Cyanobacterium Synechocystis PCC Research Articles

The cyanobacterial FtsH4 protease controls accumulation of protein factors involved in the biogenesis of photosystem I

Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1–4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

One Advantage of Being Polyploid: Prokaryotes of Various Phylogenetic Groups Can Grow in the Absence of an Environmental Phosphate Source at the Expense of Their High Genome Copy Numbers.

Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and cell division. For two polyploid prokaryotic species, the halophilic archaeon Haloferax volcanii and the cyanobacterium Synechocystis PCC 6803, it has been reported that they can grow in the absence of an external phosphate source by reducing the genome copy number per cell. To unravel whether this feature might be widespread in and typical for polyploid prokaryotes, three additional polyploid prokaryotic species were analyzed in the present study, i.e., the alphaproteobacterium Zymomonas mobilis, the gammaproteobacterium Azotobacter vinelandii, and the haloarchaeon Halobacterium salinarum. Polyploid cultures were incubated in the presence and in the absence of external phosphate, growth was recorded, and genome copy numbers per cell were quantified. Limited growth in the absence of phosphate was observed for all three species. Phosphate was added to phosphate-starved cultures to verify that the cells were still viable and growth-competent. Remarkably, stationary-phase cells grown in the absence or presence of phosphate did not become monoploid but stayed oligoploid with about five genome copies per cell. As a negative control, it was shown that monoploid Escherichia coli cultures did not exhibit any growth in the absence of phosphate. Taken together, all five polyploid prokaryotic species that have been characterized until now can grow in the absence of environmental phosphate by reducing their genome copy numbers, indicating that cell proliferation outperforms other evolutionary advantages of polyploidy.

The effect of solvent on the optical properties of myxoxanthophyll from Synechocystis sp. PCC6803

Myxoxanthophylls (myxol 2′-glycosides) are carotenoid glycosides that occur almost exclusively in cyanobacteria. In this study, a fraction containing myxoxanthophylls was isolated from the model cyanobacterium Synechocystis PCC 6803. An HPLC and LC/MS-based analysis revealed that in this organism myxoxanthophyll is present as a mixture of geometrical isomers, with a dominating all-trans form. The absorption maxima as observed in solutions of isolated myxoxanthophyll are amenable to solvent properties, displaying solvent-dependent shifts in the visible region (solvatochromism). In organic solvents, myxoxanthophyll shows the absence of any specific solvent–solute interactions, as the energy of its electronic transitions increases only with solvent polarizability. In contrast, an aqueous environment promotes intermolecular interactions, which are directly manifested as changes in the absorption spectra of myxoxanthophyll. The biological significance of the observed changes in chemical and optical properties of myxoxanthophyll molecules remains to be elucidated.

Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes

BackgroundThe robust model cyanobacterium Synechocystis PCC 6803 is increasingly explored for its potential to use solar energy, water and atmospheric CO2 for the carbon-neutral production of terpenes, the high-value chemicals that can be used for the production of drugs, flavors, fragrances and biofuels. However, as terpenes are chemically diverse, it is extremely difficult to predict whether Synechocystis is a suitable chassis for the photosynthetic production of various terpenes or only a few of them.ResultsWe have performed the first-time engineering and comparative analysis of the best-studied cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of five chemically diverse high-value terpenes: two monoterpenes (C10H16) limonene (cyclic molecule) and pinene (bicyclic), and three sesquiterpenes (C15H24) bisabolene (cyclic), farnesene (linear) and santalene (cyclic). All terpene producers appeared to grow well and to be genetically stable, as shown by the absence of changes in their production levels during the 5–9-month periods of their sub-cultivation under photoautotrophic conditions). We also found that Synechocystis PCC 6803 can efficiently and stably produce farnesene and santalene, which had never been produced before by this model organism or any other cyanobacteria, respectively. Similar production levels were observed for cells growing on nitrate (the standard nitrogen source for cyanobacteria) or urea (cheaper than nitrate). Furthermore, higher levels of farnesene were produced by cloning the heterologous farnesene synthase gene in a RSF1010-derived replicating plasmid as compared to the well-used slr0168 neutral cloning site of the chromosome.Conclusions Altogether, the present results indicate that Synechocystis PCC 6803 is better suited to produce sesquiterpenes (particularly farnesene, the most highly produced terpene of this study) than monoterpenes (especially pinene).

