Rapid evolution of a sexual reproduction gene in centric diatoms of the genus Thalassiosira

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene beta-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.     

Structural and inhibition insights into carbonic anhydrase CDCA1 from the marine diatom Thalassiosira weissflogii

Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.     

The active site structure of Thalassiosira weissflogii carbonic anhydrase 1

X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.     

Fatty acid desaturases from the microalga Thalassiosira pseudonana

Analysis of a draft nuclear genome sequence of the diatom Thalassiosira pseudonana revealed the presence of 11 open reading frames showing significant similarity to functionally characterized fatty acid front-end desaturases. The corresponding genes occupy discrete chromosomal locations as determined by comparison with the recently published genome sequence. Phylogenetic analysis showed that two of the T. pseudonana desaturase (Tpdes) sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain. Among the nine remaining gene sequences, temporal expression analysis revealed that seven were expressed in T. pseudonana cells. One of these, TpdesN, was previously characterized as encoding a Delta11-desaturase active on palmitic acid. From the six remaining putative desaturase genes, we report here that three, TpdesI, TpdesO and TpdesK, respectively encode Delta6-, Delta5- and Delta4-desaturases involved in production of the health beneficial polyunsaturated fatty acid DHA (docosahexaenoic acid). Furthermore, we show that one of the remaining genes, TpdesB, encodes a Delta8-sphingolipid desaturase with strong preference for dihydroxylated substrates.     

Gene expression induced by copper stress in the diatom Thalassiosira pseudonana

Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by approximately 500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.     

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.     

THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS

Results of past studies of the pH-dependent Si uptake kinetics of Phaeodactylum tricornutum Bohlin suggested that the anion SiO(OH)     is the chemical form of dissolved Si taken up by marine diatoms. We determined the chemical form of Si taken up by three other marine diatom species and P. tricornutum by examining the kinetics of Si use under two dramatically different SiO(OH)     :Si(OH)₄ ratios in seawater by varying pH from ≈8 to ≈9.6. Uptake rates were determined using a precise and sensitive ³²Si tracer methodology. The pH-dependent uptake kinetics obtained for all species except P. tricornutum suggest that marine diatoms transport Si(OH)₄. The half-saturation constant (K _m ) varies strongly as a function of pH for all species when the substrate of transport is assumed to be SiO(OH)     . Kinetic curves for Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, Thalassiosira weissflogii (Grunow) G. Fryxell et Hasle, and Cylindrotheca fusiformis Reimann et Lewin have statistically identical values of K _m at each pH when the substrate for transport is assumed to be Si(OH)₄ ( T. pseudonana and T. weissflogii ) or total dissolved silicon ( C. fusiformis ). In contrast, P. tricornutum exhibits unusual biphasic uptake kinetics: uptake conforms to Michaelis–Menten kinetics up to 15 to 25 μM, above which uptake increases linearly. This enigmatic response may have biased conclusions drawn from past experiments using this species. However, based on the consistency of the results for the three other species, a new model of Si transport in marine diatoms is proposed on the basis of the direct formation of a complex between the Si-transport protein and Si(OH)₄.     

ASSAY OPTIMIZATION AND REGULATION OF UREASE ACTIVITY IN TWO MARINE DIATOMS

An in vitro urease enzyme assay was developed for the marine diatoms Thalassiosira pseudonana Hasle et Heimdal (clone 3H) and T. weissflogii (Grunow) Fryxell et Hasle (clone Actin). This assay involves the colorimetric measurement of ammonium following the hydrolysis of urea in crude cell homogenates and it is the first assay to account for the rate of nitrogen assimilation in both species grown on urea as the sole nitrogen source. Urease activity was found to be present regardless of nitrogen source, although activities showed distinctly different patterns depending on the species examined and form of nitrogen supplied. Under nitrogen-replete conditions, urease activity in T. pseudonana was present constitutively when grown on NH₄⁺ and upregulated when grown on NO₃⁻ or urea. In nitrogen-replete T. weissflogii , urease activity was present at high constitutive levels regardless of the nitrogen source and showed no upregulation. Nitrogen starvation did not upregulate activity in either species.     

Characterization of a modular, cell-surface protein and identification of a new gene family in the diatom Thalassiosira pseudonana

We report the characterization of a cell-surface protein isolated from copper-stressed cells of the centric diatom Thalassiosira pseudonana Hasle and Heimdal (CCMP 1335). This protein has an apparent molecular weight of 100kDa and is highly acidic. The 100kDa protein (p100) sequence is comprised almost entirely of a novel domain termed TpRCR for T. pseudonana repetitive cysteine-rich domain, that is repeated 8 times and that contains conserved aromatic, acidic, and potential metal-binding amino acids. The analysis of the T. pseudonana genome suggests that p100 belongs to a large family of modular proteins that consist of a variable number of TpRCR domain repeats. Based on cell surface biotinylation and antibody data, p100 appears to migrate more rapidly with SDS-PAGE when extracted from cells exposed to high levels of copper; however, the discovery of a large family of TpRCR domain-containing proteins leaves open the possibility that the antibody may be cross-reacting with members of this protein family that are responding differently to copper. The response of the gene encoding p100 at the mRNA level during synchronized progression through the normal cell cycle is similar to previously characterized genes in T. pseudonana encoding cell wall proteins called silaffins.     

