surface functional group pKa values and site concentrations to accurately predict the extent of Cd adsorption onto the Synechococcus-clay mineral aggregates using the component additivity (CA) approach. We observed that the addition of cyanobacterial cells to clay mineral suspensions led to significantly larger mean aggregate sizes of clay minerals, enhancing the clay sedimentation rate. Although specifically focused on Cd, our study indicates that the ratio of bacterial plankton to clay minerals is an important determinant in terms of understanding the rate with which metals are transferred from the water column to the seafloor.

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002.

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.