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Diatom Cell Contents Assignment

Abstract

Diatoms are photosynthetic unicellular eukaryotes found in most aquatic environments. They are major players in global biogeochemical cycles, and generate as much oxygen through photosynthesis as terrestrial rainforests do. Insights into their evolutionary origins have been revealed by the whole-genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum. We now know that diatoms contain unusual assortments of genes derived from different sources, including those acquired by horizontal gene transfer from bacteria. These genes confer novel metabolic and signaling capacities that may underlie the extraordinary ecological success of diatoms on Earth today. The availability of a suite of techniques that can be used to monitor and manipulate diatom genes is enhancing our knowledge of their novel characteristics. We highlight these recent developments and illustrate how they are being used to understand different aspects of diatom biology. We also discuss the use of diatoms in commercial applications, such as for nanotechnology and biofuel production.

In the upper zone of the ocean and other water bodies, down to depths where light can penetrate, one can typically find an abundant group of eukaryotic algae known as diatoms. These microscopic, unicellular organisms are characterized by ornate, lacework-like, silicified shells and are distributed all around the world. Diatoms are photosynthetic organisms that can convert the energy from sunlight into chemical energy in the form of ATP (adenosine triphosphate). This chemical reaction confers on diatoms the ability to produce their own nutrients (sugars), thus they have an autonomous metabolism and are called photoautotrophs. Diatoms absorb and fix large amounts of atmospheric carbon dioxide (CO2) while capturing light and water to generate a major fraction of the oxygen generated on Earth by photosynthesis. They are in fact believed to contribute between 20% and 25% of global primary production, equivalent to all terrestrial rainforests combined (Falkowski et al. 1998, Field et al. 1998, Smetacek 1999), and consequently play an essential role in the well-being of our global ecosystem.

The scientist and artist Ernst Haeckel was one of the first to observe and describe diatoms in the late 19th century (Breidbach 2005). German biologist Robert Lauterborn subsequently made exquisite microscopic descriptions of subcellular events occurring during diatom cell division. A century later, Jeremy Pickett-Heaps translated Lauterborn's observations from the original German and verified his discoveries using light and electron microscopy (Pickett-Heaps et al. 1984, De Martino et al. 2009). Others began describing diatoms'habitats: Allen did research in the early 20th century (Allen WE 1926), and experimental culturing became more reliable with the finding that, in addition to light and macronutrients, certain micronutrients and vitamins were required to cultivate them (Harvey 1939). Ecological and descriptive studies continue to this day, with researchers now incorporating advanced techniques of molecular and cellular biology. Consequently, knowledge of diatoms'basic biology and their potential for a range of commercial exploitations is now advancing rapidly.

Diatoms can be recognized in the microscope by their highly ornamented silicified cell walls, known as frustules (figure 1). How diatoms generate these beautiful structures is largely unknown, although some insights are now being revealed (see below). The process is termed “biomineralization” (defined as the formation of inorganic materials under biological control), and the species-specific patterns indicate that it is genetically determined. Because marine organisms use more than 6.7 gigatons of silicon per year (Tréguer et al. 1995), it is particularly important to understand silicon uptake and deposition processes in diatoms.

Figure 1.

Electron micrograph of the elaborate silicified cell wall of a diatom (Thalassiosira oestrupii var. venrickae). The cell has a diameter of 9.5 microns. Image: Courtesy of Diana Sarno (Service for Taxonomy and Identification of Marine Phytoplankton, Stazione Zoologica Anton Dohrn, Naples, Italy).

Figure 1.

Electron micrograph of the elaborate silicified cell wall of a diatom (Thalassiosira oestrupii var. venrickae). The cell has a diameter of 9.5 microns. Image: Courtesy of Diana Sarno (Service for Taxonomy and Identification of Marine Phytoplankton, Stazione Zoologica Anton Dohrn, Naples, Italy).

Furthermore, diatoms are used as bioindicators of pollution and water quality. Because many heavy metals and organic xenobiotics inhibit diatoms'growth, other algae such as cyanobacteria come to dominate (Berland et al. 1976). It is therefore possible to determine water quality by analyzing plankton diversity. Diatoms are also used as hydrographic tracers because biogenic silica retains its primary oxygen isotopic composition after burial (Sancetta 1981). This property can be used to monitor past surface temperatures and isotopic compositions of seawater (Shemesh et al. 1992). Additionally, because the frustule can retain its structural features over geological timescales, the diatom fossil record is of high quality. These observations reveal that diatoms have been major players in marine environments for at least the past 90 million years (Kooistra et al. 2007).

