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Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Author(s): Faktorova, Drahomira; Nisbet, R Ellen R; Fernandez Robledo, Jose A; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E; Ares, Manuel; Areste, Cristina; Balestreri, Cecilia; Barbrook, Adrian C; Beardslee, Patrick; Bender, Sara; Booth, David S; Bouget, Francois-Yves; Bowler, Chris; Breglia, Susana A; Brownlee, Colin; Burger, Gertraud; Cerutti, Heriberto; Cesaroni, Rachele; Chiurillo, Miguel A; Clemente, Thomas; Coles, Duncan B; Collier, Jackie L; Cooney, Elizabeth C; Coyne, Kathryn; Docampo, Roberto; Dupont, Christopher L; Edgcomb, Virginia; Einarsson, Elin; Elustondo, Pia A; Federici, Fernan; Freire-Beneitez, Veronica; Freyria, Nastasia J; Fukuda, Kodai; Garcia, Paulo A; Girguis, Peter R; Gomaa, Fatma; Gornik, Sebastian G; Guo, Jian; Hampl, Vladimir; Hanawa, Yutaka; Haro-Contreras, Esteban R; Hehenberger, Elisabeth; Highfield, Andrea; Hirakawa, Yoshihisa; Hopes, Amanda; Howe, Christopher J; Hu, Ian; Ibanez, Jorge; Irwin, Nicholas AT; Ishii, Yuu; Janowicz, Natalia Ewa; Jones, Adam C; Kachale, Ambar; Fujimura-Kamada, Konomi; Kaur, Binnypreet; Kaye, Jonathan Z; Kazana, Eleanna; Keeling, Patrick J; King, Nicole; Klobutcher, Lawrence A; Lander, Noelia; Lassadi, Imen; Li, Zhuhong; Lin, Senjie; Lozano, Jean-Claude; Luan, Fulei; Maruyama, Shinichiro; Matute, Tamara; Miceli, Cristina; Minagawa, Jun; Moosburner, Mark; Najle, Sebastian R; Nanjappa, Deepak; Nimmo, Isabel C; Noble, Luke; Novak Vanclova, Anna MG; Nowacki, Mariusz; Nunez, Isaac; Pain, Arnab; Piersanti, Angela; Pucciarelli, Sandra; Pyrih, Jan; Rest, Joshua S | Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nitrogen assimilation is a highly regulated process requiring metabolic coordination of enzymes and pathways in the cytosol, chloroplast, and mitochondria. Previous studies of prasinophyte genomes revealed that genes encoding nitrate and ammonium transporters have a complex evolutionary history involving both vertical and horizontal transmission. Here we examine the evolutionary history of well-conserved nitrogen-assimilating enzymes to determine if a similar complex history is observed. Phylogenetic analyses suggest that genes encoding glutamine synthetase (GS) III in the prasinophytes evolved by horizontal gene transfer from a member of the heterokonts. In contrast, genes encoding GSIIE, a canonical vascular plant and green algal enzyme, were found in the Micromonas genomes but have been lost from Ostreococcus. Phylogenetic analyses placed the Micromonas GSIIs in a larger chlorophyte/vascular plant clade; a similar topology was observed for ferredoxin-dependent nitrite reductase (Fd-NiR), indicating the genes encoding GSII and Fd-NiR in these prasinophytes evolved via vertical transmission. Our results show that genes encoding the nitrogen-assimilating enzymes in Micromonas and Ostreococcus have been differentially lost and as well as recruited from different evolutionary lineages, suggesting that the regulation of nitrogen assimilation in prasinophytes will differ from other green algae.