First in vivo analysis of the regulatory protein CP12 of the model cyanobacterium Synechocystis PCC 6803: Biotechnological implications.

We report the first in vivo analysis of a canonical CP12 regulatory protein, namely the unique CP12 of the model cyanobacterium Synechocystis PCC 6803, which has the advantage of being able to grow photoautotrophically, photomixotrophically, and photoheterotrophically. The data showed that CP12 is dispensable to cell growth under standard (continuous) light and light/dark cycle, whereas it is essential for the catabolism of exogenously added glucose that normally sustains cell growth in absence of photosynthesis. Furthermore, to be active in glucose catabolism, CP12 requires its three conserved features: its AWD_VEEL motif and its two pairs of cysteine residues. Also interestingly, CP12 was found to regulate the redox equilibrium of NADPH, an activity involving its AWD_VEEL motif and its C-ter cysteine residues, but not its N-ter cysteine residues. This finding is important because NADPH powers up the methylerythritol 4-phosphate (MEP) pathway that synthesizes the geranyl-diphosphate (GPP) and farnesyl-diphosphate (FPP) metabolites, which can be transformed into high-value terpenes by recombinant cyanobacteria producing plant terpene synthase enzymes. Therefore, we have introduced into the Δcp12 mutant and the wild-type (control) strain our replicative plasmids directing the production of the monoterpene limonene and the sesquiterpene bisabolene. The photosynthetic production of both bisabolene and limonene appeared to be increased (more than two-fold) in the Δcp12 mutant as compared to the WT strain. Furthermore, the level of bisabolene production was also higher to those previously reported for various strains of Synechocystis PCC 6803 growing under standard (non-optimized) photoautotrophic conditions. Hence, the presently described Δcp12 strain with a healthy photoautotrophic growth and an increased capability to produce terpenes, is an attractive cell chassis for further gene manipulations aiming at engineering cyanobacteria for high-level photoproduction of terpenes.

REVIEW ON KNOWLEDGE GAP IN Brachiaria GRASS RESEARCH AND UTILIZATION: ETHIOPIAN PERSPECTIVE

The ability of cyanobacteria and green microalgae to synthesize gold nanoparticles is well known, as is the ability to use these nanoparticles to degrade pollutants (e.g., methyl orange, methylene blue, etc.). In this paper we present our original results on the biosynthesis of gold nanoparticles in both light and dark, by the cyanobacterium Synechocystis PCC 6803, green microalga Chlorella sorokiniana UTEX 1230, as well as our two green microalgae strains temporarily named Ra and Rd. In this way we try to find out the contribution of photosynthesis to the synthesis of nanoparticles, as well as the contribution of aerobic respiration to this process. Up to our best knowledge this is the first report concerning the possibility to influence the intracellular position of nanoparticles by varying the conditions of NP synthesis, namely in our experiments the darkness coupled with anaerobiosis. The synthesized nanoparticles were inspected by TEM, both in situ (on ultra-thin sections) and after extraction. The collected gold nanoparticles were further used as catalysts for the degradation of methylene blue.

Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production

BackgroundCyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO2 and solar energy in a direct process.ResultsHeterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L−1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5′-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L−1, an 177% increase compared to the previously reported best producing strain under identical conditions.ConclusionsThis work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.Graphic abstract

The impact of cell-bound exopolysaccharide on flocculation of the cyanobacterium Synechocystis PCC6803 with ferric chloride and chitosan

The impact of cell-bound exopolysaccharide on flocculation of the cyanobacterium Synechocystis PCC6803 with ferric chloride and chitosan

Semisynthetic [FeFe]-hydrogenase with stable expression and H2 production capacity in a photosynthetic microbe

Hydrogen (H 2 ) is a promising future chemical energy carrier and feedstock with several renewable production options, including electrolyzers and biological/bioinspired systems. The top H 2 producers in nature are [FeFe]-hydrogenases, high turnover metalloenzymes with a complex maturation process that can be circumvented by artificial synthetic activation. Here, we report the expression and activation of group A and D [FeFe]-hydrogenases in a photosynthetic host organism, the unicellular cyanobacterium Synechocystis PCC 6803. The hydrogenase from Solobacterium moorei (group A) facilitates high in vivo H 2 production from purely photoautotrophically generated substrates and unmistakably links to the metabolism of the photosynthetic host. Cells harboring the non-native, semisynthetic enzyme retain their H 2 production capacity for several days after synthetic activation. This work expands both the number and the diversity of [FeFe]-hydrogenases examined in a photosynthetic background and provides important insights for future investigations into the development and understanding of biological and biohybrid H 2 production systems. Artificial activation of newly discovered [FeFe]-hydrogenases expressed in cyanobacteria Stable expression and significant H 2 production under different environmental conditions Activated, semisynthetic [FeFe]-hydrogenase remains functional in vivo for several days [FeFe]-hydrogenases are hydrogen (H 2 )-producing metalloenzymes with excellent catalytic capacities. Photosynthetic cyanobacteria are promising chassis for large-scale H 2 production due to their fast growth and minimalistic substrate requirement. Wegelius et al. use artificial activation to enable sustainable in vivo H 2 production from newly discovered [FeFe]-hydrogenases expressed in a unicellular cyanobacterium.

The availability of neither D2 nor CP43 limits the biogenesis of photosystem II in tobacco.

The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5′-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine–Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Chloramphenicol enhances Photosystem II photodamage in intact cells of the cyanobacterium Synechocystis PCC 6803

The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the cyanobacterium Synechocystis PCC 6803. Light-induced loss of PSII activity was compared in the presence of chloramphenicol and another protein synthesis inhibitor, lincomycin, by measuring the rate of oxygen evolution in Synechocystis 6803 cells. Our data show that the rate of PSII photodamage was significantly enhanced by chloramphenicol, at the usually applied 200 μg mL−1 concentration, relative to that obtained in the presence of lincomycin. Chloramphenicol-induced enhancement of photodamage has been observed earlier in isolated PSII membrane particles, and has been assigned to the damaging effect of chloramphenicol-mediated superoxide production (Rehman et al. 2016, Front Plant Sci 7:479). This effect points to the involvement of superoxide as damaging agent in the presence of chloramphenicol also in Synechocystis cells. The chloramphenicol-induced enhancement of photodamage was observed not only in wild-type Synechocystis 6803, which contains both Photosystem I (PSI) and PSII, but also in a PSI-less mutant which contains only PSII. Importantly, the rate of PSII photodamage was also enhanced by the absence of PSI when compared to that in the wild-type strain under all conditions studied here, i.e., without addition and in the presence of protein synthesis inhibitors. We conclude that chloramphenicol enhances photodamage mostly by its interaction with PSII, leading probably to superoxide production. The presence of PSI is also an important regulatory factor of PSII photodamage most likely via decreasing excitation pressure on PSII.

A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.

Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803.

mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis, RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis, and Chlamydomonas. Moreover, Sy RNase J displays a robust 5′-exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis. We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria.

Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803

BackgroundCyanobacteria can be metabolically engineered to convert CO2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules.ResultsThe efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 ± 3.1 µg mL−1 OD−1 day−1) compared to the control strain (6.4 ± 1.4 µg mL−1 OD−1 day−1). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 ± 1.3 and 18.3 ± 3.3 µg mL−1 OD−1 day−1, respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 ± 4.48 µg mL−1 OD−1 day−1) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis.ConclusionsThis work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.

A Storable Mediatorless Electrochemical Biosensor for Herbicide Detection.

