The Florida Red Tide Dinoflagellate Karenia Brevis

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G Model HARALG-488; No of Pages 11

Harmful Algae xxx (2009) xxx–xxx

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Harmful Algae

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Review The Florida red tide dinoﬂagellate Karenia brevis: New insights into cellular and molecular processes underlying bloom dynamics

Frances M. Van Dolah a,*, Kristy B. Lidie a, Emily A. Monroe a, Debashish Bhattacharya b, Lisa Campbell c, Gregory J. Doucette a, Daniel Kamykowski d a Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Resarch, Charleston, SC, United States b Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa, Iowa City, IA, United States c Department of Oceanography, Texas A&M University, College Station, TX, United States d Department of Marine, Earth and Atmospheric Sciences, North Carolina State University, Raleigh, NC, United States

ARTICLE INFO ABSTRACT

Article history: The dinoflagellate Karenia brevis is responsible for nearly annual red tides in the Gulf of Mexico that Available online xxx cause extensive marine mortalities and human illness due to the production of brevetoxins. Although the mechanisms regulating its bloom dynamics and toxicity have received considerable attention, Keywords: investigation into these processes at the cellular and molecular level has only begun in earnest during Bacterial–algal interactions the past decade. This review provides an overview of the recent advances in our understanding of the Cell cycle cellular and molecular biology on K. brevis. Several molecular resources developed for K. brevis, including Dinoflagellate cDNA and genomic DNA libraries, DNA microarrays, metagenomic libraries, and probes for population Florida red tide genetics, have revolutionized our ability to investigate fundamental questions about K. brevis biology. Genome Two cellular processes have received particular attention, the vegetative cell cycle and vertical migration Harmful algal bloom behavior, which are of key importance due to their roles in the development of both surface populations Horizontal gene transfer Karenia brevis that constitute blooms and subsurface cell aggregations that may serve to initiate them. High throughput Population genetics sequencing of cDNA libraries has provided the first glimpse of the gene repertoire in K. brevis, with Vertical migration approximately 12,000 unique genes identified to date. Phylogenomic analysis of these genes has revealed a high rate of horizontal gene transfer in K. brevis, which has resulted in a chimeric chloroplast through the selective retention of genes of red, green, and haptophyte origin, whose adaptive significance is not yet clear. Gene expression studies using DNA microarrays have demonstrated a prevalence of post-transcriptional gene regulation in K. brevis and led to the discovery of an unusual spliced leader trans-splicing mechanism. Among the trans-spliced gene transcripts are type I polyketide synthases (PKSs), implicated in brevetoxin biosynthesis, which are unique among type I PKSs in that each transcript encodes an individual catalytic domain, suggesting a novel gene structure in this dinoflagellate. Clone libraries of 16S ribosomal DNA sequences developed from bloom waters have unveiled the temporal and spatial complexity of the microbial soup that coexists with K. brevis and its active involvement in both bloom growth and termination processes. Finally, the development and application of population genetic markers has revealed a surprisingly high genetic diversity in K. brevis blooms, long assumed to consist of essentially clonal populations. With these foundations in place, our understanding of K. brevis bloom dynamics is likely to grow exponentially in the next few years. Published by Elsevier B.V.

Contents

1. Introduction ...... 000 2. Cellular processes central to bloom growth ...... 000 2.1. Vegetative cell cycle...... 000 2.2. Vertical migration behavior ...... 000 3. The K. brevis genome...... 000 3.1. The expressed genome...... 000 3.2. K. brevis genome evolution ...... 000

* Corresponding author at: NOAA CCEHBR, 219 Fort Johnson Road, Charleston, SC 29412, United States. Tel.: +1 843 762 8529; fax: +1 843 762 8700. E-mail address: [email protected] (F.M. Van Dolah).

1568-9883/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.hal.2008.11.004

Please cite this article in press as: Van Dolah, F.M., et al., The Florida red tide dinoﬂagellate Karenia brevis: New insights into cellular and molecular processes underlying bloom dynamics. Harmful Algae (2009), doi:10.1016/j.hal.2008.11.004 G Model HARALG-488; No of Pages 11

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4. Insights into global gene expression...... 000 4.1. Photoperiodic control of gene expression ...... 000 4.2. Gene expression in response to acute stress...... 000 4.3. An RNA trans-splicing mechanism facilitates post-transcriptional RNA processing ...... 000 5. Mechanisms regulating toxicity ...... 000 6. Interactions of K. brevis with microbial community...... 000 7. Population genetics of K. brevis—what constitutes a bloom? ...... 000 8. Future directions ...... 000 References...... 000