We examined the diurnal expression of five genes encoding nitrogen-assimilating enzymes in the marine diatom Thalassiosira pseudonana (Hust.) Hasle et Heimdal following a transition from NH4 (+) - to NO3 (-) -supplemented media. The accumulation of nia transcripts (encoding nitrate reductase, NR) following the transition to NO3 (-) -supplemented media was similar to previously reported changes in NR abundance and activity. Nia mRNA levels varied diurnally, and the diurnal oscillations were abolished when cells were transferred to continuous light. Genes encoding chloroplastic (niiA) and cytosolic (nirB) nitrite reductases were identified in the genome of T. pseudonana. NiiA and nirB transcript levels increased within 2 h following the addition of NO3 (-) and varied diurnally. Patterns of diurnal variation in nia, niiA, and glnII (encoding the chloroplast-localized glutamine synthetase) mRNA abundances were similar. NirB and glnN (encoding the cytosolic-localized glutamine synthetase) mRNA levels also oscillated diurnally; however, the oscillation was out of phase with nia, niiA, and glnII. We propose that NO3 (-) is assimilated into organic molecules in both the chloroplast and cytosol of diatoms and that enzymes encoded by nirB and glnN contribute to the ecologically important dark assimilation of NO3 (-) observed in marine diatoms. As with nia, the diurnal variations in niiA, nirB, glnII, and glnN were abolished when cells were transferred to continuous light. Our results demonstrate that transcript accumulation is not circadian controlled, but, rather, changes in metabolic pools triggered by light:dark (L:D) transitions may be important in regulating the cellular mRNA levels encoding these key nitrogen assimilating enzymes.

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum-likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well-supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile-haptophyte-cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.

Glutamine synthetase (GS) is encoded by three distinct gene families (GSI, GSII, and GSIII) that are broadly distributed among the three domains of life. Previous studies established that GSII and GSIII isoenzymes were expressed in diatoms; however, less is known about the distribution and evolution of the gene families in other chromalveolate lineages. Thus, GSII cDNA sequences were isolated from three cryptophytes (Guillardia theta D. R. A. Hill et Wetherbee, Cryptomonas phaseolus Skuja, and Pyrenomonas helgolandii Santore), and GSIII was sequenced from G. theta. Red algal GSII sequences were obtained from Bangia atropurpurea (Mertens ex Roth) C. Agardh; Compsopogon caeruleus (Balbis ex C. Agardh) Mont.; Flintiella sanguinaria F. D. Ott and Porphyridium aerugineum Geitler; Rhodella violacea (Kornmann) Wehrmeyer and Dixoniella grisea (Geitler) J. L. Scott, S. T. Broadwater, B. D. Saunders, J. P. Thomas et P. W. Gabrielson; and Stylonema alsidii (Zanardini) K. M. Drew. In Bayesian inference and maximum-likelihood (ML) phylogenetic analyses, chromalveolate GSII sequences formed a weakly supported clade that nested among sequences from glaucophytes, red algae, green algae, and plants. Red algal GSII sequences formed two distinct clades. The largest clade contained representatives from the Cyanidiophytina and Rhodophytina and grouped with plants and green algae. The smaller clade (C. caeruleus, Porphyra yezoensis, and S. alsidii) nested within the chromalveolates, although its placement was unresolved. Chromalveolate GSIII sequences formed a well-supported clade in Bayesian and ML phylogenies, and mitochondrial transit peptides were identified in many of the sequences. There was strong support for a stramenopile-haptophyte-cryptophyte GSIII clade in which the cryptophyte sequence diverged from the deepest node. Overall, the evolutionary history of the GS gene families within the algae is complex with evidence for the presence of orthologous and paralogous sequences, ancient and recent gene duplications, gene losses and replacements, and the potential for both endosymbiotic and lateral gene transfers.

Nrt2 encodes a transporter (NRT2) that facilitates nitrate assimilation in most lineages of Dikarya. We used degenerate PCR to examine nrt2 sequence diversity and phylogeny in three clades of mushroom-forming basidiomycetes (Hebeloma, Hydnangiaceae and Psathyrellaceae) and tested for positive selection on nrt2 in each lineage. We investigated the relative expression of nrt2 paralogs in Hebeloma helodes and compared the patterns of expression between three Hebeloma and one Gymnopilus species, with quantitative PCR. Four lines of evidence of functional diversification in the nitrate assimilation system in mushroom-forming fungi emerged: (1) paralogs of nrt2 were recovered in Hebeloma and Hydnangiaceae, but distribution was not complete; (2) two paralogs in H. helodes appear to be differently expressed; (3) structural differences in NRT2 are apparent between paralogs and lineages; and (4) nrt2 is under significant positive selection in each lineage, and at least 6 sites are identified as under positive selection in the ectomycorrhizal family, Hydnangiaceae.

