Complex Plastids Research Articles

Were class C iron-containing superoxide dismutases of trypanosomatid parasites initially imported into a complex plastid? A hypothesis based on analyses of their N-terminal targeting signals

Trypanosomatid parasites possess 2 distinct iron-containing superoxide dismutases (Fe-SODs) designated SODA and SODC, both of which are targeted to their mitochondria. In contrast to SODAs that carry typical mitochondrial transit peptides, SODCs have highly unusual mitochondrial targeting signals. Our analyses clearly show that these pre-sequences are bipartite possessing a signal peptide-like domain followed by a transit peptide-like domain. Consequently, they resemble N-terminal extensions of proteins targeted to multi-membrane plastids, suggesting that trypanosomatids once contained a eukaryotic alga-derived plastid. Further support for this hypothesis comes from striking similarities in length, hydropathy profile, and amino acid composition of SODC pre-sequences to those of Euglena and dinoflagellate plastid proteins. To account for these data, we propose that the Trypanosomatidae initially possessed a gene encoding a mitochondrial Fe-SOD with a classical mitochondrial transit peptide. Before or after plastid acquisition, a gene duplication event gave rise to SODA and SODC. In a subsequent evolutionary step a signal peptide was linked to SODC, enabling its import into the plastid. When the trypanosomatid plastid subsequently was lost, natural selection favoured adaptation of the SODC N-terminal signal as a mitochondrial transit peptide and re-targeting to the mitochondrion.

ARIADNE'S THREAD: GUIDING A PROTEIN ACROSS FIVE MEMBRANES IN CRYPTOPHYTES(1).

Cryptophytes are the most archetypal chromalveolates, with their complex plastid having retained many features of the red algal secondary endosymbiont. Most important of these is the remnant nucleus, the nucleomorph, that is kept between the inner and outer membrane pair of the endosymbiont in the highly reduced cytosol, the periplastidial compartment (PPC). Because the nucleomorph's coding capacity is very limited, proteins need to be imported from the host cytosol across the outer two membranes into the PPC and across all four membranes into the stroma. How this is accomplished has puzzled researchers for >20 years. Recent findings show that in both cases, a bipartite topogenic signal, a signal and subsequent transit peptide (TP), is responsible for targeting proteins correctly into these two compartments. An aromatic amino acid-based motif at the +1 position of the TP holds the information determining into which compartment the precursor protein is finally transported. Together with the identification of a novel endoplasmic reticulum associated degradation (ERAD)-derived translocon in the second-outermost membrane, these findings help us to understand the sophisticated targeting mechanisms across four membranes and clarify a key innovation during chromalveolate evolution.

Tryptophan Biosynthesis in Stramenopiles: Eukaryotic Winners in the Diatom Complex Chloroplast

Tryptophan is an essential amino acid that, in eukaryotes, is synthesized either in the plastids of photoautotrophs or in the cytosol of fungi and oomycetes. Here we present an in silico analysis of the tryptophan biosynthetic pathway in stramenopiles, based on analysis of the genomes of the oomycetes Phytophthora sojae and P. ramorum and the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Although the complete pathway is putatively located in the complex chloroplast of diatoms, only one of the involved enzymes, indole-3-glycerol phosphate synthase (InGPS), displays a possible cyanobacterial origin. On the other hand, in P. tricornutum this gene is fused with the cyanobacteria-derived hypothetical protein COG4398. Anthranilate synthase is also fused in diatoms. This fusion gene is almost certainly of bacterial origin, although the particular source of the gene cannot be resolved. All other diatom enzymes originate from the nucleus of the primary host (red alga) or secondary host (ancestor of chromalveolates). The entire pathway is of eukaryotic origin and cytosolic localization in oomycetes; however, one of the enzymes, anthranilate phosphoribosyl transferase, was likely transferred to the oomycete nucleus from the red algal nucleus during secondary endosymbiosis. This suggests possible retention of the complex plastid in the ancestor of stramenopiles and later loss of this organelle in oomycetes.