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.     

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.     

光强对两种硅藻光合作用、碳酸酐酶和RubisCO活性的影响

为研究海洋浮游硅藻光合固碳能力与光强的关系, 以三角褐指藻和威氏海链藻为实验材料, 测定了不同光强培养下三角褐指藻和威氏海链藻生长、光合特性、碳酸酐酶和核酮糖-1, 5-二磷酸羧化/氧化酶活性(RubisCO)的变化, 结果显示高光强促进两种硅藻的生长, 但对威氏海链藻的影响更明显。高光强导致两种硅藻叶绿素a、c含量、光系统Ⅱ的最大光化学效率和实际光化学效率明显下降, 非光化学淬灭系数明显升高, 但对光化学淬灭系数并没有明显影响。在高光下威氏海链藻和三角褐指藻胞内外碳酸酐酶活性明显升高。在高光强下培养的威氏海链藻RubisCO活性明显高于低光下培养, 但三角褐指藻正好相反, 不管高光还是低光培养威氏海链藻RubisCO活性始终高于三角褐指藻。以上结果表明不同硅藻对光强变化的响应存在差异, 它们可以通过调节光合生理特征、光合固碳关键酶和CO2供应以适应光强的变化。       

Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.     

CARBONIC ANHYDRASE IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

The zinc metalloenzyme carbonic anhydrase plays a critical role in inorganic carbon acquisition in marine diatoms, thus conferring on zinc a key role in oceanic carbon cycling. As a first step in determining the location and function of carbonic anhydrase (CA) in Bacillariophyceae, we purified and partially sequenced CA from T. weissflogii (Gru) Fryxell et Hasle (TWCA1) and cloned the corresponding cDNA (twca1). The twca1 sequence is different from other known algal carbonic anhydrase genes, and encodes a protein of roughly 34 kDa. The amino terminal amino acids sequenced from purified TWCA1 are 72 residues downstream of the putative starting methionine predicted by twca1. This difference may be due to the presence of a short-lived signal sequence designed to guide the enzyme to the correct cellular location. The absence of any homology between TWCA1 and previously sequenced CAs from Chlorophyceae may indicate either convergent evolution or that carbon acquisition represents a fundamental physiological difference among algal phyla.     

Silica morphogenesis by alternative processing of silaffins in the diatom Thalassiosira pseudonana

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-specific, mostly porous patterns in the nano- to micrometer range. Recently, from the diatom Cylindrotheca fusiformis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and implicated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific silica structures has so far been hampered by the difficulty of obtaining structural data from these extremely complex proteins. In the present study, the five major silaffins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, functionally analyzed, and structurally characterized, mak- ing use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and post-translational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the formation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.     

Comparative genomics of the pennate diatom Phaeodactylum tricornutum

Diatoms are one of the most important constituents of phytoplankton communities in aquatic environments, but in spite of this, only recently have large-scale diatom-sequencing projects been undertaken. With the genome of the centric species Thalassiosira pseudonana available since mid-2004, accumulating sequence information for a pennate model species appears a natural subsequent aim. We have generated over 12,000 expressed sequence tags (ESTs) from the pennate diatom Phaeodactylum tricornutum, and upon assembly into a nonredundant set, 5,108 sequences were obtained. Significant similarity (E < 1E-04) to entries in the GenBank nonredundant protein database, the COG profile database, and the Pfam protein domains database were detected, respectively, in 45.0%, 21.5%, and 37.1% of the nonredundant collection of sequences. This information was employed to functionally annotate the P. tricornutum nonredundant set and to create an internet-accessible queryable diatom EST database. The nonredundant collection was then compared to the putative complete proteomes of the green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the centric diatom T. pseudonana. A number of intriguing differences were identified between the pennate and the centric diatoms concerning activities of relevance for general cell metabolism, e.g. genes involved in carbon-concentrating mechanisms, cytosolic acetyl-Coenzyme A production, and fructose-1,6-bisphosphate metabolism. Finally, codon usage and utilization of C and G relative to gene expression (as measured by EST redundance) were studied, and preferences for utilization of C and CpG doublets were noted among the P. tricornutum EST coding sequences.