We provide an analysis of the invasion and spread of the container inhabiting mosquitoes Aedes aegypti and Aedes albopictus in the Bermuda Islands. Considered eradicated in the mid-1960s, A. aegypti was redetected in 1997, and A. albopictus was first detected in 2000. Based on weekly ovitrap data collected during the early stages of the invasion, we mapped the spread of Aedes throughout the islands. We analyzed the effects of buildings and roads on mosquito density and found a significant association between density and distance to roads, but not to buildings. We discuss the potential role of human transport in the rapid spread in the islands. The temporal correlation in ovitrap collection values decreased progressively, suggesting that habitat degradation due to control efforts were responsible for local shifts in mosquito densities. We report a sharp decrease in A. aegypti presence and abundance after the arrival of A. albopictus in the year 2000. Possible mechanisms for this rapid decline at relatively low density of the second invader are discussed in the context of classical competition theory and earlier experimental results from Florida, as well as alternative explanations. We suggest that support for the competition hypothesis to account for the decline of A. aegypti is ambiguous and likely to be an incomplete explanation.

We examined the diurnal expression of five genes encoding nitrogen-assimilating enzymes in the marine diatom Thalassiosira pseudonana (Hust.) Hasle et Heimdal following a transition from NH4 (+) - to NO3 (-) -supplemented media. The accumulation of nia transcripts (encoding nitrate reductase, NR) following the transition to NO3 (-) -supplemented media was similar to previously reported changes in NR abundance and activity. Nia mRNA levels varied diurnally, and the diurnal oscillations were abolished when cells were transferred to continuous light. Genes encoding chloroplastic (niiA) and cytosolic (nirB) nitrite reductases were identified in the genome of T. pseudonana. NiiA and nirB transcript levels increased within 2 h following the addition of NO3 (-) and varied diurnally. Patterns of diurnal variation in nia, niiA, and glnII (encoding the chloroplast-localized glutamine synthetase) mRNA abundances were similar. NirB and glnN (encoding the cytosolic-localized glutamine synthetase) mRNA levels also oscillated diurnally; however, the oscillation was out of phase with nia, niiA, and glnII. We propose that NO3 (-) is assimilated into organic molecules in both the chloroplast and cytosol of diatoms and that enzymes encoded by nirB and glnN contribute to the ecologically important dark assimilation of NO3 (-) observed in marine diatoms. As with nia, the diurnal variations in niiA, nirB, glnII, and glnN were abolished when cells were transferred to continuous light. Our results demonstrate that transcript accumulation is not circadian controlled, but, rather, changes in metabolic pools triggered by light:dark (L:D) transitions may be important in regulating the cellular mRNA levels encoding these key nitrogen assimilating enzymes.

An abnormal growth form called mound has been hypothesized to be a neoplasm in the filamentous fungus Schizophyllum commune. An alternative hypothesis is that mounds represent some unusual developmental form in the fruiting body morphogenetic pathway. Hydrophobin proteins have been found in fruiting bodies where they line the surface of gas exchange pores and function to keep the pores hydrophobic. To further determine possible relationships between mounds and fruiting bodies, mound tissue was examined for gas exchange pores and the presence of hydrophobins. Cryoscanning electron microscopic images revealed the presence of channels in mound tissue and presumptive hydrophobin rodlets similar to the air channels in fruiting bodies. Hydrophobin gene expression was also measured in mound tissue using quantitative real-time PCR and showed both monokaryotic and dikaryotic mound tissue exhibited high expression of the dikaryotic specific Sc4 hydrophobin gene. In contrast, Sc4 hydrophobin expression was barely detectable in monokaryotic fruiting bodies. The expression of Sc4 hydrophobin genes in mounds suggests mound development uses this aspect of the dikaryotic fruiting developmental pathway.

To understand the enhanced ability of marine diatoms to assimilate nitrogen (N), we measured changes in the transcript abundance and enzyme activity of glutamine synthetase (GS), one of the key enzymes that link carbon (C) and N metabolism, in the common diatom Skeletonema costatum (Greville) Cleve. Transcript abundance of glnII (the gene that encodes the GSII isoenzyme), measured by quantitative reverse transcriptase-PCR, and total GS activity increased 2 to 3.5 times above background in the cells taking up nitrate (NO 3 - ) but not the cells taking up ammonium (NH 4 + ). A background level of glnII mRNA was maintained at a steady level up to 15 days of N starvation before decreasing to below detection after 21 days. These results confirm that transcription of glnII is induced to assimilate NH 4 + derived from reduction of NO 3 - . Because of this role of GSII in diatoms assimilating NH 4 + derived from NO 3 - reduction rather than from the environmental NH 4 + , quantification of glnII mRNA promises to be a useful indication of new production by phytoplankton.