1. Introduction (Van Dolah et al., 2007). In both lab and field populations, essentially all cells in a population are in G1 at the onset of the light Red tides have been documented in the Gulf of Mexico since phase or day. S-phase begins a minimum of 6 h into the light and 1530 when the Spanish explorer Cabeza de Vaca reported fish kills continues for 12–14 h, mitosis occurs between 18 and 22 h after and discolored water in what is now Tampa Bay (Steidinger et al., dawn, and the cell cycle is generally completed by the start of the 1998). In 1948 the phenomenon of water discoloration and its next day. This pattern was shown to be under circadian control, as attendant fish kills were attributed to an undescribed dinofla- it persists in continuous light (Brunelle et al., 2007). Circadian gellate, which was named Gymnodinium breve (Davis, 1948). entrainment appears to be mediated by the dark/light transition or Because of its extensive adverse impacts on coastal environments ‘‘dawn’’ cue, as delaying the onset of light results in a delay in S- and communities, the growth physiology and toxicity of this phase entry of an equivalent length of time (Van Dolah and dinoflagellate (renamed Ptychodiscus brevis and most recently Leighfield, 1999). Karenia brevis) have now been under study for almost 60 years. Given that S-phase entry is the key event controlling cell cycle However, research on the underlying molecular biology has only progression, the mechanisms that mediate this process play a been initiated in the last decade, enabled by the availability of significant role in regulating bloom growth. Using antibodies and rapidly evolving molecular technologies from the biomedical field. inhibitor studies, two conserved eukaryotic cell cycle regulators, The processes contributing to the development of K. brevis blooms cyclin and cyclin-dependent kinase, were shown to regulate both span spatial and temporal scales from the molecular mechanisms S- and M-phase entry in K. brevis (Van Dolah and Leighfield, 1999; within the dinoflagellate cell that respond to environmental cues Barbier et al., 2003). The presence of these conserved cell cycle to the oceanographic and even atmospheric processes that drive proteins provided the basis from which to begin investigating the the local environmental conditions in which K. brevis lives. signaling pathways relaying the ‘‘dawn’’ cue to the cell cycle and to Significant progress has been made towards understanding the identify cell cycle regulatory mechanisms that may lend them- large-scale oceanographic processes that support the growth of K. selves as biomarkers of bloom status or targets for control brevis blooms and dictate their movement on the west Florida strategies. In Euglena, cAMP signaling appears to mediate diel cell shelf, as reviewed by Walsh et al. (2006). However, further cycle entrainment (Carrie and Edmunds, 1993). Blocking cAMP refinement of these models requires the integration of cellular protein kinase activity inhibits cell cycle progression in the responses with these physical forcings. Bloom initiation and bloom dinoflagellate Amphidiuium operculatum the (Leighfield and Van termination processes remain only poorly understood, but are Dolah, 2001). However, it does not appear to be involved in the critical for the prediction and management of bloom impacts. Lack immediate recognition of the dark/light cue, but rather may work of information on these processes may in large part be because downstream of that event to promote the G1/S phase transition. In they require a more thorough understanding of the cellular level K. brevis, a cryptochrome blue light receptor was identified that is a decisions through which K. brevis interacts with its environment. candidate for recognition of the dawn cue, given the differential This review provides an overview of the advances made over the effects of blue and red light on S-phase entry (Brunelle et al., 2007). past decade in our understanding of the cellular and molecular However, the signaling pathways associated with this receptor are processes in K. brevis that provide the necessary underpinning to not currently known, and a role for cryptochrome in the observed further refine our understanding of bloom dynamics. blue light effects awaits confirmation.

2. Cellular processes central to bloom growth 2.2. Vertical migration behavior 2.1. Vegetative cell cycle The second cellular process that appears to play an equally Blooms of K. brevis proceed primarily through vegetative cell critical role in the growth, movement, and possibly initiation of K. division. Therefore, understanding the mechanisms that regulate brevis blooms is its diel vertical migration behavior. K. brevis cells the vegetative cell cycle and the environmental cues to which it are not simply passive particles carried by turbulence or ocean responds is important in order to understand bloom progression. currents, but rather are able to use vertical migration to maximize In many phytoplankton, the cell cycle is entrained to the carbon fixation from photosynthesis near the surface during the photoperiod, resulting in synchronous (1 division [div] d1)or day and dissolved nutrients at depth during the night. K. brevis can 1 phased cell division (<1 div day1). Cell division rates observed in attain swimming speeds of up to 1 m h (McKay et al., 2006). both lab and field populations of K. brevis average 0.3 day1 with a Maximal swimming speed is attained during the day (McKay et al., maximum of approximately 0.6 day1 (Van Dolah et al., 2007), 2006), and appears to be driven by phototaxis and geotaxis although synchronous division has been achieved in laboratory (Kamykowski et al., 1998b). columns by repeated removal of the bottom third of the culture Under nutrient replete conditions in the laboratory, K. brevis (Kamykowski et al., 1998a). Phased cell division was shown to vertical migration appears to be under circadian control, as surface occur only during the dark by Heil (1986) and the relative timing of accumulation continues during the subjective day when cells are the remaining cell cycle phases was subsequently determined in shifted to constant dark (Heil, 1986). Downward swimming does lab cultures (Van Dolah and Leighfield, 1999) and field populations not appear to be as directed: at night cells display a swimming

