The solid-state photo-CIDNP effect

Photosynthesis Research - The solid-state photo-CIDNP effect is the occurrence of a non-Boltzmann nuclear spin polarization in rigid samples upon illumination. For solid-state NMR, which can detect...     

The field-dependence of the solid-state photo-CIDNP effect in two states of heliobacterial reaction centers

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect is studied in photosynthetic reaction centers of Heliobacillus mobilis at different magnetic fields by ¹³C MAS (magic-angle spinning) NMR spectroscopy. Two active states of heliobacterial reaction centers are probed: an anaerobic preparation of heliochromatophores (“Braunstoff”, German for “brown substance”) as well as a preparation of cells after exposure to oxygen (“Grünstoff”, “green substance”). Braunstoff shows significant increase of enhanced absorptive (positive) signals toward lower magnetic fields, which is interpreted in terms of an enhanced differential relaxation (DR) mechanism. In Grünstoff, the signals remain emissive (negative) at two fields, confirming that the influence of the DR mechanism is comparably low.     

Orientation of carotenoids in the outer membrane of Synechocystis PCC 6714 (cyanobacteria)

The orientation of outer membrane carotenoids from Synechocystis PCC 6714 and Synechococcus PCC 6307 was studied by linear dichroism spectrophotometry. Uniaxially oriented, tilted outer membrane films revealed a significant linear dichroism after rotating the polarization vector of the incident light beam, indicating a predominant orientation of the carotenoid transition moments perpendicular to the outer membrane plane. Values for the reduced dichroism at the absorbance maxima presented a linear correlation to a function of the tilt angle (sin2 alpha).     

Study of the functional role of Ctp-family proteins in Synechocystis sp. PCC 6803 cyanobacteria

To understand the functional role of CtpB and CtpC proteins, which are similar to the C-terminal processing CtpA peptidase, the effect of the insertional inactivation of the ctpB and ctpC genes on the phenotypic characteristics of Synechocystis sp. PCC 6803 was studied. The inactivation of the ctpC gene was found to be lethal to the cyanobacterium, which indicates a vital role of the CtpC protein. The mutant with the inactivated ctpB gene had the same photosynthetic characteristics as the wild-type strain. The double mutant@[delta]ctpA delta ctpB with the two deleted genes was identical, in the phenotypic characteristics, to the mutant with a knock-out mutation in the ctpA gene, which was unable to grow photoautotrophically. The data obtained suggest that, in spite of the high similarity of the Ctp proteins, they serve different functions in Synechocystis sp. PCC 6803 cells and cannot compensate for each other.     

Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp.

Abstract The effect of the nitrogen source on the cellular activity level of assimilatory nitrate reductase in the cyanobacteria Anabaena variabilis (ATCC29413) and Synechocystis sp. (PCC6714) has been examined. In the filamentous N₂-fixing A. variabilis , nitrate behaved as a nutritional inducer of nitrate reductase, with ammonium acting (via products of its assimilation) as an antagonist with regard to nitrate. Ammonium-promoted repression of nitrate reductase was also evident in the unicellular non-nitrogen fixer Synechocystis , but in this strain nitrate was not required as an obligatory inducer.     

State 1-state 2 adaptation in the cyanobacteria Synechocystis PCC 6714 wild type and Synechocystis PCC 6803 wild type and phycocyanin-less mutant

The mechanism of excitation energy distribution between the two photosystems (state transitions) is studied in Synechocystis 6714 wild type and in wild type and a mutant lacking phycocyanin of Synechocystis 6803. (i) Measurements of fluorescence transients and spectra demonstrate that state transitions in these cyanobacteria are controlled by changes in the efficiency of energy transfer from PS II to PS I (spillover) rather than by changes in association of the phycobilisomes to PS II (mobile antenna model). (ii) Ultrastructural study (freeze-fracture) shows that in the mutant the alignment of the PS II associated EF particles is prevalent in state 1 while the conversion to state 2 results in randomization of the EF particle distribution, as already observed in the wild type (Olive et al. 1986). In the mutant, the distance between the EF particle rows is smaller than in the wild type, probably because of the reduced size of the phycobilisomes. Since a parallel increase of spillover is not observed we suggest that the probability of excitation transfer between PS II units and between PS II and PS I depends on the mutual orientation of the photosystems rather than on their distance. (iii) Measurements of the redox state of the plastoquinone pool in state 1 obtained by PS I illumination and in state 2 obtained by various treatments (darkness, anaerobiosis and starvation) show that the plastoquinone pool is oxidized in state 1 and reduced in state 2 except in starved cells where it is still oxidized. In the latter case, no important decrease of ATP was observed. Thus, we propose that in Synechocystis the primary control of the state transitions is the redox state of a component of the cytochrome b ₆/f complex rather than that of the plastoquinone pool.Abbreviations DCCD dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EF exoplasmic face - PQ plasto-quinone - PS photosystem - PBS phycobilisome     

A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis

A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed. pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA. Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E. coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.Service de Biochimie et Génétique Moléculaire     

Engineering a platform for photosynthetic isoprene production in cyanobacteria,using Synechocystis as the model organism

The concept of “photosynthetic biofuels” envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO₂ and H₂O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering and processing of the respective biomass.     

Mossbauer spectroscopy of cells of cyanobacteria Synechocystis sp. PCC 6803 devoid of photosystem I and containing inactive phycobilisomes]

Mossbauer spectra of the psaAB mutant of Synechocystis sp. PPC 6803 devoid of photosystem I grown in a 57Fe-containing medium were measured. The spectrum is a broadened doublet whose size (about 20%) and parameters (isomeric shift delta = 0.3 mm/s and quadrupole splitting delta = 0.8 mm/s) suggest the presence of abundant nanoclusters of Fe3+ oxides in a superparamagnetic state tightly bound to the membrane. Treatment of cells with EDTA was accompanied by a substantial (tenfold) decrease in the amount of iron nonspecifically bound to the membrane and the appearance of Fe2+ localized, probably, inside cells and/or cell membranes. In addition, the spectrum of washed cells exhibited superfine magnetic splitting due to iron oxide clusters greater in size than nanoclusters present in the membrane prior to EDTA treatment.     