Regulation and evolution of dinoflagellate luciferases are of particular interest since the enzyme is structurally unique and bioluminescence is under circadian control. In this study, three new members of the dinoflagellate luciferase gene family were identified and characterized from Pyrocystis lunula. These genes, lcfA, lcfB, and lcfC, also exhibit the unusual structure and organization previously reported for the luciferase gene of a related dinoflagellate, Lingulodinium polyedrum: three repeated domains, each encoding an active catalytic site, multiple gene copies, and tandem organization. The histidine residues involved in the pH regulation of L. polyedrum luciferase activity, and implicated in the regulation of flashing, are also fully conserved in P. lunula. The interspecific conservation between the individual luciferase domains of P. lunula and L. polyedrum is higher than among domains intramolecularly, indicating that this unique gene structure arose through duplication events that occurred prior to the divergence of these dinoflagellates. However, P. lunula luciferase genes differ from L. polyedrum in several respects, notably, the occurrence of an intron in one gene (lcfC), a 2.25-kb intergenic region connecting lcfA and lcfB, and, of particular interest, an invariant rate of synonymous (silent) substitutions along the repeat domains, in contrast to L. polyedrum luciferase, where the occurrence of synonymous substitutions is practically absent in the central region of the domains.

Regulation of antioxidant enzymes is critical to control the levels of reactive oxygen species in cell compartments highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from Lingulodinium polyedrum (formerly Gonyaulax polyedra) under different physiological conditions. A cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing high sequence similarity to cyanobacterial, algal, and plant Fe-SODs. Under standard growth conditions, on a 12:12-h light-dark cycle, Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of activity and cellular abundance with the maximum occurring during the middle of the light phase. Northern analyses showed that this rhythmicity is not related to changes in Fe-SOD mRNA levels, indicative of translational regulation. By contrast, conditions of metal-induced oxidative stress resulted in higher levels of Fe-SOD transcripts, suggesting that transcriptional control is responsible for increased protein and activity levels. Daily (circadian) and metal-induced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways, allowing biochemically distinct changes appropriate to oxidative challenges.

Background: Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results: Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion: Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function., Plymouth State University Graduate Programs Office, Woods Hole Oceanographic Institution Academic Programs Office, New Hampshire IDeA Network of Biological Research Excellence (NH-INBRE), National Center for Research Resources (U.S.) (Grant 5P20RR030360-03), National Institute of General Medical Sciences (U.S.) (Grant 8P20GM103506-03)

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4+, 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later-eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.

Multi-locus DNA fingerprints using an M13 probe were obtained for two individuals of Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California, and their laboratory-reared offspring. DNA was extracted from each of two field-collected individuals (= parents), their self-fertilized diploid offspring (three and seven individuals), and one diploid individual resulting from spores of the two parents. A total of 20 bands (4–19 kb) was detected among all individuals, ranging from 7 to 14 bands for any one individual. Two bands were present in all individuals, and three bands were unique to one parent and its three progeny. Ten bands were observed in the out-crossed individual, of which three were inherited from one parent, two from the other parent, and five were present in both parents. Genetic similarity between each parent and their self-fertilized offspring was significantly higher than similarity between the two self-fertilized groups. The data show that multi-locus DNA fingerprints can be used to assess parentage in the giant kelp and that there is consistent agreement between genetic similarity and known genetic relatedness among parents and offspring.

Multi-locus DNA fingerprints using an M13 probe were obtained for eight individuals of giant kelp Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California. For each individual, DNA was extracted from a diploid blade and from ca. 109 haploid spores that were released from four to Jive sporophylls. Viable or swimming spores from one individual were pooled and referred to as a spore group. A total of 34 bands (4–19 kb) was detected in DNA fingerprints from the eight blades and eight spore groups, with individual blade or spore groups exhibiting 7–18 bands (mean = 12.6). One band (4.5 kb) was present in all 16 samples. Eight bands were detected in 11–14 of the 16 samples. Similarity indices were calculated for all pairwise comparisons of fingerprint bands among all possible combinations of blades and spore groups. Mean similarity indices for the eight blades (0.51, SE = 0.032) and spore groups (0.56, SE = 0.031) were significantly lower than for the eight comparisons of the blade and spore groups from a single individual (0.86, SE = 0.052). The data indicate that DNA fingerprints can be used to measure genetic variation within populations of M. pyrifera because variation of DNA fingerprints associated with meiotic products (spores) of a given individual is small relative to variation observed among individuals within the population. Additionally, fingerprint variation between diploid vegetative tissue and haploid meiotic products may be a measure of genetic change due to recombination or DNA turnover mechanisms.

