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1.
Pol J Microbiol ; 68(4): 527-539, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880896

RESUMO

Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.Ulleungdo and Dokdo are volcanic islands with an oceanic climate located off the eastern coast of South Korea. In the present study, we used barcoded Illumina MiSeq to analyze eukaryotic microalgal genera collected from Seonginbong, the highest peak on Ulleungdo, and from groundwater sites on Dongdo and Seodo Islands, which are part of Dokdo. Species richness was significantly greater in the Seonginbong samples than in the Dongdo and Seodo samples, with 834 operational taxonomic units (OTUs) identified from Seonginbong compared with 203 OTUs and 182 OTUs from Dongdo and Seodo, respectively. Taxonomic composition analysis was also used to identify the dominant microalgal phyla at each of the three sites, with Chlorophyta (green algae) the most abundant phyla on Seonginbong and Dongdo, and Bacillariophyta (diatoms) the most abundant on Seodo. These findings suggest that differences in the abundances of Chlorophyta and Bacillariophyta species in the Seonginbong, Dongdo, and Seodo samples are due to variations in species richness and freshwater resources at each sampling location. To the best of our knowledge, this is the first report to detail freshwater microalgal communities on Ulleungdo and Dokdo. As such, the number of species identified in the Seonginbong, Dongdo, and Seodo samples might be an indicator of the ecological differences among these sites and varying characteristics of their microbial communities. Information regarding the microalgal communities also provides a basis for understanding the ecological interactions between microalgae species and other eukaryotic microorganisms.


Assuntos
Microalgas/isolamento & purificação , Clorófitas/classificação , Clorófitas/genética , Diatomáceas/classificação , Diatomáceas/genética , Diatomáceas/isolamento & purificação , Água Doce/análise , Ilhas , Microalgas/classificação , Microalgas/genética , Filogenia , Análise de Sequência de DNA
2.
Nat Commun ; 10(1): 4552, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591397

RESUMO

Diatoms outcompete other phytoplankton for nitrate, yet little is known about the mechanisms underpinning this ability. Genomes and genome-enabled studies have shown that diatoms possess unique features of nitrogen metabolism however, the implications for nutrient utilization and growth are poorly understood. Using a combination of transcriptomics, proteomics, metabolomics, fluxomics, and flux balance analysis to examine short-term shifts in nitrogen utilization in the model pennate diatom in Phaeodactylum tricornutum, we obtained a systems-level understanding of assimilation and intracellular distribution of nitrogen. Chloroplasts and mitochondria are energetically integrated at the critical intersection of carbon and nitrogen metabolism in diatoms. Pathways involved in this integration are organelle-localized GS-GOGAT cycles, aspartate and alanine systems for amino moiety exchange, and a split-organelle arginine biosynthesis pathway that clarifies the role of the diatom urea cycle. This unique configuration allows diatoms to efficiently adjust to changing nitrogen status, conferring an ecological advantage over other phytoplankton taxa.


Assuntos
Diatomáceas/genética , Diatomáceas/metabolismo , Redes e Vias Metabólicas/genética , Nitrogênio/metabolismo , Carbono/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Evolução Molecular , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Metabolômica/métodos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Nitratos/metabolismo , Proteômica/métodos , Água do Mar/microbiologia , Transdução de Sinais/genética
3.
Microb Cell Fact ; 18(1): 161, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547820

RESUMO

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Ácidos Graxos/metabolismo , Glucosefosfato Desidrogenase/genética , Dióxido de Carbono/análise , Diatomáceas/metabolismo , Engenharia Genética , Glucosefosfato Desidrogenase/metabolismo , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
4.
BMC Biotechnol ; 19(1): 53, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349823