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nutrients from coastal water egress or fish kills may maintain populations in near surface waters (Walsh et al., 2004). Aggregated cells can also occur near the sediment interface at night where near-bottom nutrients either associated with upwelling plumes that reside below the euphotic zone or sediment pore waters may be utilized (Janowitz and Kamykowski, 2006). Sinclair et al. (2006a) demonstrated that K. brevis cells are capable of enhanced dark nitrate uptake after 12 h of nitrate deprivation that simulated an ascent into a nitrate depleted water column, while Sinclair et al. (2006b) demonstrated that K. brevis cells aggregate in a near bottom nitrate-rich layer if the overlying water column is nitrate depleted. More recently, Sinclair and Kamykowski (2007) observed that K. brevis cells will selectively swim through a thin sediment barrier set on a 100 mm mesh that split a plastic column into a nitrate depleted upper layer and a nitrate enriched lower layer. These laboratory studies indicate that K. brevis can actively seek nutrients and support the hypothesis that offshore, near-bottom aggregates of K. brevis may provide cell populations for bloom initiation, as upwelling favorable winds transport these populations toward the coast and as nearshore fronts interact with cell 1 Fig. 1. K. brevis cell counts (cells ml ) taken during the ECOHAB 2000 cruise (2–3 migrations to serve as an aggregation point for near surface K. October) while following a near-surface drogue beginning at 00:00 h and every 2 h thereafter for 24 h at 5 m depth intervals. Based on a swimming speed of 1 m h1, brevis populations (Janowitz and Kamykowski, 2006). two subpopulations are depicted. The surface subpopulation aggregates at the surface during the day and disperses through the upper 10 m of the water column 3. The K. brevis genome at night; the near-bottom subpopulation aggregates at the bottom at night and disperses through the lower 10 m of the water column during the day. In the past decade significant investment has been made in developing the genomic resources with which to study the genetic pattern that contributes to a diffusion of cells throughout the upper structure and gene regulation critical to K. brevis bloom dynamics. water column (Kamykowski et al., 1998b). More recent work has Current molecular resources available for K. brevis are enumerated demonstrated that there is also an adaptive component to in Table 1 and their utility for furthering our understanding of K. swimming behavior that is responsive to the internal cellular brevis bloom dynamics is described below. conditions, perhaps set by threshold values for certain biochemical Like many dinoflagellates, K. brevis possesses a large haploid constituents or the C/N ratio (Yamazaki and Kamykowski, 2000). genome of 100 pg DNA cell1, or approximately 1 1011 bp (Kim Kamykowski et al. (1998a) found that approximately 50% of a K. and Martin, 1974; Rizzo et al., 1982; Sigee, 1984; Kamykowski brevis population migrated to the surface following a synchronous et al., 1998a), approximately 30 times the human genome. Unique cell division. Differences in major biochemical constituents were among eukaryotes, dinoflagellates have permanently condensed found in mid-water populations of K. brevis versus those that had chromatin which lacks nucleosomes typically involved in regulat- migrated to the surface during the day following division, with ing chromosome condensation and eukaryotic gene expression. carbohydrate, lipid, protein and chlorophyll a concentrations The condensed chromosomes of K. brevis have a characteristic generally higher in the cells found at mid-water than in surface banding pattern present in other dinoflagellates (Rizzo et al., cells (Kamykowski et al., 1998a, 1999). This suggested that 1982). This striated structure is proposed to result from formation daughter cells receive unequal shares of the parental resources of a liquid cholesteric DNA crystal in which parallel bundles of DNA and that this differential allocation influences their vertical filaments form stacked disks that make a continuous left-handed migration behavior, where the nutrient poor daughters are more twist along the chromosome’s longitudinal axis (Bouligand and positively phototactic, thus facilitating their more rapid acquisi- Norris, 2001). Basic DNA-binding proteins are probably involved in tion of biochemical stores through increased photosynthesis rates. stabilizing this structure by neutralizing local electronegative The diel patterns found in biochemical constituents do not reflect charges that would result from tightly compacted DNA filaments simple acquisition rates, however. For example, Evens et al. (2001) (Hackett et al., 2005). At the periphery of these disks are loops of showed that the diel pattern of neutral lipid content is responsive DNA that are less tightly compacted and comprise the actively to light intensity exposure, but Schaeffer (2006) found that light transcribed DNA (Sigee, 1984; Bhaud et al., 2000). This unique intensity alone was insufficient to fully explain the diel pattern chromosome structure suggests that dinoflagellates may also have among different lipid components. The internal cues that trigger evolved novel mechanisms for regulating gene expression and differential swimming behavior likely involve the integration of indeed some unusual genome characteristics and regulatory various metabolic and signaling pathways in K. brevis, that have mechanisms in K. brevis described below may relate to this only recently begun to be identified, and their further character- structure. ization will be facilitated by the molecular biological approaches described below. 3.1. The expressed genome This adaptive swimming behavior – the ‘‘choice’’ to ascend or not depending upon both external conditions and internal The large size of the K. brevis genome makes it unfeasible to biochemical stores – may explain the vertical distributions of obtain a complete genome sequence with current technologies. cells sometimes observed within K. brevis bloom patches studied However, the sequencing of cDNA libraries is an efficient approach over a diel cycle (Fig. 1). In the field, K. brevis blooms often occur in to discovering expressed genes in organisms for which genomic water columns under a broad range of light intensities to which data are unavailable, and this approach has been undertaken by they can readily adapt (Evens et al., 2001; Schaeffer et al., 2007) two research groups. Lidie et al. (2005) analyzed a collection of and in which the nutrient source is not obvious. During these 7000 expressed sequence tags (ESTs) from log phase K. brevis times, aggregated cells can occur near fronts in daylight where grown in nutrient replete conditions which provided the first

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Table 1 Molecular resources available for research on K. brevis.

Tool Description Reference cDNA libraries/EST collections Log phase, nutrient replete, Wilson isolate 15,000 50 reads; 5000 30 reads Lidie et al. (2005) Stress, Wilson isolate 10,000 50 reads; 10,000 30 reads Van Dolah et al. (unpublished) 18,000 30 reads Crawford et al. (unpublished)

DNA microarray 8 K feature oligonucleotide array 4629 unique probes; 3463 replicates Lidie et al. (2005) 11 K feature oligonucleotide array 10,192 unique gene sequences Van Dolah et al. (2007)

Metagenomics tools Bacterial 16S rDNA clone library Isolated from bloom and non-bloom water Mikulski et al. (unpublished)