Origin and Distribution of Calvin Cycle Fructose and Sedoheptulose Bisphosphatases in Plantae and Complex Algae: A Single Secondary Origin of Complex Red Plastids and Subsequent Propagation via Tertiary Endosymbioses

Sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphatase (FBPase) are essential nuclear-encoded enzymes involved in land plant Calvin cycle and gluconeogenesis. In this study, we cloned seven SBP and seven FBP cDNAs/genes and established sequences from all lineages of photosynthetic eukaryotes, in order to investigate their origin and evolution. Our data are best explained by a single recruitment of plastid-targeted SBP in Plantae after primary endosymbiosis and a further distribution to algae with complex plastids. While SBP is universally found in photosynthetic lineages, its presence in apicomplexa, ciliates, trypanosomes, and ascomycetes is surprising given that no metabolic function beyond the one in the plastid Calvin cycle is described so far. Sequences of haptophytes, cryptophytes, diatoms, and peridinin-containing dinoflagellates (complex red lineage) strongly group together in the SBP tree and the same assemblage is recovered for plastid-targeted FBP sequences, although this is less supported. Both SBP and plastid-targeted FBP are most likely of red algal origin. Including phosphoribulokinase, fructose bisphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase, a total of five independent plastid-related nuclear-encoded markers support a common origin of all complex rhodoplasts via a single secondary endosymbiosis event. However, plastid phylogenies are incongruent with those of the host cell, as illustrated by the cytosolic FBP isoenzyme. These results are discussed in the context of Cavalier-Smith's far-reaching chromalveolate hypothesis. In our opinion, a more plausible evolutionary scenario would be the establishment of a unique secondary rhodoplast and its subsequent spread via tertiary endosymbioses.

Analysis of the targeting sequences of an iron-containing superoxide dismutase (SOD) of the dinoflagellate Lingulodinium polyedrum suggests function in multiple cellular compartments

One of the proteins targeted to the peridinin plastid of the dinoflagellate Lingulodinium polyedrum is the iron-containing superoxide dismutase (LpSOD). Like dinoflagellate plastid proteins of class II, LpSOD carries a bipartite presequence comprising a signal peptide followed by a transit peptide. Our bioinformatic studies suggest that its signal peptide is atypical, however, and that the entire presequence may function as a mitochondrial targeting signal. It is possible that LpSOD represents a new class of proteins in algae with complex plastids, which are co-targeted to the plastid and mitochondrion. In addition to the ambiguous N-terminal targeting signal, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus. In accordance with a peroxisome localization of this dismutase, its mRNA has two in-frame AUG codons. Our bioinformatic analyses indicate that the first start codon resides in a much weaker oligonucleotide context than the second one. This suggests that synthesis of the plastid/mitochondrion-targeted and peroxisome-targeted isoforms could proceed through so-called leaky scanning. Moreover, our results show that expression of the two isoforms could be regulated by a 'hairpin' structure located between the first and second start codons.

Nucleus-to-Nucleus Gene Transfer and Protein Retargeting into a Remnant Cytoplasm of Cryptophytes and Diatoms

The complex plastid of the cryptophyte Guillardia theta and of the diatom Phaeodactylum tricornutum can both be traced back to an engulfed eukaryotic red alga. The eukaryotic origin of these plastids is most obvious in cryptophytes, where the organelle still possesses a remnant nucleus, the nucleomorph. The nucleomorph itself is embedded in the periplastid compartment (PPC), the remnant of the former red algal cytosol. In the cryptophyte and diatom, the complex plastid is surrounded by 4 membranes, the outer one being continuous with the host rough endoplasmatic reticulum. In a recent report, we have shown that a nuclear encoded PPC protein of G. theta expressed in P. tricornutum leads to a localization, recently described as being a "bloblike structure," which can be obtained by mutation of plastid protein-targeting sequences of the diatom itself. Here we present further nucleus-encoded PPC proteins from G. theta, such as the eukaryotic translation elongation factor-1alpha, evidence for their nucleus-to-nucleus gene transfer, and retargeting of the proteins. We also investigated the first nuclear encoded PPC-targeted protein of P. tricornutum (Hsp70) and analyzed it for in vivo localization together with the identified G. theta PPC proteins. This revealed that all localize to the bloblike structures, which we suggest is the highly reduced PPC of P. tricornutum. Furthermore, the described cryptophyte PPC proteins possibly allow the elucidation of the processes by which proteins are involved in different levels of host control over its eukaryotic organelle.

Protein Targeting into the Complex Plastid of Cryptophytes

The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost membranes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different biochemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precursor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topogenic signal of one protein localized in the periplastidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins.