RESUMO

BACKGROUND: Increasing CO2 emissions have resulted in ocean acidification, affecting marine plant photosynthesis and changing the nutrient composition of marine ecosystems. The physiological and biochemical processes of marine phytoplankton in response to ocean acidification have been reported, but have been mainly focused on growth and photosynthetic physiology. To acquire a thorough knowledge of the molecular regulation mechanisms, model species with clear genetic background should be selected for systematic study. Phaeodactylum tricornutum is a pennate diatom with the characteristics of small genome size, short generation cycle, and easy to transform. Furthermore, the genome of P. tricornutum has been completely sequenced. RESULTS AND DISCUSSION: In this study, P. tricornutum was cultured at high and normal CO2 concentrations. Cell composition changes during culture time were investigated. The 13C isotope tracing technique was used to determine fractional labeling enrichments for the main cellular components. The results suggested that when lipid content increased significantly under high CO2 conditions, total protein and soluble sugar contents decreased. The 13C labeling experiment indicated that the C skeleton needed for fatty acid C chain elongation in lipid synthesis under high CO2 conditions is not mainly derived from NaHCO3 (carbon fixed by photosynthesis). CONCLUSION: This study indicated that breakdown of intracellular protein and soluble sugar provide C skeleton for lipid synthesis under high CO2 concentration.


Assuntos
Proteínas de Algas/metabolismo , Dióxido de Carbono/metabolismo , Carbono/metabolismo , Diatomáceas/metabolismo , Lipídeos/biossíntese , Açúcares/metabolismo , Isótopos de Carbono/metabolismo , Diatomáceas/genética , Diatomáceas/fisiologia , Ecossistema , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Lipogênese , Oceanos e Mares , Fotossíntese , Água do Mar/química , Solubilidade , Açúcares/química
5.
J Biosci Bioeng ; 128(6): 755-765, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31277909

RESUMO

The diatom microalgal Phaeodactylum tricornutum accumulates a large amount of fucoxanthin. Carotenoids hydroxylases (CHYs) play key roles in fucoxanthin biosynthesis in diatoms. However, not any type of CHYs had been identified in P. tricornutum. In this study, two genes (designated Ptrcyp97b1 and Ptrcyp97b2) were cloned, identified and functionally characterized. They shared high sequence identity (50-94 %) with lutein deficient 1-like proteins from other eukaryotes. The typical catalytic active motifs of cytochrome P450s (CYP) were detected in the amino acid sequences of PtrCYP97B1 and PtrCYP97B2. The two genes were probably due to gene duplication. Ptrcyp97b1 and Ptrcyp97b2 transcriptional expression was up-regulated with distinct patterns under high light conditions. The metabolic profiles of the major carotenoids (ß-carotene, zeaxanthin, diadinoxanthin, diatoxanthin and fucoxanthin) were determined based on the high performance liquid chromatography method. The fucoxanthin and diatoxanthin contents were increased, while the ß-carotene content was decreased. By truncation of the N-terminal trans-membrane anchor or chloroplast transit peptide and addition of a 6 × His-tag, PtrCYP97B1 and PtrCYP97B2 were separately heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. Functional analysis showed that PrtCYP97B2 was able to catalyze the hydroxylation of the ß-rings of ß-carotene to produce zeaxanthin in ß-carotene-accumulating E. coli BL21(DE3) cells. PtrCYP97B1 might have the ability to catalyze the hydroxylation of other substrates other than ß-carotene. These results contribute to the further elucidation of xanthophyll biosynthesis in diatoms.


Assuntos
Carotenoides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Diatomáceas/enzimologia , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Diatomáceas/genética , Escherichia coli/genética , Hidroxilação
6.
Genes (Basel) ; 10(7)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261777

RESUMO

Sexual reproduction plays a fundamental role in diatom life cycles. It contributes to increasing genetic diversity through meiotic recombination and also represents the phase where large-sized cells are produced to counteract the cell size reduction process that characterizes these microalgae. With the aim to identify genes linked to the sexual phase of the centric planktonic diatom Skeletonema marinoi, we carried out an RNA-seq experiment comparing the expression level of transcripts in sexualized cells with that of large cells not competent for sex. A set of genes involved in meiosis were found upregulated. Despite the fact that flagellate gametes were observed in the sample, we did not detect the expression of genes involved in the synthesis of flagella that were upregulated during sexual reproduction in another centric diatom. A comparison with the set of genes changing during the first phases of sexual reproduction of the pennate diatom Pseudo-nitzschia multistriata revealed the existence of commonalities, including the strong upregulation of genes with an unknown function that we named Sex Induced Genes (SIG). Our results further broadened the panel of genes that can be used as a marker for sexual reproduction of diatoms, crucial for the interpretation of metatranscriptomic datasets.