Population genetics tools LSU rRNA markers 28S hypervariable regions D1–D2 Mikulski et al. (2005), Goodwin et al. (2005) SSU rRNA markers 18S coding region and ITS sequences Loret et al. (2002) Microsatellie markers 9 polymorphic loci Renshaw et al. (2006) 8 additional msat loci Henrichs et al. (unpublished) global insights into K. brevis genomic content. Approximately 29% because they can accommodate large insert sizes of >100 kb. In K. of the ESTs had similarity to known sequences in the GenBank brevis, however, we found that BAC insert sizes >50 kb yielded few database, which identified the presence of conserved genes viable transformants (Van Dolah et al., unpublished). This is involved in cell cycle control, intracellular signaling, stress probably due to nucleotide modifications in K. brevis DNA, such as responses, intermediary metabolism, transcription/translation hydroxymethyluracil substitutions for thymidine, which can stall machinery, and the photosystem. K. brevis ESTs have an average replication forks as the host bacterium seeks to repair this GC content of 51%, lower than that found in the dinoflagellates perceived error in the DNA strand (Otterlei et al., 1999). Consistent Alexandrium tamarense (60%, Hackett et al., 2005)orLingulodinium with this notion, end sequencing of the low frequency viable polyedrum (59%), but similar to Amphidinium (50%) and Crypthe- transformants with 100 kb inserts identified a high proportion of codinium cohnii (50%) (Codon Usage Database, Genbank Release plastid genes. The successful transformation of plastid sequences 140.0). The 7000 K. brevis ESTs analyzed formed 5280 unigene likely reflects the fact that the plastid does not utilize the modified clusters. Many gene clusters displayed single nucleotide poly- bases that are present in the dinoflagellate nuclear the genome. morphisms (SNPs), most frequently in the third codon position This was used to advantage to identify chloroplast-containing BAC such that the substitution does not alter protein sequence. Since K. clones for analysis of the K. brevis chloroplast genome, currently brevis is haploid, the presence of SNPs suggests the presence of underway. genes in multiple copies resulting from recent duplications. SNPs are similarly observed in L. polyedrum and A. carterae (Bachvaroff 3.2. K. brevis genome evolution et al., 2004) and in A. tamarense (Moustafa and Bhattacharya, unpublished), suggesting that these gene duplications may be Horizontal gene transfer (HGT) has been recognized as a major prevalent in all dinoflagellates. Few genes in dinoflagellates have force in prokaryotic evolution, contributing up to 24% of some been studied in detail, but those which have been occur in multiple bacterial genomes (Nelson et al., 1999). The recent availability of copies, and often in tandem repeats, including peridinin chlor- genomic data for eukaryotic protists has revealed a surprising ophyll binding (5000 copies in L. polyedrum; Le et al., 1997; degree of HGT also in the evolution of eukaryotic protists (e.g., Reichman et al., 2003), cyclic AMP dependent protein kinase (30 Andersson et al., 2007). In most cases the donor organism is the copies in L. polyedrum; Salois and Morse, 1997), and proliferating victim of engulfment by the recipient, sometimes leading to an cell nuclear antigen (41 copies in Pfiesteria piscicida; Zhang et al., endosymbiotic relationship. Plastids of photosynthetic eukaryotes 2006). were acquired by the endosymbiosis of a cyanobacterium (Fig. 2), To this EST collection (50 reads), an additional 5000 ESTs have whose genome was subsequently reduced by selected gene been obtained from the initial K. brevis library and 10,000 ESTs transfer to the nucleus of the host or by outright gene loss (e.g., were added from a ‘‘combined stress’’ cDNA library produced from Reyes-Prieto et al., 2006). The plastids most frequently found in cells grown under N- or P-limitation, cells exposed to heat, dinoflagellates, containing chlorophyll c2 and peridinin as the oxidative, or metal stresses, and cells in stationary phase (50 and 30 major carotenoid, were subsequently acquired through the reads). All sequences have been deposited in the Genbank dbEST secondary endosymbiosis of a red alga (i.e., the chromalveolate database. When combined with the original EST collection, the hypothesis, see Fig. 2; Bhattacharya et al., 2004). The plastid current collection of 50 ESTs clusters into 11,937 unigenes. A high genome of peridinin dinoflagellates is unlike that of any other rate of novel gene discovery in these libraries after 25,000 eukaryote, in that it is unusually highly reduced such that it is sequence reads suggests that this does not approach the entire comprised of a small number (14–16) of single-gene minicircles expressed genome. An additional K. brevis EST collection devel- (Zhang et al., 1999). In contrast, the K. brevis plastid contains oped by a second research group holds approximately 18,000 ESTs chlorophylls c1 + c2 and fucoxanthin, which are typical of representing approximately 6000 unique genes (30 reads; D. haptophyte algae, indicating that its plastid is a replacement of Crawford, unpublished). When the latter ESTs are publicly the peridinin plastid via a tertiary endosymbiosis (Tengs et al., released, the combined sequence data from these three datasets 2000; Ishida and Greene, 2002; Yoon et al., 2005). This finding will comprise the largest resource of expressed sequences from raises the question of what happened to the nuclear-encoded any dinoflagellate. plastid genes present in the ancestral peridinin dinoflagellate Although ESTs provide insight into expressed genes, a genomic genome? A comparison of the EST libraries from K. brevis with one library is an essential tool needed to gain insight into the structure from the peridinin dinoflagellate Alexandrium tamarense revealed and organization of specific genes or gene clusters. Bacterial the absence of the nuclear-encoded plastid genes that had been artificial chromosome (BAC) libraries are useful for this purpose transferred to the nucleus in the peridinin dinoflagellate (Hackett

