Mark Hildebrand

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the worlds largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research.

Diatoms, Biomineralization Processes, and Genomics

Diatoms are eukaryotic unicellular microalgae with cell walls made of a composite of organic material, and silica which is often intricately and ornately shaped (Figure 1). Diatoms take up silicon from the environment in soluble form as silicic acid, transport it into the cell, and during the period of cell wall synthesis catalyze its polymerization into silica. A striking feature is the diversity of structures that diatoms make on the nanoto microscale (Figure 1), which is indicative of the molecular control of intracellular processes by which organics facilitate mineral formation. Diatom species are classified according to their cell wall structures, and the estimated number of species is at least in the tens of thousands and perhaps more. On a global scale, diatoms are the predominant contributors to biosilica formation in the oceans, which in total is estimated at 240 × 10 mol per year. Diatoms have been a long-standing favorite of microscopists, and with the advent of electron microscopy came the ability to image at the nanoand microscale not only cell surface details by SEM but intracellular features by TEM. Two seminal publications in 1990, by Pickett-Heaps et al. and Round et al., summarized detailed observationally-based models of diatom silicification. The work described in these reviews defined processes in formation of the mineral; however, molecular-level understanding of the organics involved was minimal. The rate of advance in the field slowed thereafter because of the time required to develop molecular approaches for diatoms. Because of the time delay, there was a discontinuity in the number of research groups in the field, and as a result, the earlier literature may not be completely assimilated by current researchers. For this reason, several aspects of this article will discuss new “discoveries” of which at least the general concepts were appreciated decades ago. It is hoped that the pioneers in the field understand the need for refreshing the concepts that they may have initially developed as newer researchers begin to experience them firsthand. It is also important to appreciate the validity of earlier work, which cannot be discounted just because molecular-level characterization was not possible at that time. Current-day models and concepts should fit within the wealth of observational data that was accumulated earlier. Examination of both macroand molecular-scale * To whom correspondence should be addressed. Phone: (858) 822-0167. Fax: (858) 534-7313. E-mail: mhildebrand@ucsd.edu. Chem. Rev. 2008, 108, 4855–4874 4855

Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth

Significance As global CO2 levels rise and fossil fuel abundance decreases, the development of alternative fuels becomes increasingly imperative. Biologically derived fuels, and specifically those from microalgae, are promising sources, but improvements throughout the production process are required to reduce cost. Increasing lipid yields in microalgae without compromising growth has great potential to improve economic feasibility. We report that disrupting lipid catabolism is a practical approach to increase lipid yields in microalgae without affecting growth or biomass. We developed transgenic strains through targeted metabolic engineering that show increased lipid accumulation, biomass, and lipid yields. The target enzyme’s ubiquity suggests that this approach can be applied broadly to improve the economic feasibility of algal biofuels in other groups of microalgae. Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type–like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis: sequences, expression analysis, and identification of homologs in other diatoms.

Abstract The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.

The place of diatoms in the biofuels industry

In spite of attractive attributes, diatoms are underrepresented in research and literature related to the development of microalgal biofuels. Diatoms are highly diverse and have substantial evolutionarily-based differences in cellular organization and metabolic processes relative to chlorophytes. Diatoms have tremendous ecological success, with typically higher productivity than other algal classes, which may relate to cellular factors discussed in this review. Diatoms can accumulate lipid equivalently or to a greater extent than other algal classes, and can rapidly induce triacylglycerol under Si limitation, avoiding the detrimental effects on photosynthesis, gene expression and protein content associated with N limitation. Diatoms have been grown on production scales for aquaculture for decades, produce value-added products and are amenable to omic and genetic manipulation approaches. In this article, we highlight beneficial attributes and address potential concerns of diatoms as biofuels research and production organisms, and encourage a greater emphasis on their development in the biofuels arena.

Silicon Uptake in Diatoms Revisited: A Model for Saturable and Nonsaturable Uptake Kinetics and the Role of Silicon Transporters

The silicic acid uptake kinetics of diatoms were studied to provide a mechanistic explanation for previous work demonstrating both nonsaturable and Michaelis-Menten-type saturable uptake. Using 68Ge(OH)4 as a radiotracer for Si(OH)4, we showed a time-dependent transition from nonsaturable to saturable uptake kinetics in multiple diatom species. In cells grown under silicon (Si)-replete conditions, Si(OH)4 uptake was initially nonsaturable but became saturable over time. Cells prestarved for Si for 24 h exhibited immediate saturable kinetics. Data suggest nonsaturability was due to surge uptake when intracellular Si pool capacity was high, and saturability occurred when equilibrium was achieved between pool capacity and cell wall silica incorporation. In Thalassiosira pseudonana at low Si(OH)4 concentrations, uptake followed sigmoidal kinetics, indicating regulation by an allosteric mechanism. Competition of Si(OH)4 uptake with Ge(OH)4 suggested uptake at low Si(OH)4 concentrations was mediated by Si transporters. At high Si(OH)4, competition experiments and nonsaturability indicated uptake was not carrier mediated and occurred by diffusion. Zinc did not appear to be directly involved in Si(OH)4 uptake, in contrast to a previous suggestion. A model for Si(OH)4 uptake in diatoms is presented that proposes two control mechanisms: active transport by Si transporters at low Si(OH)4 and diffusional transport controlled by the capacity of intracellular pools in relation to cell wall silica incorporation at high Si(OH)4. The model integrates kinetic and equilibrium components of diatom Si(OH)4 uptake and consistently explains results in this and previous investigations.

Identification of Proteins from a Cell Wall Fraction of the Diatom Thalassiosira pseudonana Insights into Silica Structure Formation

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process.