ENDOBACTERIA IN THE DIATOM PINNULARIA (BACILLARIOPHYCEAE). II. HOST CELL CYCLE‐DEPENDENT TRANSLOCATION AND TRANSIENT CHLOROPLAST SCARS1

Rod‐shaped bacteria were found together with the “secondary chloroplast” in the endoplasmic reticulum (ER) in the pennate diatom Pinnularia. During the host interphase, bacteria bore cavities into the chloroplast and interacted with thylakoids, forming a functional unit with the plastid of the engulfed and reduced eukaryotic endocytobiont, without perforating the relict cell membrane and the chloroplast envelope. Fluorescently labeled antibodies directed against the large subunit of RUBISCO elicited the same pattern as DAPI–DNA complexes. In pre‐prophase, bacteria started to dissociate from the plastids and left scars at their previous locations. In profile, scars appeared as granular amorphous regions interrupting several stacks of thylakoids, whereas images of glancing sections showed foci of circumscribed spiral domains. The vortex pattern was interpreted as the result of a screwing movement of the bacteria during their withdrawal, initiated while structurally still connected to the thylakoids. Scars became effaced, and thylakoids reoriented during prophasic translocation of the complex plastid from the girdle to the valve. In the transition to prophase, bacteria were found as clusters inside ER cisternae near the nucleus, where the microtubule‐organizing center was accompanied by the spindle precursor. Microtubules were seen to be aligned with ER profiles (containing bacteria). TEM images indicated that intracellular long‐distance transport is controlled by the host cytoskeleton acting upon the ER. Restricted to this stage, bacteria appeared to divide and occasionally bore tentacle‐like appendages. Their intraluminal localization at the nucleus in prophase, near the new plasmalemma after cleavage, and their return to the chloroplasts shortly afterward revealed a cell cycle‐dependent translocation with the ER as circulatory system and, together with the obvious retardation of their division cycle, a domestication by the host.

Is Protein Import into Plastids with Four‐Membrane Envelopes Dependent on Two Toc Systems Operating in Tandem?

Abstract: Plastids with four‐membrane envelopes have evolved by several independent endosymbioses involving a eukaryotic alga as the endosymbiont and a protozoan predator as the host. It is assumed that their outermost membrane is derived from the phagosomal membrane of the host and that protein targeting to and across this membrane proceeds co‐translationally, including ER and the Golgi apparatus (e.g., chlorarachniophytes) or only ER (e.g., heterokonts). Since the two inner membranes (or the plastid envelope) of such a complex plastid are derived from the endosymbiont plastid, they are probably provided with Toc and Tic systems, enabling post‐translational passage of the imported proteins into the stroma. The third envelope membrane, or the periplastid one, originates from the endosymbiont plasmalemma, but what import apparatus operates in it remains enigmatic. Recently, Cavalier‐Smith (1999[5]) has proposed that the Toc system, pre‐existing in the endosymbiont plastid, has been relocated to the periplastid membrane, and that plastids having four envelope membranes contain two Toc systems operating in tandem and requiring the same targeting sequence, i.e., the transit peptide. Although this model is parsimonious, it encounters several serious obstacles, the most serious one resulting from the complex biogenesis of Toc75 which forms a translocation pore. In contrast to most proteins targeted to the outer membrane of the plastid envelope, this protein carries a complex transit peptide, indicating that a successful integration of the Toc system into the periplastid membrane would have to be accompanied by relocation of the stromal processing peptidase to the endosymbiont cytosol. However, such a relocation would be catastrophic because this enzyme would cleave the transit peptide off all plastid‐destined proteins, thus disabling biogenesis of the plastid compartment. Considering these difficulties, I suggest that in periplastid membranes two Toc‐independent translocation apparatuses have evolved: a porin‐like channel in chlorarachniophytes and cryptophytes, and a vesicular pathway in heterokonts and haptophytes. Since simultaneous evolution of a new transport system in the periplastid membrane and in the phagosomal one would be complicated, it is argued that plastids with four‐membrane envelopes have evolved by replacement of plastids with three‐membrane envelopes. I suggest that during the first round of secondary endosymbioses (resulting in plastids surrounded by three membranes), myzocytotically‐engulfed eukaryotic alga developed a Golgi‐mediated targeting pathway which was added to the Toc/Tic‐based apparatus of the endosymbiont plastid. During the second round of secondary endosymbioses (resulting in plastids surrounded by four membranes), phagocytotically‐engulfed eukaryotic alga exploited the Golgi pathway of the original plastid, and a new translocation system had to originate only in the periplastid membrane, although its emergence probably resulted in modification of the import machinery pre‐existing in the endosymbiont plastid.