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Fig. 2. The history of plastid endosymbioses in K. brevis. (A) The primordial algal plastid resulted from an ancient primary endosymbiosis that occurred ca. 1.5 billion years ago (Ga; Yoon et al., 2004). The cyanobacterial capture occurred in the common ancestor of the Plantae. Thereafter the common ancestor of chromalveolates (i.e., chromist + alveolate protists) putatively captured a red algal plastid through secondary endosymbiosis ca. 1.3 Ga. This was the progenitor of the broadly distributed peridinin plastid in dinoflagellates. Some dinoflagellates like K. brevis replaced the peridinin plastid with one of tertiary endosymbiotic origin (i.e., capture of a plastid from an alga containing a ‘‘secondary’’ plastid; see Bhattacharya et al., 2004). There are at least four different kinds of tertiary plastids in dinoflagellates with K. brevis containing one of haptophyte provenance. (B) The putative phylogeny of plastids described in (A) is supported by analysis of nuclear-encoded plastid-targeted proteins in Plantae and chromalveolates. This maximum likelihood tree, based on the analysis of a 5-protein concatenated data set (see Nosenko et al., 2006) shows the origin of these genes in the Plantae (RA, red algae + GR, green algae and land plants [the glaucophytes are missing]) ancestor from a cyanobacterial source (filled black circle, primary endosymbiosis), their subsequent origin in chromalveolates (HAP [haptophytes] + STR [Stramenopiles] + PD [peridinin dinoflagellates]) from a red algal source (filled blue circle, secondary endosymbiosis), and in K. brevis (FD, fucoxanthin dinoflagellate) from a haptophyte source (filled green circle, tertiary endosymbiosis). All branches have either (or both) a significant Bayesian posterior probability or a bootstrap support value >90%. et al., 2005). These results imply that the haptophyte endosym- bloom formation, the microarray was first used to investigate biosis resulted in a genome transformation in K. brevis, in which it global gene expression patterns under photoperiodic control (Van lost (or silenced) the nuclear-encoded plastid genes that had been Dolah et al., 2007). Transcript profiles identified by microarray transferred from the ancestral red algal secondary endosymbiont analysis are a composite of both changes in transcription and (Yoon et al., 2005). Phylogenomic and phylogenetic analyses show changes in RNA stability. This study found that 9.8% of genes were that the resulting proteome of the K. brevis plastid is a chimeric mix differentially expressed over the photoperiod, with most differ- of genes from red, haptophyte and green algal origin, acquired entially expressed genes exhibiting either peak or minimum from multiple sequential endosymbioses resulting in large-scale expression early in the dark. Both the overall pattern and the intracellular HGT (also known as endosymbiotic gene transfer; see proportion of genes showing differential expression is consistent Li et al., 2006; Reyes-Prieto et al., 2006) from the endosymbiont to with a microarray study carried out in Arabidopsis,inwhich11%of host nuclear genome (Nosenko et al., 2006), or HGT from external transcripts were found to be under photoperiodic control sources (Nosenko and Bhattacharya, unpublished). This unprece- (Schaffer et al., 2001). In K. brevis the differentially expressed dented genomic plastidity in dinoflagellates is likely facilitated by gene set had a surprisingly high representation (33%) of genes their myxotrophic behavior, where the ability to engulf other algae involved in post-transcriptional processing of RNA and protein and bacteria provides the opportunity to amass foreign genes in turnover, as compared to Arabidopsis (4%).Thisisofinterest the dinoflagellate nucleus. This trait may allow for rapid because of the prevalence of post-transcriptional control reported adaptation to changing environmental conditions, thereby increas- for circadian processes in dinoflagellates (Morse et al., 1989; ing their evolutionary fitness (e.g., see Nosenko et al., 2006). Mittag et al., 1998). Another predominant group of genes responding to light and dark were several photosystem genes 4. Insights into global gene expression and genes involved in energy acquisition. Most curiously, no change in the expression of cell cycle genes was observed under 4.1. Photoperiodic control of gene expression either the photoperiod (with 50% of cells cycling) or in constant light (70% of cells cycling). Given that the cell cycle is under The availability of the EST collections described above circadian control, this raises the question of whether the cell cycle facilitated the development of a DNA microarray containing genes that are in most eukaryotes under transcriptional control 4620 unique gene probes (Table 1; Lidie et al., 2005), which was are in fact post-transcriptionally regulated in K. brevis. Discerning more recently updated to include 10,263 unique gene probes from these mechanisms is critically important for the development both control and stress cDNA libraries described above. Because of of biomarkers for bloom physiology. If gene expression is its central importance to cellular processes involved in K. brevis regulated post-transcriptionally, PCR-based measurements for

6 F.M. Van Dolah et al. / Harmful Algae xxx (2009) xxx–xxx physiologically relevant markers will not be useful and antibody other stress related genes present on the array was not seen even based markers may be a more productive approach. under conditions previously shown to induce the expression at the protein level. This once again hints at post-transcriptional 4.2. Gene expression in response to acute stress regulation in K. brevis of cellular processes typically under transcriptional control in eukaryotic organisms. The ability of a K. brevis bloom to adapt to changing conditions in the coastal environment dictates its longevity and thus the 4.3. An RNA trans-splicing mechanism facilitates post-transcriptional severity of its negative impacts. Therefore, understanding RNA processing the physiological basis for adaptation of K. brevis to changing environmental conditions is a prerequisite for understanding The lack of transcriptional regulation of the cell cycle genes and bloom longevity and bloom termination. Cellular responses to acute stress responses suggests that K. brevis may utilize post- acute stress consist of a phylogenetically conserved gene network transcriptional mechanisms for regulating gene expression. termed the ‘‘environmental stress response’’ (Gasch et al., 2000). Trypanosomes, kinetoplastid protists only distantly related to Key components of this response include a temporary shutoff of dinoflagellates, provide a model for how such post-transcriptional general transcription, concurrent with the induction of tran- regulation may take place. Like dinoflagellates, trypanosomes lack scription and translation of conserved stress proteins that serve identifiable transcriptional regulatory elements such as TATA to maintain other proteins in their properly folded configuration boxes. Trypanosome RNA polymerase II transcription is constitu- and to repair or recycle damaged proteins while the cell adapts to tive and genes are transcribed in polycistronic units. To produce its altered conditions. Using Western blotting, Miller-Morey and mature mRNAs from these pre-RNAs, a short non-coding RNA Van Dolah (2004) identified several conserved stress proteins in sequence termed the spliced leader (SL) is trans-spliced onto a K. brevis including heat shock proteins and superoxide dismu- message at a splice acceptor site located in intergenic regions of the tases, which were responsive to heat or oxidative stress. Their polycistronic messages (Fig. 3). As a result, diverse mRNAs acquire presence and their induction over a time course typical for a common 50-sequence and, simultaneously, messages become eukaryotic cells suggested K. brevis possesses a typical environ- polyadenylated via a separate mechanism. The presence of mental stress response network. Lidie and Van Dolah (2007a) tandemly repeated genes in dinoflagellates and the demonstration used the K. brevis microarray to query global transcriptional that at least some of these are transcribed into polycistronic units responses to acute stresses including heat, peroxide, lead, and (e.g., Rubisco; Zhang and Lin, 2003) suggested that a similar sodium nitrite. In all cases, a hallmark decrease was seen in gene mechanism might exist in dinoflagellates. Indeed, Lidie and Van transcripts involved in ribosome generation, translation, and Dolah (2007a, 2007b) identified an identical 22 bp sequence on the energy acquisition, consistent with a general stress response that 50 end of 100 diverse ESTs in the libraries described above. includes a transient shut-off of general mRNA transcription. Concurrently, Zhang et al. (2007) identified the identical sequence However, transcription of stereotypical heat shock proteins and in several species of dinoflagellates from all taxonomic orders,

Fig. 3. The spliced leader trans-splicing mechanism in trypanosomes. A. (1) Polycistrinic message with three open reading frames (ORFs; thick bars) and intergenic regions (thin lines). (2) The spliced leader (dashed line) is added at a splice signal located 100 bp upstream from the start codon for each ORF, (3) simultaneous addition of a poly-A tail results in mature messages containing an identical 50cap and spliced leader, 50 UTR, coding sequence, 30 UTR, and poly-A tail. B. Twelve ESTs from K. brevis possessing identical 50 ends that represent the spliced leader. Sequences are variably truncated due to incomplete reverse transcription or cloning artifacts. The AG splice donor site is underlined.