A novel mediatorless photo-bioelectrochemical sensor operated with a biofilm of the cyanobacterium Synechocystis PCC6803 wt. for herbicide detection with long term stability (>20 days) was successfully developed and tested. Photoanodic current generation was obtained in the absence of artificial mediators. The inhibitory effect on photocurrent of three commonly used herbicides (i.e., atrazine, diuron, and paraquat) was used as a means of measuring their concentrations in aqueous solution. The injection of atrazine and diuron into the algal medium caused an immediate photocurrent drop due to the inhibition of photosynthetic electron transport. The detected concentrations were suitable for environmental analysis, as revealed by a comparison with the freshwater quality benchmarks set by the Environmental Protection Agency of the United States (US EPA). In contrast, paraquat caused an initial increase (~2 h) of the photocurrent effect of about 200%, as this compound can act as a redox mediator between the cells and the anode. A relatively long-term stability of the biosensor was demonstrated, by keeping anodes colonized with cyanobacterial biofilm in the dark at 4 °C. After 22 days of storage, the performance in terms of the photocurrent was comparable with the freshly prepared biosensor. This result was confirmed by the measurement of chlorophyll content, which demonstrated preservation of the cyanobacterial biofilm. The capacity of this biosensor to recover after a cold season or other prolonged environmental stresses could be a key advantage in field applications, such as in water bodies and agriculture. This study is a step forward in the biotechnological development and implementation of storable mediatorless electrochemical biosensors for herbicide detection.

From Cyanobacteria to Human, MAPEG-Type Glutathione-S-Transferases Operate in Cell Tolerance to Heat, Cold, and Lipid Peroxidation.

The MAPEG2 sub-family of glutathione-S-transferase proteins (GST) has been poorly investigated in vivo, even in prokaryotes such as cyanobacteria the organisms that are regarded as having developed glutathione-dependent enzymes to protect themselves against the reactive oxygen species (ROS) often produced by their powerful photosynthesis. We report the first in vivo analysis of a cyanobacterial MAPEG2-like protein (Sll1147) in the model cyanobacterium Synechocystis PCC 6803. While Sll1147 is dispensable to cell growth in standard photo-autotrophic conditions, it plays an important role in the resistance to heat and cold, and to n-tertbutyl hydroperoxide (n-tBOOH) that induces lipid peroxidation. These findings suggest that Sll1147 could be involved in membrane fluidity, which is critical for photosynthesis. Attesting its sensitivity to these stresses, the Δsll1147 mutant lacking Sll1147 challenged by heat, cold, or n-tBOOH undergoes transient accumulation of peroxidized lipids and then of reduced and oxidized glutathione. These results are welcome because little is known concerning the signaling and/or protection mechanisms used by cyanobacteria to cope with heat and cold, two inevitable environmental stresses that limit their growth, and thus their production of biomass for our food chain and of biotechnologically interesting chemicals. Also interestingly, the decreased resistance to heat, cold and n-tBOOH of the Δsll1147 mutant could be rescued back to normal (wild-type) levels upon the expression of synthetic MAPEG2-encoding human genes adapted to the cyanobacterial codon usage. These synthetic hmGST2 and hmGST3 genes were also able to increase the Escherichia coli tolerance to heat and n-tBOOH. Collectively, these finding indicate that the activity of the MAPEG2 proteins have been conserved, at least in part, during evolution from (cyano)bacteria to human.

First in vivo Evidence That Glutathione-S-Transferase Operates in Photo-Oxidative Stress in Cyanobacteria.