Record-density genome enclosed by multiple membranes

Plastids of eukaryotic plant cells originated from endosymbiotic cyanobacteria. As mitochondria, higher plant plastids are bounded by double membranes and possess autonomous genomes. Most plastid genes were transferred to the nucleus during symbiotic evolution, resulting in tight coordination of the former symbionts.The evolution of such coordination can be studied in algae harboring complex plastids that are surrounded by multiple membranes. These plastids arose when heterotrophic eukaryotes engulfed plastid-bearing eukaryotic autotrophs. In the unicellular cryptomonads, endosymbiogenesis seems stuck in an early phase. The plastid, bounded by a double membrane, is enclosed in the periplastid membrane (equivalent to the plasma membrane of the symbiont), which also contains a vestigial nucleus, the nucleomorph. The whole complex is located within the endoplasmic reticulum of the host. The recently completed sequence 1xThe highly reduced genome of an enslaved algal nucleus. Douglas, S. et al. Nature. 2001; 410: 1091–1096Crossref | PubMed | Scopus (306)See all References1 of the three chromosomes of the Guillardia theta nucleomorph now reveal the highest gene density known from a eukaryotic genome. 464 putative genes are contained within not more than 551 kbp, with negligible amounts of noncoding regions. Multiple gene copies are rare. Spliceosomal introns occur in less than 4% of the genes, and 53% have homologs of known function. The vast majority of these genes have functions that are related to gene expression and chromosome replication. Only 30 genes encode plastid proteins, and even fewer have ‘cytoplasmic’ functions within the endosymbiont. Thus, at least a thousand different nuclear encoded proteins must be imported into the complex plastid 1xThe highly reduced genome of an enslaved algal nucleus. Douglas, S. et al. Nature. 2001; 410: 1091–1096Crossref | PubMed | Scopus (306)See all

Amyloplasts that sediment in protonemata of the moss Ceratodon purpureus are nonrandomly distributed in microgravity.

Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates for g-induced drag while still allowing for regulated amyloplast sedimentation.

Nucleomorphs as genomic tools for photosynthesis research

Beside the nucleus, the plastid and mitochondria, cryptomonads possess a fourth DNA-containing semiautonomous organelle, the nucleomorph. This organell was shown to be the remnant of the nucleus of a former free-living rhodophytic alga, which was engulfed and reduced to a complex plastid by a heterotrophic host cell. By sequencing the three minute nucleomorph chromosomes of the cryptomonad Guillardia theta, we present evidence that the purpose of the nucleomorph is to perpetuate itself, the periplastid space it is embedded in and a eukaryotic expression machinery for the synthesis of 30 nucleomorph-encoded chloroplast proteins (Douglas et al. (2001), Nature 410, 1091-1096). Some of these chloroplast located proteins have homologous sequences in the cyanobacterium Synechocystis sp. PCC6803 but differ from those by an amino-terminal extension which functions as a transit peptide for the translocation to the plastid. Beside proteins of known function (FtsZ, Hlip, CbbX), we identified 11 gene products whose homologs in cyanobacteria do not have any known function. These are the genes of interest we have in part characterized. By knock-out experiments in cyanobacteria and physiological investigations we analyzed the functions of the nucleomorph-encoded proteins in respect to the photosynthesis machinery. Major results so far are the detection of components attached to photosystem II and the creation of mutants with a reduced phycocyanin content.

Transport of Proteins into Cryptomonads Complex Plastids

Complex plastids, found in many alga groups, are surrounded by three or four membranes. Therefore, proteins of the complex plastids, which are encoded in the cell nucleus, must cross three or four membranes during transport to the plastid. To study this process we have developed a method for isolating transport-competent two membrane-bound plastids derived from the complex plastids of the cryptophyte Guillardia theta. This in vitro protein import system provides the first non-heterologous system for studying the import of proteins into four-membrane complex plastids. We use our import system as well as canine microsomes to demonstrate in the case of cryptomonads how nuclear proteins pass the first nucleomorph-encoded proteins the third and fourth membrane and discuss the potential mechanisms for protein transport across the second membrane.