Assuntos
Diatomáceas/genética , Flagelos/genética , Expressão Gênica , Meiose/genética , Reprodução/genética , Reprodução/fisiologia , Comportamento Sexual/fisiologia , Transcriptoma/genética
7.
Genome Biol Evol ; 11(7): 1829-1837, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31218358

RESUMO

Mutations are the origin of genetic diversity, and the mutation rate is a fundamental parameter to understand all aspects of molecular evolution. The combination of mutation-accumulation experiments and high-throughput sequencing enabled the estimation of mutation rates in most model organisms, but several major eukaryotic lineages remain unexplored. Here, we report the first estimation of the spontaneous mutation rate in a model unicellular eukaryote from the Stramenopile kingdom, the diatom Phaeodactylum tricornutum (strain RCC2967). We sequenced 36 mutation accumulation lines for an average of 181 generations per line and identified 156 de novo mutations. The base substitution mutation rate per site per generation is µbs = 4.77 × 10-10 and the insertion-deletion mutation rate is µid = 1.58 × 10-11. The mutation rate varies as a function of the nucleotide context and is biased toward an excess of mutations from GC to AT, consistent with previous observations in other species. Interestingly, the mutation rates between the genomes of organelles and the nucleus differ, with a significantly higher mutation rate in the mitochondria. This confirms previous claims based on indirect estimations of the mutation rate in mitochondria of photosynthetic eukaryotes that acquired their plastid through a secondary endosymbiosis. This novel estimate enables us to infer the effective population size of P. tricornutum to be Ne∼8.72 × 106.


Assuntos
Diatomáceas/genética , Evolução Molecular , Mutagênese/genética , Mutagênese/fisiologia , Taxa de Mutação
8.
Am J Bot ; 106(4): 560-572, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30958893

RESUMO

PREMISE OF THE STUDY: Loss of photosynthesis is a common and often repeated trajectory in nearly all major groups of photosynthetic eukaryotes. One small subset of "apochloritic" diatoms in the genus Nitzschia have lost their ability to photosynthesize and require extracellular carbon for growth. Similar to other secondarily nonphotosynthetic taxa, apochloritic diatoms maintain colorless plastids with highly reduced plastid genomes. Although the narrow taxonomic breadth of apochloritic Nitzschia suggests a single loss of photosynthesis in their common ancestor, previous phylogenetic analyses suggested that photosynthesis was lost multiple times. METHODS: We analyzed genes from the nuclear, plastid, and mitochondrial genomes for a broad set of taxa to test whether photosynthesis was lost one or multiple times in Bacillariales. We also sequenced and characterized the plastid genome of a nonphotosynthetic Nitzschia species. KEY RESULTS: Phylogenetic analyses showed that genes from all three genetic compartments either supported or failed to reject monophyly of apochloritic Nitzschia species, consistent with a single loss of photosynthesis in this group. The plastid genomes of two apochloritic Nitzschia are highly similar in all respects, indicating streamlining of the plastid genome before the split of these two species. CONCLUSIONS: A better understanding of the phylogeny and ecology of apochloritic Nitzschia, together with emerging genomic resources, will help identify the factors that have driven and maintained the loss of photosynthesis in this group of diatoms. Finally, some habitats host diverse communities of co-occurring nonphotosynthetic diatoms, reflecting resource abundance or resource partitioning in ecologically favorable habitats.


Assuntos
Diatomáceas/genética , Fotossíntese/genética , Filogenia , Diatomáceas/metabolismo , Genomas de Plastídeos
9.
Plant Physiol ; 179(4): 1779-1795, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30733257