Sulphated exopolysaccharides produced by two unicellular strains of cyanobacteria,Synechocystis PCC 6803 and 6714

The exopolysaccharides (EPS) of two unicellular strains of cyanobacteria Synechocystis PCC 6803 and 6714, formed labile, radial structures, uniformly distributed on the cell surface, and stainable by specific dyes for acidic polysaccharides. The two strains produced EPS at similar rates, which depended, along with the duration of the producing phase, on the incubation conditions. The exopolysaccharides from both strains were constituted of at least 11–12 mono-oses, probably forming several types of polymers. They contained about 15–20% (w/w) uronic derivatives and 10–15% (w/w) osamines. Proteins represented 20–40% of total weight. A most interesting feature was the presence of 7–8% (molar ratio) sulphate residues, a characteristic that is otherwise limited to exopolysaccharides produced by eukaryotic algae.Abbreviations EPS exopolysaccharides - KDO 3-deoxy-d-mannooctulosonate - LPS lipopolysaccharides     

Site-directed mutagenesis on the heme axial-ligands of cytochrome b559 in photosystem II by using cyanobacteria Synechocystis PCC 6803

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the alpha or the beta subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kalpha mutant grew photoautotrophically, and accumulated stable PSII reaction centers ( approximately 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Ybeta mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers ( approximately 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559alpha and beta polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559beta polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YbetaPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YbetaPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YbetaPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kalpha mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kalpha mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kalpha and H22YbetaPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.     

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Summary Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10^–4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation. Correspondence to: K. Sode     

Targeted, PCR-based gene disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid synthase genes in Synechocystis sp. PCC6803

A PCR-based method is described for the efficient construction of targeted gene disruptions and gene fusions in the cyanobacterium Synechocystis sp. PCC6803. In a simple two-step PCR approach, a gene conversion cassette was synthesized targeting the polyhydroxyalkanoic acid (PHA) synthase genes. Upon transformation, PHA production in Synechocystis under normal as well as high production culture conditions was undetectable. The application of this method to the genetic inactivation of the phaE-C _Syn gene cluster demonstrates its potential for genetic engineering of cyanobacteria and the study of functional genomics in Synechocystis. Received: 3 March 2000 / Received revision: 1 June 2000 / Accepted: 9 June 2000     

Cloning the alpha-amylase gene of Bacillus amyloliquefaciens in cyanobacteria cells

The recombinant plasmids of pIAH4amy series were constructed containing the alpha-amylase gene of Bacillus amyloliquefaciens A50 with its own promoter and leading sequence within an integrative vector plasmid pIAH4 (CmR) for cyanobacterium Anacystis nidulans R2. At Anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all CmR transformants produce alpha-amylase. Expression of bacillar alpha-amylase gene in cyanobacterium cells is independent of the cloned gene orientation in the vector plasmid. Secretion of alpha-amylase into the cyanobacterial periplasm has been demonstrated.     

Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria

A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled alpha-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote.     

Blooming of cyanobacteria in turbulent water with steep light gradients: The effect of intermittent light and dark periods on the oxygen evolution capacity of Synechocystis sp. PCC 6803

Abstract: The influence of intermittent high-light dosage on Synechocystis sp. PCC 6803 with respect to oxygen evolution capacity, fluorescence yield and carotenoid pigment pattern was investigated, using high-light- and low-light-adapted cultures. The results showed that this cyanobacterium was able to survive high light stress for a full day if this stress was applied on and off with intermittently presented recovery periods in darkness. Enhanced respiratory activity in the high-light adapted cells was detected and this may be an important factor in preventing photodamage under high light stress. Cyanobacterial photosynthetic and respiratory electron transfer pathways are both present within the same membrane, and share common electron carriers. The role of respiratory activity in preventing overexcitation of photosystem 2 is discussed with regard to cyanobacterial ecology.     

Production of H2 by sulphur-deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or at various extracellular pH

AIMS: To examine sulphur (S) deprivation in combination with the presence of methane (CH4) and changes in extracellular pH as a method to enhance in situ hydrogen (H2) generation during fermentation in the unicellular non-diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803. METHODS AND RESULTS: The level of H2 production, measured using a gas chromatography, was determined in S-deprived cells of G. alpicola and Synechocystis PCC 6803 during fermentation. Starvation on S enhanced the rate of H2 production by more than fourfold in both strains. S-deprived cyanobacteria were able to maintain maximum rate of H2 production during at least 8 h of fermentation representing the entire dark period of a day. Increased H2 production was observed during dark anoxic incubation with a gas phase of 100% CH4 (up to four times) at lower pH of the medium (5.0-5.5). CONCLUSIONS: S-deprivation in combination with CH4, added or maybe produced by another micro-organisms, and changes in the pH of the media can be used to further increase the specific capacity of unicellular non-N2-fixing cyanobacteria to produce H2 during fermentation with the overall aim of applying it for outdoor photobiological H2 production. SIGNIFICANCE AND IMPACT OF THE STUDY: S-deprivation with respect to H2 production is well studied in the green algae Chlamydomonas reinhardtii while its application for H2 production in cyanobacteria is novel. Similarly, the stimulation of H2 generation in the presence of CH4 opens up new possibilities to increase the H2 production. Natural gas enriched with H2 seems to be a perspective fuel and may be an intermediate step on the pathway to the exploitation of pure biohydrogen.