Abstract: Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl- and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and they were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process that was also dependent on the CAAD domain.
Abstract: Pulp and paper mills typically generate significant quantities of non-hazardous solid waste which require management as a waste material or as a by-product. Most of these solids are removed after primary mechanical treatment, resulting in a sludge that contains large quantities of fibers, papermaking fillers, or both. Although this primary sludge is commonly landfilled, it could be recycled into production on-site, reused in other pulp and paper mills, or used in other products. In an effort to explore these possibilities, the fiber content, fiber quality, and key physical and chemical properties (humidity, ash content, abrasiveness, drainability, and O2 uptake) of 20 different primary sludges sampled in European mills are reviewed in this paper. Although sludge characteristics are highly variable across pulp and paper mill processes, sludges can be considered to fall into two main types: high-ash sludge (>30% dry weight) and low-ash sludge (<30% dry weight). Results of paper tests (caliper, breaking length, tear index, elongation, bursting strength, stiffness, opacity, whiteness, and porosity) and board tests (ring crush test, Concora medium test, corrugated crush test) suggest that at least 12 of the sludges studied could be reused in the paper and board industry.
The results make it possible to differentiate three primary sludge grades: the first needs little cleaning and has appropriate strength properties to be a component of printing and writing papers, tissues, and wrapping papers; the second requires cleaning, bleaching, or both and has appropriate strength properties for applications that do not require high brightness, such as corrugated board, boxboard, and some tissue grades; the third requires cleaning and has limited strength properties, but could be used in some mills that operate using closed water cycles because the final product can tolerate a certain degree of dirt and contamination, as in some packaging and construction-paper grades. Primary sludges share several features in common with recycled paper, and therefore these two materials could be managed together.
Abstract: A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.
Abstract: This contribution is a synthesis of the final technical report of the Project âApplications of recycled paper mills effluents to wood substitutive products -RESPRO-â which was funded by the European Commission under the Brite/Euram program. The Project increased alternative management of primary sludge from pulp and paper mills. Particularly, those non-destructive alternatives related to the reutilization of sludges in pulp and paper industry and to the recycling of this waste in the wood sector. The main objective of the project was to study the technical feasibility of these alternatives at a precompetitive level.
Abstract: Adenylyl (ACs) and guanylyl cyclases (GCs) synthesize the cyclic nucleotides cAMP and cGMP, playing a key role in signal transduction both in prokaryotes and eukaryotes. Some cyanobacteria produce cAMP and cGMP and their concentrations fluctuate depending on nutrients and oxygen availability, as well as on light conditions. Advances in cyanobacterial genome-sequencing projects reveal the existence of an increasing number of putative ACs and GCs which all belong to the so-called universal Class III of AC/GCs. We describe the complete sets of these purine nucleotide cyclases found in the available genomes, and analyze them by primary sequence alignment, molecular architecture and phylogenetic methods. As a rule, ACs and GCs are more abundant i) in fresh water than in marine cyanobacteria, ii) in filamentous than in unicellular cyanobacteria and iii) in diazotrophic than in non-diazotrophic ones. The filamentous diazotrophic marine cyanobacterium Trichodesmium erythraeum IMS101 contains 11 putative ACs and 2 putative GCs. This suggests a relationship between the ecophysiology of this bacteria and the cyclic nucleotide transduction pathway that they contain. Our analysis allows us to derive a systematic nomenclature for cyanobacterial purine nucleotide cyclases that will require a further precise biochemical characterization, in particular for the most divergent sequences, to confirm their putative functions. In cyanobacteria, the repertoire of ACs and GCs is likely the result of deletions, gene duplication, protein fusion and fission events, as well as mutation in the residues that determine the specificity for ATP and GTP. As a consequence, AC isoforms are frequently present in cyanobacteria as well as the presence of ACs and GCs domains at the C-terminal of multienzyme polypeptides. Altogether, the results suggest that cyanobacteria could be the simplest model organisms to study the system complexity of cyclic nucleotide signaling pathways.
Abstract: In unicellular non-diazotrophic cyanobacteria, NblA is a small polypeptide required for phycobilisome degradation during macronutrient limitation. In the filamentous N(2)-fixing Tolypothrix sp., a nblA gene (nblAI) lies upstream of the cpeBA operon that encodes phycoerythrin apoproteins. Using a specific anti-NblAI antibody it was found that in strains of Tolypothrix sp. NblAI abundance increases under nitrogen-limiting conditions but the protein is also present in cells grown in nitrogen-replete medium. Gold immunolabelling experiments showed that, upon a nitrogen shift-down, NblAI is preferentially located in the differentiated heterocysts, where O(2) evolution has to be shut off for nitrogenase to operate. The results lead to the proposal that NblAI is a necessary 'cofactor' but not the triggering factor that governs phycobilisome degradation in Tolypothrix sp.