RESUMO

The ecological success of diatoms, key contributors to photosynthesis, is partly based on their ability to perfectly balance efficient light harvesting and photoprotection. Diatoms contain higher numbers of antenna proteins than vascular plants for light harvesting and for photoprotection. These proteins are arranged in fucoxanthin-chlorophyll protein (FCP) complexes. The number of FCP complexes, their subunit composition, and their interactions in the thylakoid membranes remain elusive in different diatoms. We used the recently available genome sequence of the centric diatom Cyclotella cryptica to analyze gene sequences for putative light-harvesting proteins in C. meneghiniana, and to elucidate the FCP complex composition. We analyzed two pools of FCP complexes that were trimeric (FCPa) and nonameric (FCPb). FCPa was composed of four different trimeric subtypes. Two different nonameric FCPb complexes were present. All were distinguished by their polypeptide composition and partly by pigmentation. With use of a milder purification method, two fractions composed of different FCP complexes were isolated. One was enriched in FCPs incorporating the photoprotective subunit Lhcx1, such as the newly identified nonameric FCPb2 and the major trimeric FCPa4 complex, which are predetermined to be involved in energy-dependent nonphotochemical quenching. The other fraction contained mainly FCPs that were devoid of Lhcx1, FCPa3, and FCPb1. Both fractions also included small amounts of trimeric FCPa complexes with the centric diatom-specific Lhcx protein, Lhcx6_1, as subunit. Thus, the antenna organization of centric diatoms, as well as the distribution of different photoprotective Lhcx proteins, differs from that of other diatoms, as well as from plants.


Assuntos
Proteínas de Ligação à Clorofila/química , Diatomáceas/genética , Diatomáceas/metabolismo , Filogenia , Subunidades Proteicas/química , Tilacoides/metabolismo
10.
BMC Mol Biol ; 20(1): 7, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808304

RESUMO

BACKGROUND: A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and L-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. L-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms. RESULTS: Several genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that L-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor. CONCLUSIONS: Pseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for L-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.


Assuntos
Diatomáceas/genética , Diatomáceas/metabolismo , Ácido Caínico/análogos & derivados , Toxinas Marinhas/genética , Toxinas Marinhas/metabolismo , Redes e Vias Metabólicas/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Herbivoria , Ácido Caínico/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos Açúcares/metabolismo
11.
Mar Genomics ; 45: 72-78, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30792089

RESUMO

Previous phylogenomic analyses of diatoms have discovered some plastid-targeted genes apparently coming from green algae. Number of these genes varied from less than a half of EGT-compatible genes to an overwhelming majority, and their presence was treated as an evidence of cryptic green plastid. We have performed such an analysis with a novel weighted approach on an extended dataset of diatom genomes and proteomes. Approximately equal evidence was found for red and green algal origins for diatoms genes. Considering that very similar results were obtained on other secondary photosynthetic groups whose endosymbioses were independent from that of the diatom ancestors, we consider the serial plastid replacements unparsimonious. A better explanation of these data can be provided by the shopping bag model, where a future host switches numerous endosymbionts and acquires some genes from each of them. Eventually the host loses the ability to replace endosymbionts (e.g. through the loss of phagotrophy) and whatever symbiont was present at the moment gets fixed and reduced to an organelle.


Assuntos
Clorófitas/genética , Diatomáceas/genética , Evolução Molecular , Genoma , Rodófitas/genética , Simbiose/genética , Proteínas de Algas/análise , Filogenia
12.
Forensic Sci Int ; 297: 81-89, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30784950

RESUMO

In forensic science, the determination of diatoms bears several goals, for example to estimate the post mortem interval (PMI), to determine the cause of death of a corpse found in water as well as to further locate the suspected drowning site. However, to identify diatoms using morphological method beyond the genus level is difficult and requires expert knowledge. During the last decade, a new concept of DNA barcode is becoming a promising approach to identify the taxa of diatoms. In the present study, we performed a diatom morphological analysis covering 10 different water areas in Nanjing section of the Yangtze River. The diatoms were classified to the 'genus' level by using optical microscope and electron microscope. According to the morphological study results, we further designed and analyzed the 18S rDNA sequences as barcodes at the 'species' level. Our study established the morphological and DNA barcoding profile for the diatoms in Nanjing section of the Yangtze River for the first time which will be of great significance for the using of diatoms in forensic application. The results indicated that detailed analysis of selected diatom DNA barcodes can be used as a useful tool for forensic applications.