F.M. Van Dolah et al. / Harmful Algae xxx (2009) xxx–xxx 7 primitive to derived, suggesting that this is a conserved feature of include keto reductase (KR), dehydratase (DH), and enoyl dinoflagellates that is absent from their nearest relatives the reductase (ER) domains that carry out reduction of the b-keto apicomplexans. The gene for this spliced leader was identified from group, and a thioesterase (TE) domain that terminates chain K. brevis genomic DNA and found to be associated with the 5S rRNA elongation. Each module catalyzes one Claisen condensation gene, consistent with the location of SL genes in other organisms and various reductions or modifications depending upon what known to use this mechanism (Drouin and Monis de Sa, 1995). catalytic domains are present in that module. Modular type I PKSs These findings suggest that K. brevis uses SL trans-splicing to build polyketides in an assembly line manner where each module process polycistrionic messages into mature mRNAs. The extent to is used once and the growing polyketide chain is passed to the which this mechanism is used by K. brevis or other dinoflagellates subsequent module for the next chain elongation (Khosla et al., is not certain because most of the sequences in cDNA libraries do 1999). not represent full-length transcripts. However, using PCR with SL- Stable isotope incorporation studies found unusual truncations and gene specific-primers, all K. brevis transcripts investigated to of acetyl groups in brevetoxin as well as methyl side chains, date, with the exception of plastid encoded genes, appear to derived from methionine, that indicate brevetoxin biosynthesis include the 50 SL sequence, including a number of the stress and may require atypical polyketide synthases (Lee et al., 1986, 1989; cell cycle genes described above. Since its discovery a decade ago, Chou and Shimizu, 1987; Shimizu et al., 2001). The carbon spliced leader trans-splicing has been identified in a variety of truncations could be carried out on a PKS via an epoxidation eukaryotic protists and primitive chordates, which utilize this followed by thioesterification, which would require the addition of mechanism to varying degrees (Davis, 1996). The precise role of epoxidase and thioesterase genes to the PKS gene or gene cluster this mechanism in dinoflagellates has not yet been elucidated, but (Shimizu et al., 2001). in trypanosomes the presence of the spliced leader appears to The first studies to identify the genes for brevetoxin biosynth- modulate mRNA stability or translation initiation (Maroney et al., esis in dinoflagellates used degenerate PCR primers to type I and 1995). Given the absence of nucleosomes and the compact type II KS domains (Snyder et al., 2003). PCR products from structure of dinoflagellate chromosomes, regulation of gene multiple dinoflagellate species were homologous with known type expression at the post-transcriptional level may be more efficient I PKSs. Phylogenetic analysis placed three of four sequences than regulation of transcription. obtained from K. brevis within a eukaryotic clade that included sequences of closely related apicomplexans (Snyder et al., 2003). 5. Mechanisms regulating toxicity The fourth sequence appeared to be of bacterial origin. Fluorescent in situ hybridization (FISH) was used to localize PKS genes K. brevis blooms are associated with extensive fish kills, marine following flow cytometry-sorting of cultures to separate K. brevis mammal, bird, and turtle mortalities and human respiratory and cells from bacterial cells. FISH localization suggested the bacterial food poisoning (Backer, this issue; Landsburg, this issue). These origin of some PKS genes and dinoflagellate origin of other PKS effects are attributed to the production of a suite of potent genes previously identified through PCR strategies (Snyder et al., excitatory neurotoxins, the brevetoxins (PbTx, after Pychodiscus 2005). brevis). At least nine brevetoxin congeners have been characterized Using a different approach, Monroe and Van Dolah (2008) in K. brevis that are derived from two carbon backbones, termed A identified eight additional PKS sequences in K. brevis through and B. PbTx congener composition changes during the course of a screening of the EST collections described above, including six KS growth curve, with PbTx2 dominating during log phase and PbTx3 domains, one KR domain and one sequence containing both ACP and PbTx1 increasing as cells move into stationary phase (Roszell and KS domains. Phylogenetic analysis indicated that these et al., 1990). Furthermore, during log phase growth PbTx is largely sequences were clearly of eukaryotic, and not bacterial origin. intracellular, whereas in stationary phase cultures, extracellular Full length gene transcripts were obtained from the ESTs by 50 and toxin becomes more prevalent and the total toxin concentration in 30 RACE (RNA amplification of cDNA ends), which demonstrated the culture increases (Roth et al., 2007). A similar trend is seen in the presence of eukaryotic signatures (30 untranslated region, natural blooms as they age (Pierce et al., 2001; Tester et al., 2008). poly(A+) tail) and the spliced leader sequence, described above, at Variation in cellular toxin quota of 10–70 pg cell1 in different the 50 terminus. These sequences are unusual in that each clonal isolates grown under identical conditions suggests genetic transcript encodes an individual domain, rather than a multi- variability in toxin biosynthetic capacity. Cellular toxin levels have domain protein seen in typical type I PKSs. The presence of the been reported to respond to environmental conditions, with spliced leader suggests PKS expression may be under post- approximately a threefold increase in cultures under either N- or P- transcriptional control. This is consistent with microarray studies limitation relative to nutrient replete conditions (Greene et al., carried out on K. brevis under N- and P-limitation (Monroe et al., 2000). Similarly, light (Greene et al., 2000) and salinity (Maier 2005) which showed no significant change in transcript abun- Brown et al., 2006) are reported to alter cellular toxin concentra- dance. The presence of separate transcripts for individual catalytic tions. domains further suggests a novel gene structure in dinoflagellates Brevetoxins are ladder-like all-trans-fused polycyclic ether (Fig. 4). Whereas type I PKS genes characterized to date encode polyketide compounds, a structure unique to dinoflagellates (Lin multiple modules containing many domains, the gene structure and Risk, 1981; Shimizu et al., 1986). Polyketides are synthesized suggested by the K. brevis PKS transcripts consists of separate PKS by complex enzymes called polyketide synthases (PKSs) that, genes encoding for discrete domains. These domains therefore beginning with acetyl CoA, carry out successive Claisen condensa- occur on separate polypeptide chains, more similar to the structure tions with additional acetate groups, to produce a growing of type II PKSs but with sequence similarity closest to type I. polyketide chain. PKSs are typically classified as type I, II, or III. Confirmation of the gene structure and organization of K. brevis Early work in dinoflagellate PKS (Snyder et al., 2003 see discussion PKSs now requires analysis of the genomic DNA libraries described below) suggests dinoflagellate polyketides are synthesized by type above. I enzymes. Type I PKSs are large multifunctional proteins organized into ‘‘modules’’, each containing multiple catalytic domains. The 6. Interactions of K. brevis with microbial community core catalytic domains within each module include the ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) It has become increasingly clear that the complex microbial that are necessary for chain elongation, while ‘‘optional’’ domains community within which K. brevis and other harmful algal blooms