Diatoms belong to the heterokont branch of the eukaryotes. This group lies within the hypothesized Chromalveolata kingdom within which several major lineages, including algae, can be found (Harper et al. 2005). These lineages were originally defined using morphological and developmental characters, and have subsequently been refined using molecular approaches—for example, sequence analysis of ribosomal RNA genes and highly conserved proteins such as RuBisCo (ribulose-1, 5-bisphosphate carboxylase oxygenase) and elongation factor Tu (Baldauf et al. 1996, 2000). More recent, larger-scale phylogenomics approaches based on multiple sequence alignments are providing further insights into the evolutionary relationships between diatoms (Baldauf 2003, Li et al. 2006). Algal chloroplasts are believed to be derived from photosynthetic prokaryotes that invaded or were engulfed by a eukaryotic cell and then became endosymbionts more than 1.5 billion years ago (Gibbs 1981, Cavalier-Smith 1982, 1986). This event subsequently gave rise to the green and red algal lineages. The chromalveolates are thought to have derived from a second endosymbiotic event that occurred around 1 billion years ago (Yoon et al. 2004), in which a red, algal-like organism became associated a second time with a heterotrophic eukaryote (figure 2). The most striking evidence for this is the presence of four membranes surrounding the chloroplasts in many photosynthetic chromalveolates such as the diatoms (Gibbs 1981). Diatoms are further divided into two groups, the centrics and pennates, on the basis of their radial and bilateral symmetry, respectively. Diatom fossils representing centric species date from the Cretaceous, whereas pennate diatoms appear to have arisen later, around 90 million years ago.

Figure 2.

Schematic representation of the secondary endosymbiotic process thought to have given rise to the diatoms. An autotrophic red algal—like ancestor was endocytosed by a heterotrophic host cell. In the resulting cell, gene transfer occurred between the endosymbiont nucleus and the host nucleus, and probably also from the plastid and mitochondrial genomes. The resulting diatom cell contains the endosymbiont chloroplast, surrounded by four membranes, the host nucleus, and the host mitochondria. New genes have also been acquired by horizontal gene transfer from bacteria. Nuclei are shown in blue. Abbreviations: D, diatom; HGT, horizontal gene transfer; m, mitochondria; pp, primary plastid; SE, secondary endosymbiosis; sp, secondary plastid.

Figure 2.

Schematic representation of the secondary endosymbiotic process thought to have given rise to the diatoms. An autotrophic red algal—like ancestor was endocytosed by a heterotrophic host cell. In the resulting cell, gene transfer occurred between the endosymbiont nucleus and the host nucleus, and probably also from the plastid and mitochondrial genomes. The resulting diatom cell contains the endosymbiont chloroplast, surrounded by four membranes, the host nucleus, and the host mitochondria. New genes have also been acquired by horizontal gene transfer from bacteria. Nuclei are shown in blue. Abbreviations: D, diatom; HGT, horizontal gene transfer; m, mitochondria; pp, primary plastid; SE, secondary endosymbiosis; sp, secondary plastid.

Studies of diatom biology have gone through a paradigm shift following the recent incorporation of molecular and cellular methods to dissect their biology. Most of these studies have been performed on two species, Thalassiosira pseudonana and Phaeodactylum tricornutum, now considered model species for the centrics and pennates, respectively, because of the availability of whole-genome sequences and molecular tools to assess gene function (Armbrust et al. 2004, Poulsen et al. 2007, Siaut et al. 2007, Bowler at al. 2008).

Diatom genome sequencing confirms novel evolutionary histories

Both diatom genomes have been sequenced by the Joint Genome Institute in California. The sequence from the centric diatom T. pseudonana

Culture conditions

T. pseudonana (clone CCMP 1335) was grown at 20 °C and 24 h light at 100–140 μE, in artificial seawater medium (NEPCC) according to the North East Pacific Culture Collection protocol (http://www3.botany.ubc.ca/cccm/NEPCC/esaw.html). NEPCC medium contains 100 μM concentration of Na2SiO4. For silica starvation growth experiments, this concentration was reduced to 50 μM and all other nutrients were added at 2 × concentrations, except for vitamin solution that remained at 0.296 μM thiamine, 4.09 nM biotin and 1.48 nM vitamin B12 in all growth media. For nitrate starvation experiments, NaNO3 was reduced from 0.55 mM to 0.1 mM, or was completely omitted from the NEPCC with all other nutrients added at 2 × concentrations, except for vitamin solution as above.