Glutamine synthetase (GS), which catalyzes the formation of glutamine from ammonium and glutamate in the presence of ATP, is encoded by three distinct gene families: GSI, GSII, and GSIII. Genes encoding GSI are found in the Bacteria and Archaea, whereas GSII genes are found in eukaryotes and a few species of Bacteria. Members of the third family, GSIII, have been described from a limited number of bacteria; however, recent biochemical and molecular data suggest that this type of enzyme is broadly distributed among the algae. Peptide fragments obtained from GS purified from the marine diatom Skeletonema costatum (Greville) Cleve are 77% identical to a partial sequence of GSIII from Chaetoceros compressum Lauder, which permits the unambiguous assignment of the biochemically characterized enzyme to the GSIII gene family. The N-terminal sequence was 43% identical to the GSIII-like enzyme purified from the haptophyte Emiliania huxleyi (Lohm.) Hay et Miller and several residues were conserved among bacterial and eukaryotic GSIII enzymes. The presence of genes encoding GSIII in diatoms and haptophytes indicates that this enzyme family is more broadly distributed in eukaryotes than previously suspected.

We report the sequence-based characterization and expression patterns of three manganese peroxidase genes from the white rot fungus and grape vine pathogen Fomitiporia mediterranea (Agaricomycotina, Hymenochaetales), termed Fmmnp1 , Fmmnp2 , and Fmmnp3 . The predicted open reading frames (ORFs) are 1,516-, 1,351-, and 1,345-bp long and are interrupted by seven, four, and four introns, respectively. The deduced amino acid sequences encode manganese peroxidases (EC 1.11.1.13) containing 371, 369, and 371 residues, respectively, and are similar to the manganese peroxidases of the model white rot organism Phanerochaete chrysosporium . The expression of the genes is most likely differentially regulated, as revealed by real-time PCR analysis. Phylogenetic analysis reveals that other members of the order Hymenochaetales harbor mnp genes encoding proteins that are related only distantly to those of F. mediterranea . Furthermore, multiple partial lip - and mnp -like sequences obtained for Pycnoporus cinnabarinus (Agaricomycotina, Polyporales) suggest that lignin degradation by white rot taxa relies heavily on ligninolytic peroxidases and is not efficiently achieved by laccases only.

THE taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins. Prochlorophytes were originally proposed (with concomitant scepticism1–3) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts4–6. Results from molecular sequence phytogenies, however, have suggested that Prochlorothrix hollandica7 is not on a lineage that leads to plastids8–12. Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus mariniis13,14 and theLissoclinum patella symbiont Prochloron sp.15, indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts. This implies that the three prochlorophytes and the green chloro-plast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events. We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A (ref. 16)), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans17–19.