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The strong representation of Bacteroidetes in the 16S rDNA library from K. brevis bloom water is of interest because many of the known algicidal bacteria are members of this group, primarily Cytophaga (nomenclature under revision) and Flavo- bacterium. Representatives of both Cytophaga (strain 41-DBG2; Doucette et al., 1999) and the Flavobacteriaceae (strain S03; Roth et al., 2008a) isolated from Gulf of Mexico waters have been demonstrated to exhibit algicidal activity against K. brevis. Characterization of algicidal activity demonstrated that the latter (S03) required direct contact with K. brevis in order to elicit its algicidal effects while the former (41-DBG2), which produced a Fig. 4. A. The structure of a modular type I polyketide synthase transcript encoding dissolved, heat labile algicide(s) (Twiner et al., 2004), did not. multiple modules, each containing several catalytic domains. B. Structure of K. Regardless of the mode of algicidal attack, lysis of K. brevis cells brevis polyketide synthase transcripts, which have sequence homology to type I PKSs, but generally encode only a single domain on a trans-spliced, polyadenylated by these two bacterial strains caused a release of dissolved message (KS, ketosynthase; AT, acyl transferase; DH, dehydratase; ER, enoyl brevetoxin (ca. 60% of the total toxin) into the surrounding water. reductase; KR, ketoreductase; ACP, acyl carrier protein). The majority of this dissolved toxin comprised open A-ring PbTx- 2 and -3 derivatives that were 100-fold less potent than their occur has significant effects on their development and termination respective parent toxins (Roth et al., 2007). These findings (Fukami et al., 1991; Doucette, 1995; Doucette et al., 1998; Skerratt suggest that attack of K. brevis by algicidal bacteria can lead to a et al., 2002). K. brevis exists in a sea of bacteria, viruses, fungi, and reduction in toxicity and affect the fate and trophic transfer other microbes, all of which may exert an influence on its growth routes of brevetoxins during bloom events. Amongst the K. brevis and toxicity either directly or indirectly. Of these interactions, the clones tested against algicidal strains of bacteria, both suscep- roles of bacteria in regulating bloom dynamics have received the tible and resistant clones have been identified. Resistance to most attention over the past decade. Bacterial communities attack was determined to be a function of the ambient bacterial associated with algal blooms are not simply random assemblages flora present rather than an inherent trait of the dinoflagellate of marine bacterial species, but rather have a characteristic profile, clone (Mayali and Doucette, 2002). Recently, a bacterium capable commonly dominated by alpha-Proteobacteria, particularly those of preventing algicidal strains from attaining the threshold of the Roseobacter clade, some of which have been proposed to density needed to induce algal cell lysis (ca. 106 cells ml1)was have growth promoting capability (Jasti et al., 2005). Another isolated from a resistant K. brevis culture and identified as a constituent, the gamma-Proteobacteria, has been alternatively member of the Flavobacteriaceae (Roth et al., 2008b). This work attributed to both growth stimulation and bloom decline in was the first to highlight the complex nature of microbial different situations (Kodama et al., 2006). Members of the beta- interactions and their potential consequences to K. brevis bloom Proteobacteria are encountered less commonly, but dominate the dynamics. intracellular flora of Gymnodinium instriatum (Alverca et al., 2002). Viruses have also been implicated in the lysis of dinoflagellates A fourth group, the Cytophaga–Flavobacterium (CFB) clade, is also and termination of blooms (for review, Salomon and Imai, 2006). frequently represented within this community. Many CFB taxa Viruses infecting microalgae are usually host specific, such that occur in association with particles in the marine environment and their abundance peaks during blooms, but falls to undetectable are active in the remineralization of high molecular weight levels during non-bloom periods. Paul et al. (2002) isolated a heat dissolved organic matter (Kirchman, 2001), which may lead to labile ‘‘filterable lytic agent’’ from 0.2 mm filtered K. brevis bloom algal growth promotion. Conversely, certain members of the CFB water that caused lysis of K. brevis cultures. Following cell lysis, are also included among the approximately 60 algicidal bacteria high numbers of virus-like particles (4–7 109 ml1) were present identified to date (Fukuyo et al., 2002; Mayali and Azam, 2004). in the culture supernatant. However, no identifiable virus particles Natural bacterial populations have also been documented to were found in association with lysing cells; thus, the involvement produce iron binding ligands known as siderophores, which are of lytic viruses in K. brevis bloom termination remains an unsolved capable of making this frequently growth-limiting trace metal question. available to phytoplankton (Soria-Dengg et al., 2001). Many marine bacterial species are unculturable; therefore, 7. Population genetics of K. brevis—what constitutes a bloom? understanding the composition and associated functional ecology of these microbes is being facilitated by the development of 16S The genetic diversity within and among blooms of K. brevis is of rDNA community libraries. To date, one such library has been significance in understanding both the origin of blooms that developed to date for K. brevis, including 16s rDNA sequences from emerge in novel locations and the mechanisms driving the several depths in waters containing various concentrations of K. evolution of this species. The life cycle of K. brevis has not been brevis cells, as well as areas where this dinoflagellate is undetectable entirely resolved (Steidinger et al., 1998); however, the presence of (Mikulski et al., unpublished data). This library shows a clear a sexual cycle is evident from the existence of planozygotes. dominance of alpha-Proteobacteria (primarily members of the Nonetheless, a resting cyst stage has not been documented, so the Rhodobacterales, including Roseobacter spp.) and a frequent rate of genetic recombination remains entirely unexplored. presence of the Bacteroidetes when K. brevis cells are present in Because blooms appear to result largely from vegetative cell the water column at bloom concentrations. Interestingly, pre- division of a haploid organism, it might be expected that K. brevis liminary analysis of the library also suggests a potential relationship blooms display little or no genetic variability, and that natural of bacteria capable of DMSP utilization with K. brevis presence, the selection should be driven largely by genetic drift. In dinoflagel- latter well-known as a DMSP producer. While providing the first lates for which sexual reproduction plays a known role in bloom molecular-based snapshot of a K. brevis associated bacterial cycles, high levels of genetic diversity are seen (Bolch et al., 1999; consortium, it is clear that data on the spatio-temporal variability Nagai et al., 2006; Alpermann et al., 2006). Consistent with this and the functional attributes of dominant taxa present in bloom notion, clonal isolates of K. brevis grown under identical conditions associated microbial communities are needed to fully understand display significant phenotypic variability, as assessed by salinity how they may influence bloom progression. tolerance, growth rates, and toxin production.