Abstract: Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His-tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co-purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non-inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.
Abstract: In the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red-light-grown cells as an alkaline phosphatase-sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli-overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co-transcription of both genes was demonstrated by reverse transcription (RT)-PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red-light shifts. RcaD seems to be a co-activator co-ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.
Abstract: Cyanobacteria modulate intracellular levels of cAMP and cGMP in response to environmental conditions (light, nutrients and pH). In an attempt to identify components of the cAMP and cGMP signalling pathways in Synechocystis PCC 6803, the authors screened its complete genome sequence by using bioinformatic tools and data from sequence-function studies performed on both eukaryotic and prokaryotic cAMP/cGMP-dependent proteins. Sll1624 and Slr2100 were tentatively assigned as being two putative cyclic nucleotide phosphodiesterases. Five proteins were identified as having all the determinants required to be cyclic nucleotide receptors, two of them being probably more specific for cGMP (an element of two-component regulatory systems - Slr2104 - and a putative cyclic-nucleotide-gated cation channel - Slr1575), the three others being probably more specific for cAMP: (i) a protein of unidentified function (Slr0842); (ii) a putative cyclic-nucleotide-modulated permease (Slr0593), previously annotated as a kinase A regulatory subunit; and (iii) a putative transcription factor (CRP-SYN: =Sll1371), which possesses cAMP- and DNA-binding determinants homologous to those of the cAMP receptor protein of Escherichia coli (CRP-EC:). This homology, together with the presence in Synechocystis of CRP-EC:-like binding sites upstream of crp, cya1, slr1575, and several genes encoding enzymes involved in transport and metabolism, strongly suggests that CRP-SYN: is a global regulator.
Abstract: Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.
Abstract: In the non-N2-fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH-I-pCl1), N starvation induced an increase in the rate of respiration and a decrease in the rate of O2 evolution. When NO3â was added to illuminated N-starved cells, O2 evolution immediately increased to levels shown by NO3â grown cells, even though N-starved cells had lost most of their in vitro photosynthetic activities. Stimulation of noncyclic electron flow was maximal under light-saturating conditions and after 2â3 days of N starvation. The respiratory rate of N-starved cells was stimulated by the addition of NO3â or NH4+ and partially inhibited at very low irradiances, even in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Results indicate that N-starved cells obtain the energy supply for N assimilation through a process different from that used by N-sufficient cells. N-starved cells were able to take up NO3â in the dark and when illuminated in the presence of DCMU under anaerobiosis. Following NO3â addition, the photosynthetic yield of the in vivo noncyclic electron transport slightly increased, whereas it decreased after NH4+ addition. Addition of NO3â or NH4+ favored photoinhibition of photosystem II, the effect being faster after NH4+ addition.
Abstract: In an attempt to clarify the interactions between the available nitrogen source and the photosystems in cyanobacteria, O2 exchange and fluorescence emission were monitored in spheroplasts and intact cells of the non N2-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl1) growing on different nitrogen sources or in the absence of nitrogen. Short-term (time scale of seconds to minutes), NH+4 addition to NOâ3-growing or N-starved cells and, to a minor extent, NOâ3addition to N-starved cells, induced state 2 transitions both in light and dark. Long term (time scale of days), the fluorescence yield of PSII relative to that of PSII at 77 K was higher in NOâ3- than in NH+4 growing cells, and even higher in N-starved cells. In the dark, the plastoquinone pool was more reduced in NH+4- than in NOâ3-growing cells. Both PSII and PSI activities and the degree of linking between both photosystems were affected in the long term, so that non-cyclic electron transport decreased in parallel to the ferredoxin requirement to assimilate each nitrogen source. Results indicate that nitrogen metabolism exerts short- and long-term control over the photosynthetic apparatus, which acclimates to the energy requirement of the available nitrogen source.
Abstract: In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.