NITRATE TRANSPORTER GENES FROM THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE): mRNA LEVELS CONTROLLED BY NITROGEN SOURCE AND BY THE CELL CYCLE

The molecular characterization of components involved in nitrate uptake and assimilation in phytoplankton is likely to provide new insights into these processes, their regulation, and their effect on primary production. We report the cloning and initial characterization of the first nitrate transporter genes in a marine organism, from the diatom Cylindrotheca fusiformis Reimann et Lewin. A clone isolated from a silicon‐responsive cDNA library was shown by sequence comparison to encode a homolog of high‐affinity nitrate transporters. The C. fusiformis nitrate transporter cDNA was named NAT (NitrAte Transporter). The NAT cDNA was used to isolate a genomic clone that contained two additional nitrate transporter genes, NAT1 and NAT2, arranged in tandem. The cDNA and two genomic sequences were highly conserved, and only 18 of 1446 nucleotides in the coding region differed. At least four copies of NAT genes were present in C. fusiformis and as shown by hybridization, multiple copies were present in other diatom species. The transcript abundance of NAT genes in cultures with different nitrogen sources was monitored by RNase protection assays. NAT mRNA levels were high in the presence of nitrate, at nearly the same level during nitrogen starvation, and also high in urea‐containing cultures. Lower mRNA levels occurred in nitrite‐grown cultures. NAT transcript levels were highly repressed with NH4Cl or NH4NO3 as the nitrogen source, although very low amounts were detected. These results suggested that monitoring NAT mRNA levels could serve as a marker for (1) nitrate uptake in nitrate medium, (2) nitrogen starvation, and (3) ammonium use by virtue of absence of expression. NAT mRNA levels were not directly regulated by light or dark, but were apparently related to cellular growth and protein synthesis. Using light/dark synchronized cultures to monitor cell cycle responses, NAT mRNA levels were high in early G1 phase, decreased through the remainder of G1, then increased during DNA synthesis in S phase and into G2, and finally decreased after M phase. In silicon‐starvation synchronized cultures, levels were high at the G1/S phase boundary, high throughout S and G2, and finally decreased after M phase. It was clear that NAT expression, and by inference nitrate uptake, did not occur at continuous levels throughout the cell cycle. The results of the RNase protection experiments suggested that transcriptional regulation is a major contributing factor in the control of diatom nitrate uptake. The cloning of the C. fusiformis nitrate transporter genes provides a new tool for investigating diatom nitrogen uptake and metabolism. In addition, the regulation of NAT expression by nitrogen source is likely to be useful in developing techniques to specifically control the expression of genes fused to NAT regulatory sequences in transgenic diatoms.

Biological processing of nanostructured silica in diatoms

Abstract The most outstanding example of biological processing of silicon occurs in the unicellular algae known as diatoms. The diatom cell wall contains nanostructured silica with features exceeding current manufacturing capabilities, reproduced with exactness in vast numbers. Such structures must result from specific molecular interactions between cellular components and silicon, and larger scale movements of the intracellular compartment where silica polymerization occurs, the silica deposition vesicle (SDV). New insights into diatom silicification have arisen from recent characterization of the molecular components involved. We have isolated genes encoding silicic acid transporters (SITs) responsible for transport across the cell’s lipid bilayer membrane. The SITs are the only proteins shown to specifically interact with silicon, and a major goal is to identify amino acids responsible for silicic acid recognition and binding. Long-chain polyamines, both free and peptide-attached, apparently induce silica polymerization in diatoms. These compounds have not been shown to have direct control over the formation of larger-scale structure, but observations suggest some involvement. Movements and molding of the SDV during silicification, driven by the cytoskeleton, are major determinants of silica macrostructure. We have applied and are developing techniques to elucidate the molecular mechanisms underlying diatom silicification. This investigation could inspire biomimetic approaches, or lead to the specific manipulation of silicified diatom structures for direct application in nanostructured materials syntheses. These materials are not limited to being silica-based; recent work using shape-preserving displacement reactions has converted diatom silica into an inorganic metal oxide, while maintaining the detailed silica morphology.

COMPARATIVE SEQUENCE ANALYSIS OF DIATOM SILICON TRANSPORTERS: TOWARD A MECHANISTIC MODEL OF SILICON TRANSPORT†

Silicon is an important element in biology, for organisms ranging from unicellular algae to humans. It acts as a structural material for both plants and animals, but can also function as a metabolite or regulator of gene expression, affecting a wide range of cellular processes. Molecular details of biological interaction with silicon are poorly understood. Diatoms, the largest group of silicifying organisms, are a good model system for studying this interaction. The first proteins shown to directly interact with silicon were diatom silicon transporters (SITs). Because the basis for substrate recognition lies within the primary sequence of a protein, identification of conserved amino acid residues would provide insight into the mechanism of SIT function. Lack of SIT sequences from a diversity of diatoms and high sequence conservation in known SITs has precluded identification of such residues. In this study, PCR was used to amplify partial SIT sequences from eight diverse diatom species. Multiple gene copies were prevalent in each species, and phylogenetic analysis showed that SITs generally group according to species. In addition to partial SIT sequences, full‐length SIT genes were identified from the pennate diatom, Nitzschia alba (Lewin and Lewin), and the centric diatom Skeletonema costatum (Greville) Cleve. Comparing these SITs with previously identified SITs showed structural differences between SITs of centrics and pennates, suggesting differences in transport mechanism or regulation. Comparative amino acid analysis identified conserved regions that may be important for silicon transport, including repeats of the motif GXQ. A model for silicon uptake and efflux is presented that is consistent with known aspects of transport.