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A number of population genetic markers have been assessed in signature identify conditions conducive to bloom initiation or K. brevis in the past decade. Ribosomal large subunit (LSU) probes bloom termination? Finally, the application of the recently from the D1, D2, and 28s hypervariable regions were found to be developed population genetics tools will shed light on genetic identical between geographically distinct populations of K. brevis, variation within and between blooms, elucidate the contribution making them unsuitable for assessing population level variability. of K. brevis and other recently identified Karenia species to blooms However, they are unique to K. brevis, making them useful for in the Gulf of Mexico, aid in defining the origin of blooms identification at the species level, which is particularly useful in in different geographic regions, and potentially the assist in mixed bloom assemblages (Mikulski et al., 2005; Goodwin et al., characterizing the life cycle of K. brevis. 2005). Similarly the ribosomal small subunit (SSU) 18S coding region and ITS sequences were found to be identical among isolates References from Texas and Florida displaying phenotypic differences (Loret et al., 2002). In contrast, microsatellite markers, short stretches of Alverca, E., Biegala, I.C., Kennaway, G.M., Lewis, J., Franca, S., 2002. In situ identifica- DNA containing tandemly di- and tri-nucleotide repeat motif of tion and localization of bacteria associated with Gyrodinium instriatum (Gym- nodiniales, Dinophyceae) by electron and confocal microscopy. Eur. J. Phycol. variable length, have been successfully used to uncover genetic 37, 523–530. diversity among phenotypically distinct clonal isolates. Nine Alpermann, T.J., John, U.E., Medlin, L.K., Edwards, K.J., Hayes, P.K., Evans, K.M., 2006. polymorphic loci have been characterized and two to six alleles Six new satellite markers for the toxic dinoflagellate Alexandrium tamarense. Mol. Ecol. Notes 6, 1057–1059. found at each locus. For a sample of 13 K. brevis cultures (five from Andersson, J.O., Sjogren, A.M., Horner, D.S., Murphy, C.A., Dyal, P.L., Svard, S.G., the same 1999 bloom sample), gene diversity ranged from 0.15 to Logsdon Jr., J.M., Ragan, M.A., Hirt, R.P., Roger, A.J., 2007. A genomic survey of the 0.75 (Renshaw et al., 2006). This variability may suggest that fish parasite Spironucleus salmonicida indicates genomic plasticity among diplo- monads and significant lateral gene transfer in eukaryote genome evolution. bloom populations are genetically highly diverse, or this genetic BMC Genomics 8, 51. pool may provide different subpopulations that bloom preferen- Bachvaroff, T.R., Concepcion, G.T., Rogers, C.R., Herman, E.M., Delwiche, C.F., 2004. tially in response to different environmental conditions. To address Dinoflagellate expressed sequence tag data reveal massive transfer of chloroplast genes to the nuclear genome. Protist 155, 65–78. this question, Campbell et al. (2004) have applied microsatellite Barbier, M., Leighfield, T.A., Soyer-Gobillard, M.-O., Van Dolah, F.M., 2003. Expres- markers to characterize the variability among clonal cultures sion of a cyclin B homologue in the cell cycle of the dinoflagellate, Karenia brevis. isolated from a bloom off the Texas coast. More recently, the J. Eukaryot. Microbiol. 50, 123–131. advent of single cell genotyping permits the evaluation of genetic Bhattacharya, D., Yoon, H.S., Hackett, J.D., 2004. Chromalveolates unite: endosymbiosis connects the dots. BioEssays 26, 50–60. variability within a bloom without the labor-intensive establish- Bhaud, Y., Guillebault, D., Lennon, J.-F., Defacque, H., Soyer-Gobillard, M.-O., Mor- ment of clonal isolates (Henrichs et al., 2007). This approach is eau, H., 2000. Morphology and behavior of dinoflagellate chromosomes during currently being used to examine the temporal patterns of genetic the cell cycle and mitosis. J. Cell Sci. 113, 1231–1239. Bolch, C.J.S., Blackburn, S.I., Hallegraeff, G.M., Vaillancourt, R.E., 1999. Genetic diversity in bloom populations of K. brevis collected weekly during variation among strains of the toxic dinoflagellate Gymnodinium catenatum the 2005 and 2006 blooms off the Texas coast. (Dinophyceae). J. Phycol. 35, 356–367. Bouligand, Y., Norris, V., 2001. Chromosome separation and segregation in dinoflagellates and bacteria may depend on liquid crystalline states. Biochimie 83, 8. Future directions 187–192. Brunelle, S.A., Hazard, S., Sotka, E., Van Dolah, F.M., 2007. Characterization of a The past decade has produced remarkable progress in the dinoflagellate cryptochrome blue light receptor with a possible role in circadian control of the cell cycle. J. 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