Targeted silacidin gene deregulation (TSD) vectors (Supplementary Figure S1) were constructed using standard cloning techniques. A 256 bp fragment of the silacidin gene was amplified from T. pseudonana complementary DNA using the primers SILASF (containing NotI and HindIII sites) and SILASR (containing NotI and EcoRV sites) and inserted into the vectors pTpfcp and pTpNR (Poulsen et al., 2006) using the NotI site (additional sites were added to aid in future cloning). The resulting vectors, pTpNRSILAS and pTpFCPSILAS, were sequenced and those containing the silacidin fragment in the antisense orientation were used.

The plasmids were introduced into T. pseudonana using the Biolistic PDS-1000/He particle delivery system (BIORAD, Hercules, CA, USA) using M10 tungsten particles according to the method reported by Poulsen et al., 2006. The pTpNRSILAS vector does not contain the antibiotic resistant gene ‘NAT’, conveying resistance to nourseothricin, and thus was cotransformed with the vector pTpFCPNAT (Poulsen et al., 2006; Supplementary Figure 1). Transformed cells were plated onto 50% NEPC medium with 0.8% agar, supplemented with 100 μg ml−1 nourseothricin (Werner Bioagent, Jena, Germany). Control cell lines were produced by transforming with the pTpFCPNAT vector only.

Screening of nourseothricin-resistant TSD transformants

Screening of TSD mutants was initially performed using light microscopy (Olympus BX40) and coulter counter (Beckman multisizer 3 with 100 μm aperture; Fullerton, CA, USA) to observe physical differences between transformant, control (nourseothricin-resistant) and wild-type (WT) cell lines.

The presence of TSD cassettes was confirmed in transformants by PCR using the SILASR primer in combination with either primer pTpFCPt, yielding a 705 bp product, or pTpNRt, yielding an 844 bp product targeting the nitrate reductase or FCP terminator as appropriate.

Isolation of silacidins

Two different harvesting procedures were used. Cells grown in 20 l silicic acid replete NEPCC medium were harvested during mid-late exponential growth phase by flow-through centrifugation in CEPA High Speed centrifuge Z41 (Carl Padberg Zentrifugenbau GmbH, Lahr/Schwarzwald, Germany). Cultures were harvested from 20 l silicic deplete NEPCC following 48 h silicic acid starvation by filtration onto Isopore 1.2 μm pore-sized membrane filters (Millipore, Billerica, MA, USA). The harvested cells were boiled twice in a lysis buffer containing 0.1 m ethylenediaminetetraacetic acid and 2% sodium dodecyl sulphate. The suspension was centrifuged and washed until the supernatant remained colourless. Diatom silica was dissolved in an acidified ammonium fluoride solution (8 m NH4F, 2 m HF, pH 4–5) at room temperature for 25 min. The extract was centrifuged and the supernatant was desalted on a HiTrap column (GE Healthcare, Chicago, IL, USA). The eluate was dried in vacuo, dissolved in 250 μl 2 m NaCl and after centrifugation, size fractionated on a Superose 12 10/300 GL column (GE Healthcare; running buffer 200 mM ammonium formate, pH 7.7; flow rate 0.4 ml min−1; detection at 226 nm). Fractions eluting between 35 and 38 min (containing silaffin 1/2 l and silacidins) were combined, dried in vacuo, dissolved in 2 m NaCl and loaded onto a Superdex-Peptide HR 10/30 column (GE Healthcare; running buffer 10 mM Tris-HCl, 2 m NaCl, pH 7.5; flow rate 0.3 ml min−1; detection at 220 nm). Fractions eluting between 33 and 40 min contained silacidins. Recombinant silacidin A’ produced in Escherichia coli BL 21 DE3 was used as a standard (Richthammer et al., 2011).