Assuntos
Código de Barras de DNA Taxonômico , DNA Ribossômico , Diatomáceas/genética , RNA Ribossômico 18S , China , Humanos , Microscopia , Microscopia Eletrônica , Filogenia , Reação em Cadeia da Polimerase , Rios , Análise de Sequência de DNA
13.
World J Microbiol Biotechnol ; 35(2): 35, 2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30712106

RESUMO

As major primary producers in marine environments, diatoms are considered a valuable feedstock of biologically active compounds for application in several biotechnological fields. Due to their metabolic plasticity, especially for light perception and use and in order to make microalgal production more environmentally sustainable, marine diatoms are considered good candidates for the large-scale cultivation. Among physical parameters, light plays a primary role. Even if sunlight is cost-effective, the employment of artificial light becomes a winning strategy if a high-value microalgal biomass is produced. Several researches on marine diatoms are designed to study the influence of different light regimens to increase biomass production enriched in biotechnologically high-value compounds (lipids, carotenoids, proteins, polysaccharides), or with emphasised photonic properties of the frustule.


Assuntos
Produtos Biológicos/metabolismo , Biotecnologia , Diatomáceas/metabolismo , Diatomáceas/efeitos da radiação , Microalgas/metabolismo , Microalgas/efeitos da radiação , Diatomáceas/genética , Diatomáceas/crescimento & desenvolvimento , Luz , Microalgas/genética , Microalgas/crescimento & desenvolvimento
14.
J Basic Microbiol ; 59(5): 504-510, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30811614

RESUMO

We examined the dynamic of periphytic diatom colonization and litter decompositional pattern in an intermittent stream of South India. Litter bags containing 5 g of fresh fallen leaves (belongs to five different species of angiosperms) were fixed in the five stream pools and they were collected after 7, 14, 21, and 28 days of incubation. This experiment showed the diversity, richness, and abundance of diatoms developed on leaf litter. In total, 131 µl-1 cells of diatoms under four species in three families were collected from decomposing leaves in benthic area of stream pools. During experiment, 142 µl-1 cells representing 11 taxa in 10 families were observed in litter bags. The colonization of epiphytic diatom was rapid in early stage of litter decomposition. The higher number of colonization made by Asterionella and Stenopterobia found in litter bags of all experimental weeks. These findings suggest that the rapid microbial colonization (ephiphytic algae) was occurred in the early stage of litter decomposition in an intermittent tropical stream and a comprehensive ecological investigation in perennial natural streams in India is needed.


Assuntos
Diatomáceas/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Rios , Biodegradação Ambiental , Biodiversidade , Diatomáceas/classificação , Diatomáceas/genética , Índia , Magnoliopsida/metabolismo , Fatores de Tempo
16.
FEMS Microbiol Lett ; 366(2)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629176

RESUMO

Diatom diazotroph associations (DDAs) are a widespread marine planktonic symbiosis between several diatom genera and di-nitrogen (N2)-fixing bacteria. Combining single cell confocal microscopy observations and molecular genetic approaches on individual field collected cells, we determined the phylogenetic diversity, distribution and evolution of the DDAs. Confocal analyses coupled with 3-D imaging re-evaluated the cellular location of DDA symbionts. DDA diversity was resolved by paired gene sequencing (18S rRNA and rbcL genes, 16S rRNA and nifH genes). A survey using the newly acquired sequences against public databases found sequences with high similarity (99-100%) to either host (18S rRNA) or symbiont (16S rRNA) in atypical regions for DDAs (high latitudes, anoxic basin and copepod gut). Concatenated phylogenies were congruent for the host and cyanobacteria sequences and implied co-evolution. Time-calibrated trees dated the appearance of N2 fixing planktonic symbiosis from 100-50Mya and were consistent with the symbiont cellular location: symbioses with internal partners are more ancient. An ancestral state reconstruction traced the evolution of traits in DDAs and highlight that the adaptive radiation to the marine environment was likely facilitated by the symbiosis. Our results present the evolutionary nature of DDAs and provide new genetic and phenotypic information for these biogeochemically relevant populations.