Although the endosymbiotic evolution of chloroplasts through primary and secondary associations is well established, the evolutionary timing and stability of the secondary endosymbiotic events is less well resolved. Heterokonts include both photosynthetic and nonphotosynthetic members and the nonphotosynthetic lineages branch basally in phylogenetic reconstructions. Molecular and morphological data indicate that heterokont chloroplasts evolved via a secondary endosymbiosis, involving a heterotrophic host cell and a photosynthetic ancestor of the red algae and this endosymbiotic event may have preceded the divergence of heterokonts and alveolates. If photosynthesis evolved early in this lineage, nuclear genomes of the nonphotosynthetic groups may contain genes that are not essential to photosynthesis but were derived from the endosymbiont genome through gene transfer. These genes offer the potential to trace the evolutionary history of chloroplast gains and losses within these lineages. Glutamine synthetase (GS) is essential for ammonium assimilation and glutamine biosynthesis in all organisms. Three paralogous gene families (GSI, GSII, and GSIII) have been identified and are broadly distributed among prokaryotic and eukaryotic lineages. In diatoms (Heterokonta), the nuclear-encoded chloroplast and cytosolic-localized GS isoforms are encoded by members of the GSII and GSIII family, respectively. Here, we explore the evolutionary history of GSII in both photosynthetic and nonphotosynthetic heterokonts, red algae, and other eukaryotes. GSII cDNA sequences were obtained from two species of oomycetes by polymerase chain reaction amplification. Additional GSII sequences from eukaryotes and bacteria were obtained from publicly available databases and genome projects. Bayesian inference and maximum likelihood phylogenetic analyses of GSII provided strong support for the monophyly of heterokonts, rhodophytes, chlorophytes, and plants and strong to moderate support for the Opisthokonts. Although the phylogeny is reflective of the unikont/bikont division of eukaryotes, we propose based on the robustness of the phylogenetic analyses that the heterokont GSII gene evolved via endosymbiotic gene transfer from the nucleus of the red-algal endosymbiont to the nucleus of the host. The lack of GSIII sequences in the oomycetes examined here further suggests that the GSIII gene that functions in the cytosol of photosynthetic heterokonts was replaced by the endosymbiont-derived GSII gene.

Gonyaulax luciferase is a single-chain ( approximately 137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each). Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments ( approximately 35 kDa) of luciferase are active. The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8. While the activity loss at higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual domains. The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves similar to that of the full-length enzyme. For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-activity profiles mimicked that of the proteolytic fragments reported earlier. Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at high pH. This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence.

Assimilation of nitrogen into structural and functional macromolecules is a critical process in all living organisms. Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH4+ and glutamate thus coupling nitrogen assimilation with carbon metabolism. The GS superfamily is comprised of three gene families: GSI, GSII, and GSIII. Representatives of the three gene families are found in prokaryotes, suggesting the genes are paralogous. In contrast, knowledge of the distribution and evolution of the GS gene families in eukaryotes is limited. The endosymbiotic origin of plastids is well established. Phylogenetic analyses of genes distributed among photosynthetic and non-photosynthetic organisms may provide insight into the timing and stability of the endosymbiotic events. GSII has been characterized from a variety of prokaryotic and eukaryotic species, including the stramenopiles, Skeletonema costatum and Phytophthora infestans (AAN31463). I evaluated several a priori hypotheses regarding the placement of the stramenopile GSII sequences in addition to performing unconstrained analyses. In all analyses, GSII from S. costatum and P. infestans grouped together. Also, there was strong to moderate support for a ‘protist+plant’ clade, which included the vascular plants, photosynthetic algae, and the non-photosynthetic P. infestans. Although it remains unresolved whether GSII was transferred from the nucleus of the endosymbiont early in the evolution of stramenopiles, the continued accumulation of GSII sequences from diverse eukaryotes should allow us to rigorously evaluate this hypothesis in the future.

Bioluminescence is broadly distributed in marine dinoflagellates and has been intensively studied in Lingulodinium (Gonyaulax) polyedra. In this species, bioluminescence is regulated in a circadian fashion; the enzyme (luciferase) and the luciferin (substrate)-binding protein are synthesized and degraded on a daily basis. Synthesis of both proteins is regulated at the level of translation. The L. polyedra luciferase gene is composed of three contiguous domains that are greater than 75% identical at the nucleic acid level. Possible explanations for the high degree of sequence conservation include: (1) the domains evolved through a recent duplication event; (2) the sequence similarity is maintained by a molecular process such as gene conversion; or (3) there is a functional role associated with the primary nucleic acid sequence, such as in the translational regulation of luciferase expression. The phylogenetic relationship of dinoflagellates predicted from 18S rDNA genes provides a framework for examining the molecular evolution of the regulation of luciferase expression and of genes encoding luciferase and the luciferin-binding protein. In particular, we are examining the evolution of the circadian rhythm of bioluminescence and of luciferase abundance, the presence/absence of the luciferin-binding protein, and the molecular structure of the luciferase gene. We anticipate that this approach will distinguish between regions of the luciferase molecule that are conserved for enzyme function versus those concerned with the regulation of protein expression. In addition, it will provide insight into the evolution of the regulatory processes and pathways.