Transcript level silacidin expression analysis

qRT-PCR was used to establish whether the silacidin gene deregulation was effective at the RNA level as well as at the protein level. 100 ml cultures were concentrated onto Isopore 1.2 μm pore size RTTP filters (Millipore) and flash frozen before RNA extraction with Directzol RNA miniprep Kit (Zymo Research, Irvine, CA, USA). Isolated RNA was treated, complementary DNA synthesized and qRT-PCR performed according to Durkin et al. (2009). The primers (SILqPCR-F and SILqPCR-R) were designed to target a region of the silacidin mRNA outside of the antisense fragment encoded by the gene deregulation vectors. Primers used are shown in Supplementary Table S2.

Imaging and cell measurements

Light microscope images of live cultures were taken using a Zeiss AxioPlan 2ie widefield microscope equipped with an AxioCam HRm CCD camera. For scanning electron microscopy, 15 ml samples of cell cultures were concentrated by centrifugation before treatment with 30% H2O2, samples were pelleted by centrifugation and washed with deionised water five times before 25 μl resuspended material was mounted onto round glass cover slips mounted on stubs and dried overnight. Stubs were coated in gold particles using a sputter coater and imaged with a Zeiss Supra 55 CP FEG scanning electron microscope (John Innes Centre Bioimaging Facility). For transmission electron microscopy, the diatom cell samples were frozen in liquid propane at −175 °C, then substituted with 2% osmium tetroxide (OsO4) in acetone and 2% distilled water at −80 °C for 48 h, before warming to −20 °C for 4 h and 4 °C for 1 h. Samples were then dehydrated twice each in anhydrous acetone and ethanol for 30 min at room temperature. Samples were then continuously dehydrated in ethanol at room temperature overnight before being infiltrated with PO (propylene oxide) twice for 30 min each, and put into a 70:30 mixture of PO and an epoxy resin (Quetol-651; Nisshin EM Co., Tokyo, Japan) for 1 h. Then, PO was volatilized overnight. The samples were transferred to a fresh 100% resin and polymerized at 60 °C for 48 h. The resins were ultra-thin sectioned at 70 nm with a diamond knife using an ultramicrotome (Ultracut UCT; Leica, Vienna, Austria), and mounted on copper grids. They were stained with 2% uranyl acetate at room temperature for 15 min, washed with distilled water, and secondary-stained with lead stain solution (Sigma-Alderich Co., Tokyo, Japan) at room temperature for 3 min. The grids were observed by a transmission electron microscope (JEM-1400Plus: JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV. Images from light, scanning and tranmission electron microscopy were used to measure cell dimensions and frustule thickness using ImageJ software. Measurements of cell diameter and length from light microscope images of cells in girdle-band orientation were used to calculate surface area and volume, and surface area to volume ratios for individual cells.

Silicon quantification

Cell samples corresponding to 4 or 6 × 108 cells were collected by centrifugation for each of three replicate samples of WT and TSD cells, respectively. The cells were transferred to wells of an AcroPrep Advance 350 plate (0.2 μm Supor, Pall, Port Washington, NY, USA) and washed four times with MilliQ water before extracting with 100% methanol until residue remained yellowish or colourless. Samples were washed a further four times with MIlliQ water and silica was dissolved at 95 °C for 1 h using 60 μl of a 2 m NaOH solution. Samples were centrifuged (3220 g, 15 min, room temperature) and the flow-through was collected in a new 96-well plate. Another incubation with 20 μl 2 m NaOH solution was conducted to achieve complete silica dissolution and after centrifugation, the flow-through collected in the same plate. The volume of the combined fractions was volumetrically determined using microliter pipette. The amount of dissolved silica in these samples was determined by the molybdenum blue test (Ramachandran and Gupta, 1985). Resulting silica concentrations were divided by cells per sample to give values for silica per cell.

Growth experiments

Cell lines were grown in batch cultures of 250 ml in triplicate for each growth experiment. Cultures were grown according to culture conditions above. Daily measurements were taken for cell counts (coulter counter, Beckman), photosynthesis based on the quantum yield of photosystem II (Fv/Fm; Phyto-PAM-ED, Walz), and light microscopy (Olympus BX40) was used to assess the average number of cells per particle in order to adjust cell counts to allow for cell aggregation. Growth rates were calculated as the slope of the natural logarithm of cell numbers during exponential phase growth.

Si(OH)4 uptake

Samples were taken daily during a growth experiment for analysis, and cells were removed by filtration through isopore 1.2 μm pore size RTTP filters (Millipore), before a second filtration through 0.2 μm pore size Minisart filters (Sigma-Aldrich). Samples were analysed using a Skalar SAN++ continuous flow analyser.

