Increase in the rate of photosynthetic linear electron transport in cyanobacteruim <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

Spectrally decomposed dark-to-light transitions in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB–PSII complexes with closed (saturated) RCs, a quenched or open PB–PSII complex, and a PB–PSII ‘not fully closed.’ For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB–PSII–PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.     

Transition from exponential to linear photoautotrophic growth changes the physiology of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Phototrophic microorganisms like cyanobacteria show growth curves in batch culture that differ from the corresponding growth curves of chemotrophic bacteria. Instead of the usual three phases, i.e., lag-, log-, and stationary phase, phototrophs display four distinct phases. The extra growth phase is a phase of linear, light-limited growth that follows the exponential phase and is often ignored as being different. Results of this study demonstrate marked growth phase-dependent alterations in the photophysiology of the cyanobacterium Synechocystis sp. PCC 6803 between cells harvested from the exponential and the linear growth phase. Notable differences are a gradual shift in the energy transfer of the light-harvesting phycobilisomes to the photosystems and a distinct change in the redox state of the plastoquinone pool. These differences will likely affect the result of physiological studies and the efficiency of product formation of Synechocystis in biotechnological applications. Our study also demonstrates that the length of the period of exponential growth can be extended by a gradually stronger incident light intensity that matches the increase of the cell density of the culture.     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

Efficient harvesting of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 with filamentous fungal pellets

Cyanobacteria play a major role as direct producers of biofuels, such as ethanol and butanol, with the aid of genetic engineering. However, development of a new harvesting-technology is essential to achieve economic viability of biofuel production from cyanobacteria. In this study, we demonstrated the feasibility of harvesting the unicellular cyanobacterium Synechocystis sp. PCC 6803 using pre-made filamentous fungal pellets and investigated key factors affecting efficiency of harvest, including fungal strain, pellet quantity (number of pellets), initial pH, and organic carbon source. Synechocystis sp. PCC 6803 cells attached to Aspergillus oryzae pellets, indicating that this fungal pellet had a desirable harvesting effect, while Rhizopus oryzae pellets had no effect on harvesting. Increasing pellet quantity and adding organic carbon sources, such as glucose and xylose, improved the harvesting efficiency of Aspergillus oryzae pellet; efficiency was not affected by the initial pH.     

Comparison of the Photosynthetic Yield of Cyanobacteria and Green Algae: Different Methods Give Different Answers

The societal importance of renewable carbon-based commodities and energy carriers has elicited a particular interest for high performance phototrophic microorganisms. Selection of optimal strains is often based on direct comparison under laboratory conditions of maximal growth rate or additional valued features such as lipid content. Instead of reporting growth rate in culture, estimation of photosynthetic efficiency (quantum yield of PSII) by pulse-amplitude modulated (PAM) fluorimetry is an often applied alternative method. Here we compared the quantum yield of PSII and the photonic yield on biomass for the green alga Chlorella sorokiniana 211-8K and the cyanobacterium Synechocystis sp. PCC 6803. Our data demonstrate that the PAM technique inherently underestimates the photosynthetic efficiency of cyanobacteria by rendering a high F₀ and a low F_M, specifically after the commonly practiced dark pre-incubation before a yield measurement. Yet when comparing the calculated biomass yield on light in continuous culture experiments, we obtained nearly equal values for both species. Using mutants of Synechocystis sp. PCC 6803, we analyzed the factors that compromise its PAM-based quantum yield measurements. We will discuss the role of dark respiratory activity, fluorescence emission from the phycobilisomes, and the Mehler-like reaction. Based on the above observations we recommend that PAM measurements in cyanobacteria are interpreted only qualitatively.     

Production of gold nanoparticles by biogenesis using bacteria

Diazotrophic cyanobacteria Anabaena sp. PCC 7120, four Nostoc strains, and two Azotobacter species (A. vinelandii and A. chroococcum) were found to produce gold nanoparticles (GNP) under nitrogen fixation conditions. GNP biogenesis occurred at AuHCl₄ concentrations from 0.1 to 1 mM. In the cultures of unicellular cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 incapable of nitrogen fixation, no GNP were formed at the same concentrations of gold salts. The plasmon resonance band peak was located at 552 nm. This position is characteristic of spherical GNP 10 to 30 nm in size. Small amounts of GNP were also formed in the culture liquid supernatants of the tested nitrogen-fixing bacteria at AuHCl₄ concentrations from 0.25 to 0.5 mM.     

Co-regulation of photosynthetic processes under potassium deficiency across CO<Subscript>2</Subscript> levels in soybean: mechanisms of limitations and adaptations

The slow kinetic phases of the chlorophyll a fluorescence transient (induction) are valuable tools in studying dynamic regulation of light harvesting, light energy distribution between photosystems, and heat dissipation in photosynthetic organisms. However, the origin of these phases are not yet fully understood. This is especially true in the case of prokaryotic oxygenic photoautotrophs, the cyanobacteria. To understand the origin of the slowest (tens of minutes) kinetic phase, the M–T fluorescence decline, in the context of light acclimation of these globally important microorganisms, we have compared spectrally resolved fluorescence induction data from the wild type Synechocystis sp. PCC 6803 cells, using orange (λ?=?593 nm) actinic light, with those of mutants, ΔapcD and ΔOCP, that are unable to perform either state transition or fluorescence quenching by orange carotenoid protein (OCP), respectively. Our results suggest a multiple origin of the M–T decline and reveal a complex interplay of various known regulatory processes in maintaining the redox homeostasis of a cyanobacterial cell. In addition, they lead us to suggest that a new type of regulatory process, operating on the timescale of minutes to hours, is involved in dissipating excess light energy in cyanobacteria.     

Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells

Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. The intracellular thylakoid membranes of cyanobacteria harbor the protein complexes of the photosynthetic electron transport chain (Nowaczyk et al., 2010; Bernat and Rögner, 2011). The photosynthetic electron transport chain is composed of three large membrane protein complexes, i.e. PSII, the cytochrome b₆f complex, and PSI. Excitation energy trapping by PSII results in water splitting at the PSII donor side within the thylakoid lumen and transport of electrons to the primary and secondary electron accepting quinone molecules Q_A and Q_B, respectively. Following double reduction and protonation, Q_B is released from PSII into the plastoquinone (PQ) pool and delivers electrons to the cytochrome b₆f complex. The cytochrome b₆f complex transfers the electrons to the soluble electron carrier plastocyanin or cytochrome c₆, which subsequently reduces PSI. For efficient light harvesting, cyanobacteria contain soluble light-harvesting antenna proteins, the phycobilisomes (PBSs), which transfer light energy to the photosynthetic reaction centers. In cyanobacteria, the PSI-to-PSII ratio is controlled by light and by the redox state of the PQ/PQH₂-pool (Fujita et al., 1987), and under high-light growth conditions, typically less PSI is present in cyanobacterial thylakoid membranes compared with low-light conditions (Fujita et al., 1994).Thylakoid membranes and photosynthetic electron transport are essential for phototrophic growth of cyanobacterial cells. Despite their importance for survival of cyanobacteria, biogenesis of thylakoid membranes is yet not well understood. It still is an ongoing debate whether the internal membrane systems (cytoplasmic and thylakoid membranes) are connected in cyanobacteria or not, and thus whether thylakoids represent a completely separated membrane entity (Liberton et al., 2006; van de Meene et al., 2006, 2012; Schneider et al., 2007). Up to now, only few proteins have been described to be involved in thylakoid membrane biogenesis. Among them the Vipp1 protein (vesicle inducing protein in plastids1) seems to play an important role in thylakoid membrane biogenesis, as in chloroplasts of Arabidopsis (Arabidopsis thaliana) and in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), depletion of Vipp1 results in a reduced thylakoid membrane system (Kroll et al., 2001; Westphal et al., 2001). While the exact physiological function of the protein is not yet known (Vothknecht et al., 2012), depletion of Vipp1 in Synechocystis not only results in reduced thylakoid membrane formation, but also affects the activity and structure of components of the photosynthetic electron transport chain (Fuhrmann et al., 2009; Gao and Xu, 2009).As complexes of the respiratory electron transport chain are also localized in cyanobacterial thylakoids, the photosynthetic and respiratory electron transport pathways are highly interconnected and both contribute to formation of an electrochemical gradient across the thylakoid membrane and energy production. Due to this, Synechocystis is able to grow completely heterotrophically under light-activated photoheterotrophic growth (LAHG) conditions in the presence of high Glc concentrations (Anderson and McIntosh, 1991; Smart et al., 1991).In this study, we have used dark-grown Synechocystis cells to investigate “greening” of Synechocystis cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions. Following transfer of Synechocystis cells into the light, complete restoration of a typical thylakoid membrane system was observed within 24 h. While dark-grown Synechocystis cells contained only rudimentary thylakoid membranes, they still contained a high concentration of PBSs, active PSI as well as inactive PSII complexes. Activation of PSII complexes appears to be linked to the biogenesis of organized thylakoid membrane pairs.     

<Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803 cells heterologously expressing bacterial tyrosine ammonia lyase can use exogenous tyrosine for <Emphasis Type="Italic">p</Emphasis>-coumaric acid production

Phototrophic cyanobacteria may be considered as an alternative host for producing numerous bioactive compounds. We demonstrate that the Synechocystis PCC 6803 expressing tyrosine ammonia-lyase from Rhodobacter sphaeroides under Ptrc1O promoter produce p-coumaric acid at a rate three times higher than that under Ptrc1Ocore promoter, accounting for 18.4 ± 1.5 μg of p-coumaric acid per 10⁸ cells (0.36 mg L^?1). Additionally, our study is the first report to show the biotransformation of tyrosine to p-coumaric acid reaching a maximum 2.4-fold increase when 0.5 mM tyrosine was supplemented to the growth medium. Liquid chromatography-mass spectrometry analysis revealed the occurrence of diverse patterns of metabolites under different concentrations of supplemented tyrosine, suggesting that it is used in additional metabolic pathways.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Biogenesis of chlorophyll-binding proteins under iron stress in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesize chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or-deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Isolation and Characterization of Toxic Cyanobacteria from Different Natural Sources

We isolated seven algologically and five bacteriologically pure cultures of toxin-producing cyanobacteria from Turgen gorge (Kazakhstan), Karlovy Vary (Czech Republic), and Shar-Nuur Lake, Bayan Ulgiiregion (Mongolia) springs. According to the Daphnia magna test, Desertifilum sp. and Nostoc sp. strains were the most toxic in the test of isolated strains (complete death of all test organisms was detected after 48 h). These strains possessed the highest inhibitory effect on proliferation of the HeLa cancer cell line. The Anabaena sp. 35 and Nostoc sp. 4 strains were also high toxic. Model strains Synechocystis PCC 6803 and Synechococcus elongates PCC 7942, as well as the strain isolated in the present work, Synechococcus sp. 55, were less toxic. Mass spectrometry made it possible to assign cyanobacterial toxins to cyclic depsipeptides. Two cyclic depsipeptides, micropeptin T and oscillapeptin, were detected in Desertifilum sp. extracts. Cryptophycin and small amounts of cyclic depsipeptide micropeptin SD were detected in Nostoc sp. extract.