Assuntos
Evolução Biológica , Cianobactérias/genética , Diatomáceas/genética , Variação Genética , Simbiose , Cianobactérias/classificação , Cianobactérias/isolamento & purificação , Cianobactérias/fisiologia , Diatomáceas/classificação , Diatomáceas/microbiologia , Diatomáceas/fisiologia , Fixação de Nitrogênio , Filogenia , Água do Mar/microbiologia , Água do Mar/parasitologia
17.
Appl Microbiol Biotechnol ; 103(4): 1837-1850, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30617536

RESUMO

The effect of ultraviolet radiation (UVR) on photosynthetic efficiency and the resulting mechanisms against UV exposure employed by phytoplankton are not completely understood. To address this knowledge gap, we developed a novel close-coupled, wavelength-configurable platform designed to produce precise and repeatable in vitro irradiation of Corethron hystrix, a member of a genera found abundantly in the Southern Ocean where UV exposure is high. We aimed to determine its metabolic, protective, and repair mechanisms as a function of varying levels of specific electromagnetic energy. Our results show that the physiological responses to each energy level of UV have a negative linear decrease in the photosynthetic efficiency of photosystem II proportional to UV intensity, corresponding to a large increase in the turnover time of quinone reoxidation. Gene expression changes of photosystem II-related reaction center proteins D1, CP43, and CP47 showed coordinated downregulation whereas the central metabolic pathway demonstrated mixed expression of up and downregulated transcripts after UVR exposure. These results suggest that while UVR may damage photosynthetic machinery, oxidative damage may limit production of new photosynthetic and electron transport complexes as a result of UVR exposure.


Assuntos
Diatomáceas/genética , Diatomáceas/efeitos da radiação , Regulação para Baixo , Regulação da Expressão Gênica/efeitos da radiação , Fotossíntese/efeitos da radiação , Raios Ultravioleta
18.
Plant Biotechnol J ; 17(1): 75-87, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29754445

RESUMO

Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.


Assuntos
Diatomáceas/genética , Engenharia Genética , Triterpenos Pentacíclicos/metabolismo , Terpenos/metabolismo , Triterpenos/metabolismo , Reatores Biológicos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diatomáceas/metabolismo , Engenharia Genética/métodos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Lotus/enzimologia , Lotus/genética , Medicago truncatula/enzimologia , Medicago truncatula/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
19.
Nat Microbiol ; 4(2): 226-233, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30478288

RESUMO

Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms1-3. The steroid biosynthetic pathway is highly conserved and is key in eukaryote evolution4-7. The flavoprotein squalene epoxidase (SQE) catalyses the first oxygenation reaction in this pathway and is rate limiting. However, despite its conservation in animals, plants and fungi, several phylogenetically widely distributed eukaryote genomes lack an SQE-encoding gene7,8. Here, we discovered and characterized an alternative SQE (AltSQE) belonging to the fatty acid hydroxylase superfamily. AltSQE was identified through screening of a gene library of the diatom Phaeodactylum tricornutum in a SQE-deficient yeast. In accordance with its divergent protein structure and need for cofactors, we found that AltSQE is insensitive to the conventional SQE inhibitor terbinafine. AltSQE is present in many eukaryotic lineages but is mutually exclusive with SQE and shows a patchy distribution within monophyletic clades. Our discovery provides an alternative element for the conserved steroid biosynthesis pathway, raises questions about eukaryote metabolic evolution and opens routes to develop selective SQE inhibitors to control hazardous organisms.


Assuntos
Eucariotos/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Esteroides/biossíntese , Vias Biossintéticas , Coenzimas , Diatomáceas/enzimologia , Diatomáceas/genética , Diatomáceas/metabolismo , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/química , Filogenia , Conformação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esqualeno/análogos & derivados , Esqualeno/metabolismo , Esqualeno Mono-Oxigenase/química , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Terbinafina/farmacologia
20.
ACS Synth Biol ; 8(1): 57-69, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30525458

RESUMO

Genetic engineering of Phaeodactylum tricornutum as a model organism for diatoms is the basis of molecular and biochemical research, and can also be used in biotechnological approaches. So far, integration of foreign DNA into the genome happens randomly by nonhomologous end joining (NHEJ), if the classical method of particle bombardment is used, with the danger of negative physiological side effects. Here we show that a putative gene for a DNA ligase IV homologue ( ligIV) in P. tricornutum codes for a functional LigIV. The knock-down of ligIV in P. tricornutum via antisense RNA drastically enhances homologous recombination (HR) by interfering with the NHEJ pathway at its central DNA ligation step done by LigIV. This enables a specific integration of DNA at desired locations, greatly enhanced transformation rates and provides a new way of specifically altering the genome of P. tricornutum.


Assuntos
Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Diatomáceas/genética , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Ligase Dependente de ATP/fisiologia , Recombinação Homóloga/genética
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