Deepened isotherms associated with El Nino resulted in severe nutrient limitation and very low kelp productivity during the last half of 1983. Frond growth rates were so low that terminal blades formed before reaching the surface, eliminating the canopy. Frond initiation rates were also extremely low, resulting in significant reductions in mean plant size. Plants growing above 10 m were more severely affected than plants at 20 m. These results suggest that nutrient pulses associated with internal waves are critical for survival of Macrocystis pyrifera in nutritionally marginal habitats in southern California.

Macrofauna living on subtidal rocks reefs in southern California excrete ammonium, a potentially important nutrient for benthic algae. Ammonium excretion rates of eleven macroinvertebrate and five fish taxa were determined from a total of 324 in situ incubations conducted between October 1984 and August 1985 at 14 to 17 m depths off Santa Catalina Island, California. Total ammonium excretion ranged from over 100 μmol h-1 by the kelp bass Paralabrax clathratus to less than 0.1 μmol h-1 by the gastropod Conus californicus. Weight-specific ammonium excretion generally ranged from 0.5 to 4 μmol g-1 h-1 in invertebrates and from 3 to 7 μmol g-1 h-1 in fishes. Intraspecific excretion rates varied substantially. Coefficient of variation of excretion rates were higher than reported for laboratory studies and multiple regression indicated that 50 to 90% of the variation in ammonium excretion rates of five species studied in detail could not be explained by the combined variation in dry weight, water temperature, time of day, and incubation dates. The excretion data, along with estimates of population densities and size-frequency distributions, indicate that benthic macrofauna release a total of 25 to 30 μmol NH 4 + m-2 h-1 both day and night. The species that generally make the largest contributions are a gobiid fish (Lythrypnus dalli), followed by three gastropods (Astraea undosa, Tegula eiseni, and T. aureotincta) and a sea urchin (Centrostephanus coronatus). The amount of ammonium excreted by these macrofauna on rocky reefs is insignificant compared to our previously published data on the nighttime excretion of blacksmith (Chromis punctipinnis), a pomacentrid fish that feeds in the water column during the day and shelters on the reef at night. Including blacksmiths, we estimate that the amount released by rocky-reef macrofauna at night is >280 μmol m-2 h-1, a rate that is similar to that for many other marine communities. Additional studies are required to determine if benthic algae utilize ammonium released by these macrofauna, especially at night.

Background Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida. Results GSII sequences were isolated from four species of green algae (Trebouxiophyceae), and additional green algal (Chlorophyceae and Prasinophytae) and streptophyte (Charales, Desmidiales, Bryophyta, Marchantiophyta, Lycopodiophyta and Tracheophyta) sequences were obtained from public databases. In Bayesian and maximum likelihood analyses, eubacterial (GSIIB) and eukaryotic (GSIIE) GSII sequences formed distinct clades. Both GSIIB and GSIIE were found in chlorophytes and early-diverging streptophytes. The GSIIB enzymes from these groups formed a well-supported sister clade with the γ-Proteobacteria, providing evidence that GSIIB in the Chloroplastida arose by horizontal gene transfer (HGT). Bayesian relaxed molecular clock analyses suggest that GSIIB and GSIIE coexisted for an extended period of time but it is unclear whether the proposed HGT happened prior to or after the divergence of the primary endosymbiotic lineages (the Archaeplastida). However, GSIIB genes have not been identified in glaucophytes or red algae, favoring the hypothesis that GSIIB was gained after the divergence of the primary endosymbiotic lineages. Duplicate copies of the GSIIB gene were present in Chlamydomonas reinhardtii, Volvox carteri f. nagariensis, and Physcomitrella patens. Both GSIIB proteins in C. reinhardtii and V. carteri f. nagariensis had N-terminal transit sequences, indicating they are targeted to the chloroplast or mitochondrion. In contrast, GSIIB proteins of P. patens lacked transit sequences, suggesting a cytosolic function. GSIIB sequences were absent in vascular plants where the duplication of GSIIE replaced the function of GSIIB. Conclusions Phylogenetic evidence suggests GSIIB in Chloroplastida evolved by HGT, possibly after the divergence of the primary endosymbiotic lineages. Thus while multiple GS isoenzymes are common among members of the Chloroplastida, the isoenzymes may have evolved via different evolutionary processes. The acquisition of essential enzymes by HGT may provide rapid changes in biochemical capacity and therefore be favored by natural selection.