RESUMO
Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.
Assuntos
Proteínas de Algas/metabolismo , Clorófitas/metabolismo , Amido/metabolismo , Proteínas de Algas/genética , Sequência de Aminoácidos , Amilopectina/metabolismo , Amilose/metabolismo , Clorófitas/enzimologia , Clorófitas/genética , Clonagem Molecular , Plastídeos , Ligação Proteica , Alinhamento de SequênciaRESUMO
THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.
Assuntos
Proteínas de Algas/metabolismo , Lisina/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas de Algas/química , Proteínas de Algas/genética , Chlamydomonas reinhardtii/química , Teoria da Densidade Funcional , Ferro/química , Ferro/metabolismo , Lisina/química , Modelos Químicos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMO
Cyanobacteria and microalgae are oxygen-producing photosynthetic unicellular organisms encompassing a great diversity of species, which are able to grow under all types of extreme environments and exposed to a wide variety of predators and microbial pathogens. The antibacterial compounds described for these organisms include alkaloids, fatty acids, indoles, macrolides, peptides, phenols, pigments and terpenes, among others. This review presents an overview of antibacterial peptides isolated from cyanobacteria and microalgae, as well as their synergism and mechanisms of action described so far. Antibacterial cyanopeptides belong to different orders, but mainly from Oscillatoriales and Nostocales. Cyanopeptides have different structures but are mainly cyclic peptides. This vast peptide repertoire includes ribosomal and abundant non-ribosomal peptides, evaluated by standard conventional methodologies against pathogenic Gram-negative and Gram-positive bacteria. The antibacterial activity described for microalgal peptides is considerably scarcer, and limited to protein hydrolysates from two Chlorella species, and few peptides from Tetraselmis suecica. Despite the promising applications of antibacterial peptides and the importance of searching for new natural sources of antibiotics, limitations still persist for their pharmaceutical applications.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Cianobactérias/química , Microalgas/química , Proteínas de Algas/química , Proteínas de Algas/isolamento & purificação , Proteínas de Algas/farmacologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Anti-Hipertensivos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Sinergismo Farmacológico , Eucariotos/química , Humanos , Técnicas de Síntese em Fase SólidaRESUMO
In plant-pathogen interactions, plant immunity through pathogen-associated molecular pattern receptors (PAMPs) and R proteins, also called pattern recognition receptors (PRRs), occurs in different ways depending on both plant and pathogen species. The use and search for a structural pattern based on the presence and absence of characteristic domains, regardless of their disposition within a sequence, could be efficient in identifying PRRs proteins. Here, we develop a method mainly based on text mining and set theory to identify PRR and R genes that classify them into 13 categories based on the presence and absence of the main domains. Analyzing 24 plant and algae genomes, we showed that the RRGPredictor was more efficient, specific and sensitive than other tools already available, and identified PRR proteins with variations in size and in domain distribution throughout the sequence. Besides an easy identification of new plant PRRs proteins, RRGPredictor provided a low computational cost.
Assuntos
Proteínas de Plantas/genética , Receptores de Reconhecimento de Padrão/genética , Software , Proteínas de Algas/genética , Mineração de Dados , Genoma de Planta , Genômica/métodos , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Domínios Proteicos , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/classificaçãoRESUMO
Dye-sensitized solar cells (DSSCs) have been highlighted as the promising alternative to generate clean energy based on low pay-back time materials. These devices have been designed to mimic solar energy conversion processes from photosynthetic organisms (the most efficient energy transduction phenomenon observed in nature) with the aid of low-cost materials. Recently, light-harvesting complexes (LHC) have been proposed as potential dyes in DSSCs based on their higher light-absorption efficiencies as compared to synthetic dyes. In this work, photo-electrochemical hybrid devices were rationally designed by adding for the first time Leu and Lys tags to heterologously expressed light-harvesting proteins from Chlamydomonas reinhardtii, thus allowing their proper orientation and immobilization on graphene electrodes. The light-harvesting complex 4 from C. reinhardtii (LHC4) was initially expressed in Escherichia coli, purified via affinity chromatography and subsequently immobilized on plasma-treated thin-film graphene electrodes. A photocurrent density of 40.30 ± 9.26 µA/cm2 was measured on devices using liquid electrolytes supplemented with a phosphonated viologen to facilitate charge transfer. Our results suggest that a new family of graphene-based thin-film photovoltaic devices can be manufactured from rationally tagged LHC proteins and opens the possibility to further explore fundamental processes of energy transfer for biological components interfaced with synthetic materials.
Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Técnicas Eletroquímicas/métodos , Grafite/química , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Algas/genética , Corantes/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Energia SolarRESUMO
BACKGROUND: The marine alga Ulva compressa is the dominant species in copper-polluted coastal areas in northern Chile. It has been shown that the alga tolerates micromolar concentrations of copper and accumulates copper at the intracellular level. Transcriptomic analyses were performed using total RNA of the alga cultivated with 10 µ M copper for 0, 1, 3 and 5 days using RNA-seq in order to identify processes involved in copper tolerance. RESULTS: The levels of transcripts encoding proteins belonging to Light Harvesting Complex II (LHCII), photosystem II (PSII), cytochrome b6f, PSI, LHCI, ATP synthase and proteins involved in repair of PSII and protection of PSI were increased in the alga cultivated with copper. In addition, the level of transcripts encoding proteins of mitochondrial electron transport chain, ATP synthase, and enzymes involved in C, N and S assimilation were also enhanced. The higher percentages of increase in the level of transcripts were mainly observed at days 3 and 5. In contrast, transcripts involved protein synthesis and degradation, signal transduction, and replication and DNA repair, were decreased. In addition, net photosynthesis and respiration increased in the alga cultivated with copper, mainly at days 1 to 3. Furthermore, the activities of enzymes involved in C, N and S assimilation, rubisco, glutamine synthase and cysteine synthase, respectively, were also increased, mainly at days 1 and 3. CONCLUSIONS: The marine alga U. compressa tolerates copper excess through a concomitant increase in expression of proteins involved in photosynthesis, respiration, and C, N and S assimilation, which represents an exceptional mechanism of copper tolerance.
Assuntos
Cobre/efeitos adversos , Fotossíntese/efeitos dos fármacos , Ulva/efeitos dos fármacos , Poluentes Químicos da Água/efeitos adversos , Proteínas de Algas/análise , Carbono/metabolismo , Perfilação da Expressão Gênica , Nitrogênio/metabolismo , Oxigênio/metabolismo , Enxofre/metabolismo , Ulva/metabolismo , Ulva/fisiologiaRESUMO
In this work, transcripts encoding three metallothioneins from Ulva compressa (UcMTs) were amplified: The 5'and 3' UTRs by RACE-PCR, and the open reading frames (ORFs) by PCR. Transcripts encoding UcMT1.1 (Crassostrea-like), UcMT2 (Mytilus-like), and UcMT3 (Dreissena-like) showed a 5'UTR of 61, 71, and 65 nucleotides and a 3'UTR of 418, 235, and 193 nucleotides, respectively. UcMT1.1 ORF encodes a protein of 81 amino acids (MW 8.2 KDa) with 25 cysteines (29.4%), arranged as three motifs CC and nine motifs CXC; UcMT2 ORF encode a protein of 90 amino acids (9.05 kDa) with 27 cysteines (30%), arranged as three motifs CC, nine motifs CXC, and one motif CXXC; UcMT3 encode a protein of 139 amino acids (13.4 kDa) with 34 cysteines (24%), arranged as seven motifs CC and seven motifs CXC. UcMT1 and UcMT2 were more similar among each other, showing 60% similarity in amino acids; UcMT3 showed only 31% similarity with UcMT1 and UcMT2. In addition, UcMTs displayed structural similarity with MTs of marine invertebrates MTs and the terrestrial invertebrate Caenorhabtidis elegans MTs, but not with MTs from red or brown macroalgae. The ORFs fused with GST were expressed in bacteria allowing copper accumulation, mainly in MT1 and MT2, and zinc, in the case of the three MTs. Thus, the three MTs allowed copper and zinc accumulation in vivo. UcMTs may play a role in copper and zinc accumulation in U. compressa.
Assuntos
Proteínas de Algas/metabolismo , Metalotioneína/metabolismo , Ulva/enzimologia , Proteínas de Algas/química , Proteínas de Algas/genética , Sequência de Aminoácidos , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/metabolismo , Metalotioneína/química , Metalotioneína/genética , Fases de Leitura Aberta/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Zinco/metabolismoRESUMO
Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.
Assuntos
Proteínas de Bactérias/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxirredutases/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética/métodos , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosfitos/farmacologia , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Seleção Genética , Transformação GenéticaRESUMO
The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.
Assuntos
Proteínas de Algas/genética , Chlamydomonas reinhardtii/genética , Glicerolfosfato Desidrogenase/genética , Microalgas/genética , Regiões Promotoras Genéticas , Transgenes/genética , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/enzimologia , Clonagem Molecular , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Microalgas/química , Microalgas/enzimologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Elementos de Resposta , Cloreto de Sódio/química , Cloreto de Sódio/farmacologiaRESUMO
Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.