Aggregation analysis

During the same growth experiment, a sub-sample was taken from each replicate culture and viewed using light microscopy (Olympus BX40). At least 100 cells were counted per sample in triplicate and the number of cells per aggregate was recorded. In addition, the number of cells counted was divided by the number of aggregates counted to give the average number of cells per particle. These data were used to normalise cell abundances obtained by coulter counter for the same cultures.

Transcriptome sequencing

RNA was extracted from triplicate cultures of nourseothricin cassette alone (NAT) and two independent TSD cell lines harvested during late exponential phase and following 48 h silicon starvation as described under ‘Transcript level silacidin expression analysis’ (Supplementary Figure S2). RNA sequencing was performed according to the Illumina TruSeq RNA protocol by The Earlham Institute (Norwich Research Park). Reads were aligned to the assembled T. pseudonana genome using the Tophat program (https://ccb.jhu.edu/software/tophat/index.shtml). Differentially expressed genes between the NAT control and TSD cell lines were retrieved according to twofold (1 × log2), P<0.01 differential regulation criteria (Supplementary Table S1).

Testing for overrepresented Interpro domains and GO terms was performed using the default Wallenius approximation method using a 0.05 false discovery rate cutoff (Benjamini and Hochberg, 1995). Overrepresented interpro domains are given in Supplementary Table S2. Protein family (pfam domains) assignment was used from the T. pseudonana JGI genome website (http://genome.jgi-psf.org/Thaps3/Thaps3.info.html) to designate predicted functions for differentially regulated genes.

Silacidin and size regulation in T. pseudonana under batch cultivation vs experimental evolution

T. pseudonana was cultivated semi-continuously at 22 °C and 9 °C, by performing transfers every third day, before cultures reached stationary phase. Prior to this, cultures were maintained in batch culture conditions. Samples were taken from the T. pseudonana used to inoculate the experimental cultures before the start of the experiment (T0, batch cultivation), as well as after 300 generations (T300, experimental evolution) at each of two temperature regimes (T0-22 °C; T300-22 °C; T300-9 °C). Light microscope imaging and transcriptome sequencing were performed as described above. Transcript abundances were calculated as reads per kilobase of gene model per million mapped reads. Abundances for the silacidin gene ID 268311, silaffin 1 and 3 (IDs 11 366 and 25 921, respectively), actin-like housekeeping gene (ID 269504, Durkin et al., 2009), and genes associated with silacidin deregulation in TSD1 and 3 under both late exponential and silicon-starved conditions identified by transcriptome sequencing (IDs 23685, 8616, 7349, 23671, 23686, 7435, 9840, 7353, 264048, 263350, 3898, 8776, 12137, 6886, 6681, 7687, 8615 and 9371) were extracted for each sample. Extensive analysis of this experimental evolution study is in preparation for further publication (Schmidt et al., in prep).

Sequencing of silacidin homologues from centric diatom species

Primers were designed based on the T. pseudonana mRNA sequence from RACE-PCR (Richthammer et al., 2011) and targeted the 5’ and 3’ untranslated regions as well as internal exon sequences. PCR was challenging owing to the highly repetitive nature of the silacidin gene sequence. Primers (shown in Supplementary Table S3) were used in each of the four possible combinations. Hot-start, touch-down PCRs in a final volume of 50 μl with 1 mg ml−1 bovine serum albumin, were performed with an initial denaturation of 95 °C for 10 min, after which Taq polymerase, dNTPs and primers were added, followed by a touch-down phase of either 15 or 45 cycles of 95 °C for 30 s, 65–50 °C for 30 s and 72 °C for 1 min, followed by 15 or 45 cycles of of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min bringing the total number of cycles to 60. A final extension step of 72 °C for 10 min before amplicons were electrophoresed on a 2% agar TBE gel, and the longest amplicons for each species were selected for sequencing. Amplicons were cloned into the PCR2.1 sequencing vector using the Invitrogen original TA cloning kit for sequencing (Eurofins, Luxembourg, Europe). Obtained sequences were translated to amino acid sequence using the ExPASy translate tool (http://web.expasy.org/translate/) and aligned with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Ribosomal 18 S PCRs were performed according to Jahn et al. (2014) for all species to confirm that all samples were compatible with PCR.

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