Although glutathione (GSH) and GSH-dependent enzymes, such as glutathione transferases (GSTs), are thought to have been developed by cyanobacteria to cope with the reactive oxygen species (ROS) that they massively produced by their active photosynthesis, there had been no in vivo analysis of the role of GSTs in cyanobacteria so far. Consequently, we have analyzed two of the six GSTs of the model cyanobacterium Synechocystis PCC 6803, namely Sll1545 (to extend its in vitro study) and Slr0236 (because it is the best homolog to Sll1545). We report that Sll1545 is essential to cell growth in standard photo-autotrophic conditions, whereas Slr0236 is dispensable. Furthermore, both Sll1545 and Slr0236 operate in the protection against stresses triggered by high light, H2O2, menadione and methylene blue. The absence of Slr0236 and the depletion of Sll1545 decrease the tolerance to methylene blue in a cumulative way. Similarly, the combined absence of Slr0236 and depletion of Sll1545 decrease the resistance to high light. Attesting their sensitivity to high-light or methylene blue, these Δslr0236-sll1545 cells transiently accumulate ROS, and then reduced and oxidized glutathione in that order. In contrast, the absence of Slr0236 and the depletion of Sll1545 increase the tolerance to menadione in a cumulative way. This increased menadione resistance is due, at least in part, to the higher level of catalase and/or peroxidase activity of these mutants. Similarly, the increased H2O2 resistance of the Δslr0236-sll1545 cells is due, at least in part, to its higher level of peroxidase activity.

A generalized computational framework to streamline thermodynamics and kinetics analysis of metabolic pathways

Metabolic engineering is a critical biotechnological approach in addressing global energy and environment challenges. Most engineering efforts, however, consist of laborious and inefficient trial-and-error of target pathways, due in part to the lack of methodologies that can comprehensively assess pathway properties in thermodynamics and kinetics. Metabolic engineering can benefit from computational tools that evaluate feasibility, expense and stability of non-natural metabolic pathways. Such tools can also help us understand natural pathways and their regulation at systems level. Here we introduce a computational toolbox, PathParser, which, for the first time, integrates multiple important functions for pathway analysis including thermodynamics analysis, kinetics-based protein cost optimization and robustness analysis. Specifically, PathParser enables optimization of the driving force of a pathway by minimizing the Gibbs free energy of least thermodynamically favorable reaction. In addition, based on reaction thermodynamics and enzyme kinetics, it can compute the minimal enzyme protein cost that supports metabolic flux, and evaluate pathway stability and flux in response to enzyme concentration perturbations. In a demo analysis of the Calvin–Benson–Bassham cycle and photorespiration pathway in the model cyanobacterium Synechocystis PCC 6803, the computation results are corroborated by experimental proteomics data as well as metabolic engineering outcomes. This toolbox may have broad application in metabolic engineering and systems biology in other microbial systems.

Sequential deletions of photosystem II assembly factors Ycf48, Ycf39 and Pam68 result in progressive loss of autotrophy in the cyanobacterium Synechocystis PCC 6803.

The biogenesis of the cyanobacterial photosystem II (PSII) complex requires a number of auxiliary assembly factors that improve efficiency of the process but their precise function is not well understood. To assess a possible synergic action of the Ycf48 and Ycf39 factors acting in early steps of the biogenesis via interaction with the nascent D1 subunit of PSII, we constructed and characterised a double mutant of the cyanobacterium Synechocystis PCC 6803 lacking both these proteins. In addition, we also deleted the ycf39 gene in the double mutant lacking Ycf48 and Pam68, the latter being a ribosomal factor promoting insertion of chlorophyll (Chl) into the CP47 subunit of PSII. The resulting double ΔYcf48/ΔYcf39 and triple ΔYcf48/ΔPam68/ΔYcf39 mutants were deficient in PSII and total Chl, and in contrast to the source mutants, they lost the capacity for autotrophy. Interestingly, autotrophic growth was restored in both of the new multiple mutants by enhancing Chl biosynthesis using a specific ferrochelatase inhibitor. Taking together with the weak radioactive labelling of the D1 protein, these findings can be explained by inhibition of the D1 synthesis caused by the lack and/or incorrect binding of Chl molecules. The results emphasise the key importance of the sufficient Chl supply for the PSII biogenesis and also support the existence of a so far enigmatic regulatory mechanism leading to the reduced overall Chl biosynthesis/accumulation when the PSII assembly is impaired.