Deborah L. Robertson

Different regulatory mechanisms modulate the expression of a dinoflagellate iron-superoxide dismutase.

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) fromLingulodinium 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.

Aedes aegypti and Aedes albopictus in Bermuda: extinction, invasion, invasion and extinction

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.

UNRAVELING THE REGULATION OF NITROGEN ASSIMILATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE): DIURNAL VARIATIONS IN TRANSCRIPT LEVELS FOR FIVE GENES INVOLVED IN NITROGEN ASSIMILATION 1

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.

Isolation and Characterization of Glutamine Synthetase from the Marine Diatom Skeletonema costatum

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.

Characterization of Three mnp Genes of Fomitiporia mediterranea and Report of Additional Class II Peroxidases in the Order Hymenochaetales

ABSTRACT 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.

GLUTAMINE SYNTHETASE IN MARINE ALGAE: NEW SURPRISES FROM AN OLD ENZYME

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.

Ammonium excretion by macroinvertebrates and fishes on a subtidal rocky reef 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 NH4+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.

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

BackgroundGlutamine 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.ResultsGSII 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, Volvoxcarteri f. nagariensis, and Physcomitrellapatens. 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.ConclusionsPhylogenetic 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.

Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

BackgroundPseudo-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.ResultsThrough 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.ConclusionOur 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.

Molecular Evolution of Nitrogen Assimilatory Enzymes in Marine Prasinophytes

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.