Development of a molecular-based index for assessing iron status in bloom-forming pennate diatoms

Iron availability limits primary productivity in large areas of the world's oceans. Ascertaining the iron status of phytoplankton is essential for understanding the factors regulating their growth and ecology. We developed an incubation-independent, molecular-based approach to assess the iron nutritional status of specific members of the diatom community, initially focusing on the ecologically important pennate diatom Pseudo-nitzschia. Through a comparative transcriptomic approach, we identified two genes that track the iron status of Pseudo-nitzschia with high fidelity. The first gene, ferritin (FTN), encodes for the highly specialized iron storage protein induced under iron-replete conditions. The second gene, ISIP2a, encodes an iron-concentrating protein induced under iron-limiting conditions. In the oceanic diatom Pseudo-nitzschia granii (Hasle) Hasle, transcript abundance of these genes directly relates to changes in iron availability, with increased FTN transcript abundance under iron-replete conditions and increased ISIP2a transcript abundance under iron-limiting conditions. The resulting ISIP2a:FTN transcript ratio reflects the iron status of cells, where a high ratio indicates iron limitation. Field samples collected from iron grow-out microcosm experiments conducted in low iron waters of the Gulf of Alaska and variable iron waters in the California upwelling zone verify the validity of our proposed Pseudo-nitzschia Iron Limitation Index, which can be used to ascertain in situ iron status and further developed for other ecologically important diatoms

Doing synthetic biology with photosynthetic microorganisms

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.Peer reviewe

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing

Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes

Inter- and intraspecific relationships between nuclear DNA content and cell size in selected members of the centric diatom genus <i>Thalassiosira</i> (Bacillariophyceae)

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species-rich and well-studied centric diatom genera. Both minimum and maximum species-specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000-fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3-fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, beta-tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance

Oogamous reproduction, with two-step auxosporulation, in the centric diatom <i>Thalassiosira punctigera</i> (Bacillariophyta)

Thalassiosira species are common components of marine planktonic communities worldwide and are used intensively as model experimental organisms. However, data on life cycles and sexuality within the genus are fragmentary. A clone of the cosmopolitan marine diatom Thalassiosira punctigera Cleve emend. Hasle was isolated from the North Sea and oogamous sexual reproduction was observed in culture. Cells approximately 45 µm and smaller became sexualized. Oogonia were produced preferentially and spermatogenesis was infrequent. Unfertilized oogonia always aborted and their development was apparently arrested at prophase of meiosis I. Further progression through meiosis and auxospore formation occurred only after a sperm had penetrated into the oocyte. Many cells of the new large-celled generation (approximately 90–120 µm in size) immediately became sexualized again but only oogonia were produced. A few of the large oogonia became auxospores and produced initial cells 132–153 µm in diameter. The second step of auxosporulation probably involved fertilization of large-celled oocytes by the sperm of the small-celled spermatogonangia that were still present in the culture. An F1 clone obtained after selfing within the small-celled auxosporulation size range was investigated. Like the parent clone, the F1 clone was homothallic but no auxosporulation was observed: spermatogonangia were unable to produce viable sperm, apparently because of inbreeding depression. Aggregation and interaction of oogonia were documented, and may be relevant for understanding the mechanisms of signaling and recognition between sexualized cells and the evolution of sexuality in pennate diatoms

Iron and silicic acid effects on phytoplankton productivity, diversity and chemical composition in the central equatorial Pacific Ocean

A microcosm nutrient-amendment experiment using central equatorial Pacific Ocean (0u, 140uW) mixed-layer waters was conducted to determine biogeochemical controls on phytoplankton with an emphasis on post-iron enrichment nutrient uptake dynamics and species composition. The addition of either Fe (termed Fe-only) or Fe and Si(OH)4 (termed FeSi) to on-deck incubations resulted in growth primarily of pennate diatoms, with statistically equivalent increases relative to the control in maximum photochemical efficiency, chlorophyll a (Chl a) concentrations, particulate organic carbon and nitrogen concentrations, and dissolved inorganic carbon uptake rates. In contrast, at peak Chl a concentrations, there was a 3.4-fold higher abundance of large diatoms and a 3.9- fold lower abundance of small pennate diatoms in FeSi relative to Fe-only, which translated into a 3.5-fold higher Si(OH)4 uptake rate and a 2.1-fold higher biogenic silica concentration. Fourier transform infrared spectroscopy indicated that relative to cells from Fe-only, cells from FeSi possessed the lowest protein : carbohydrate ratios, and ratios of lipids, proteins, and carbohydrates relative to silica, consistent with differences in diatom C allocation or increased silicification or both. Our results suggest that after Fe addition, diatom organic matter accumulation rates (i.e., C and N uptake rates) are enhanced but the low, ambient [Si(OH)4] retards cell division rates, resulting in fewer large diatoms with relatively high C and N contents. After the simultaneous addition of Fe and Si(OH)4, enhanced rates of diatom organic matter accumulation and cell division results in more large, heavily silicified diatoms with relatively low C and N contents

Development of a molecular‐based index for assessing iron status in bloom‐forming pennate diatoms

Iron availability limits primary productivity in large areas of the world's oceans. Ascertaining the iron status of phytoplankton is essential for understanding the factors regulating their growth and ecology. We developed an incubation-independent, molecular-based approach to assess the iron nutritional status of specific members of the diatom community, initially focusing on the ecologically important pennate diatom Pseudo-nitzschia. Through a comparative transcriptomic approach, we identified two genes that track the iron status of Pseudo-nitzschia with high fidelity. The first gene, ferritin (FTN), encodes for the highly specialized iron storage protein induced under iron-replete conditions. The second gene, ISIP2a, encodes an iron-concentrating protein induced under iron-limiting conditions. In the oceanic diatom Pseudo-nitzschia granii (Hasle) Hasle, transcript abundance of these genes directly relates to changes in iron availability, with increased FTN transcript abundance under iron-replete conditions and increased ISIP2a transcript abundance under iron-limiting conditions. The resulting ISIP2a:FTN transcript ratio reflects the iron status of cells, where a high ratio indicates iron limitation. Field samples collected from iron grow-out microcosm experiments conducted in low iron waters of the Gulf of Alaska and variable iron waters in the California upwelling zone verify the validity of our proposed Pseudo-nitzschia Iron Limitation Index, which can be used to ascertain in situ iron status and further developed for other ecologically important diatoms

Erstellung sowie Untersuchung des Betriebsverhaltens eines Windenergiekonverters. T. 4 Versuchsaufbau und Messergebnisse

See also BMFT-FB-T--82-204; 82-205; 82-206SIGLEDEGerman