Abstract: The effect of NH4+ addition to NO3â-growing cells of the non-N2-fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH-1-pCl1) on photo-synthetic and respiratory electron transport as well as on the intracellular levels of amino acids and some organic acids was studied. Addition of ammonium to nitrate-growing cells resulted in substantial increases in the pool size of most amino acids and a transient decrease in the pool size of organic acids. The high demand for organic acids was partially overcome by degradation of stored carbohydrates, more than by newly fixed carbon, as indicated by the large stimulation of the respiration rate upon ammonium addition. Following ammonium addition, the photosynthetic yield of the in vivo noncyclic electron transport decreased, and the sensitivity of photosystem II to photodamage increased. Results indicate that cells balance their photosynthetic and respiratory activities depending on nitrogen availability and point to an important involvement of respiration in providing energy for ammonium assimilation until adaptation of bioenergetic processes to the new nitrogen source is complete
Abstract: Experiments of turbidity decrease induced by detergents were systematically performed to characterize the solubilization of Phormidium laminosum membrane fragments. SDS, Triton X-100 and a mixture of octyl glucoside/decyl maltoside/lithium dodecyl sulfate (OG/DM/LiDS, in a molar ratio of 4.19:2.54:1) were used. The detergent concentration in the aqueous phase (DW) and the effective detergent/chlorophyll ratio in mixed aggregates (Re) were determined. Both parameters increased during the solubilization and in an exponential way in the range from 10 to 90% solubilization. At detergent concentrations which caused the complete solubilization, Dw values were close to the described critical micellar concentrations (cmc), but solubilization started at concentrations well below the cmc. At the onset of solubilization five molecules of SDS, one of Triton X-100 and three of the mixture OG/DM/LiDS, per chlorophyll molecule, saturated the membrane fragments. The increase of Dw and Re values was characterized by two constants. This permits the design of a model to predict the detergent concentration which produces a desired solubilization of thylakoid membrane fragments for a given chlorophyll concentration.
Abstract: A method has been designed for the quantification of 2-oxoglutarate after ion-exclusion high performance liquid chromatography of samples containing high levels of citrate plus isocitrate, which interfere in the quantification of 2-oxoglutarate. The amount of 2-oxoglutarate in a mixed chromatographic peak containing citrate plus isocitrate has been estimated using a diode array detector and calculating the extinction coefficient of these molecules at three wavelengths. The reliability of the method has been ascertained using mixtures of standard organic acids. This procedure has been applied to determine the intracellular content of 2-oxoglutarate in the cyanobacterium Phormidium laminosum.
Abstract: This document is a synthesis report from a research project carried out under CRAFT which is the area of the Industrial and Materials Technologies programme providing assistance to small and medium-sized enterprises (SMEs). It has been submitted directly by the project's consortium. It details the results of the project and sketches the consortium's follow-up intentions. For more information on this project prior to down-loading the synthesis report, click on the hyperlinked project ref. number in the table below to go directly to the relevant record in the CORDIS Projects database.
Abstract: LA DEFICIENCIA DE NITROGENO MODIFICA EL TRANSPORTE
ELECTRONICO FOTOSINTETICO. LA PRINCIPAL MODIFICACION SE
EJERCE SOBRE EL FOTOSISTEMA II, QUE ES INACTIVADO A DOS
NIVELES. EL PRIMERO ES LA DISMINUCION DE LA SECCION
OPTICA TRANSVERSAL DE ABSORCION LUMINOSA, PROMOVIDA
FUNDAMENTALMENTE POR LA DEGRADACION DE LOS FICOBILISOMAS,
Y EL SEGUNDO ES LA DISMINUCION DE LA VELOCIDAD DE
RECAMBIO ENTRE LA QB REDUCIDA Y LA PQ OXIDADA. LA
FOTOSINTESIS NO CICLICA ES SUSTITUIDA POR UN TRANSPORTE
ELECTRONICO FOTOSINTETICO QUE TRANSCURRIRIA DESDE EL
PODER REDUCTOR (NAD (P) H) GENERADO DURANTE LA GLUCOLISIS
Y LA RUTA OXIDATIVA DE PENTOSAS FOSFATO, HASTA LA
FERREDOXINA, A TRAVES DEL PSI. MEDIANTE ESTE TRANSPORTE
ELECTRONICO LA CELULA DISPONE DE UN SISTEMA MUY EFICAZ DE
PRODUCCION DE PODER REDUCTOR Y ATP, FAVORECIDO POR LA
DEGRADACION DE LOS CARBOHIDRATOS ACUMULADOS DURANTE EL
PERIODO DE DEFICIENCIA DE NITROGENO. LA ALTA AFINIDAD DEL
PSI POR LA LUZ POSIBILITA QUE LA ASIMILACION DE NITRATO
EN CELULAS DEFICIENTES SE LLEVE A CABO A BAJAS
INTENSIDADES LUMINOSAS. LA DISMINUCION DE LA SECCION
OPTICA TRANSVERSAL DEL PSII IMPIDE QUE SE PRODUZCA LA
FOTOINHIBICION DE ESTE. LAS VARIACIONES EN EL METABOLISMO
DEL NITROGENO EJERCEN UN EFECTO, TANTO A CORTO COMO A
LARGO PLAZO, SOBRE LAS CARACTERISTICAS ESPECTRALES DEL
APARATO FOTOSINTETICO, MODULANDO LA LUZ QUE ABSORBE CADA
FOTOSISTEMA Y, EN CONSECUENCIA, EL TRANSPORTE ELECTRONICO
FOTOSINTETICO.