Phylogenetic Relationships among the Cryptophyta: Analyses of Nuclear-Encoded SSU rRNA Sequences Support the Monophyly of Extant Plastid-Containing Lineages

The Cryptophyta comprise photoautotrophic protists with complex plastids which harbor a remnant eukaryotic nucleus (nucleomorph) and a few heterotrophic taxa which either lack a plastid (Goniomonas) or contain a complex plastid devoid of pigments (Ieucoplast; Chilomonas). To resolve the phylogenetic relationships between photosynthetic, leucoplast-containing and aplastidial taxa, we determined complete nuclear-encoded SSU rRNA-sequences from 12 cryptophyte taxa representing the genera Cryptomonas, Chilomonas, Rhodomonas, Chroomonas, Hemiselmis, Proteomonas and Teleaulax and, as an outgroup taxon, Cyanoptyche gloeocystis (Glaucocystophyta). Phylogenetic analyses of SSU rRNA sequences from a total of 24 cryptophyte taxa rooted with 4 glaucocystophyte taxa using distance, parsimony and likelihood methods as well as LogDet transformations invariably position the aplastidial genus Goniomonas as a sister taxon to a monophyletic lineage consisting of all plastid containing cryptophytes including Chilomonas. Among the plastid-containing taxa, we identify six major clades each supported by high bootstrap values: clade I (Cryptomonas and Chilomonas), clade II (Rhodomonas, Pyrenomonas, Rhinomonas and Storeatula), clade III (Guillardia and the 'unidentified cryptophyte' strain CCMP 325), clade IV (Teleaulax and Geminigera), clade V (Proteomonas) and clade VI (Hemiselmis, Chroomonas and Komma). Clade I (Cryptomonas and Chilomonas) represents a sister group to clades II-VI which together form a monophyletic lineage; the phylogenetic relationships between clades II-VI remain largely unresolved. Chilomonas is positioned within the Cryptomonas clade and thus presumably evolved from a photosynthetic taxon of this genus. In our analysis the characters blue and red pigmentation do not correspond with a basal subdivision of the phylum, thus rejecting this character for higher-level classification of cryptophytes. However, different spectroscopic subtypes of phycoerythrin (PE I-III) and phycocyanin (PC II-IV) represent informative characters at a lower taxonomic level. Phycocyanin types are confined to the later diverging clade VI and within Hemiselmis, a species with phycocyanin is monophyletic with two species containing phycoerythrin. This supports previous molecular studies which demonstrated that the β subunit of all cryptophyte biliproteins, regardless of spectroscopic type, is phylogenetically derived from the red algal β-phycoerythrin gene family, therefore the cryptophyte phycocyanins presumably originated by chromophore replacement from phycoerythrin. Our phylogenetic analysis does not support a previous suggestion that the aplastidial cryptophyte Goniomonas evolved from an ancestor containing a complex cryptomonadtype plastid by nucleomorph and plastid loss.

Mechanism of Protein Targeting to the Chlorarachniophyte Plastids and the Evolution of Complex Plastids with Four Membranes — A Hypothesis

Abstract:Chlorarachniophyta are phototrophic amoeboflagellates, with plastids surrounded by four membranes. Contrary to other plastids of this type which occur in chromists, their outermost membrane bears no ribosomes. It is argued that the nuclear‐encoded chlorarachniophyte plastid proteins are first transported into the ER, then to the Colgi apparatus, and finally to the plastids. The same import mechanism could be originally present in the chromist ancestor, prior to the fusion of their plastids with the RER membranes. According to the most recent concept, the complex plastids of Chromista and Chlorarachniophyta have evolved through replacement of the cyanobacterial plastids. The assumption that these plastids had an envelope composed not of two, but of three membranes makes it possible to avoid the erlier discerned difficulties with conversion of a eukaryotic alga into a complex plastid. My scenario provides an additional support to the hypothesis on polyphy‐letic origin of four‐membraned plastids.

How to Evolve a Complex Plastid? ‐ A Hypothesis

AbstractThe evolution of complex plastids in general and the phylogeny of the intermediate forms present in chlorarachniophytes and cryptophytes are considered. By comparing possible protein transport mechanisms in these algae, also taking into account problems in explaining horizontal gene transfer between eukaryotic nuclei, we arrive at the hypothesis that the initial step in the evolution of complex plastids was a primary endocytobiosis of the host (!) cell. Our hypothesis also explains why the nucleomorph (the reduced nucleus of a secondary endocytobiont) is not eliminated.

PsbD sequences of Bumilleriopsis filiformis (Heterokontophyta, Xanthophyceae) and Porphyridium purpureum (Rhodophyta, Bangiophycidae): evidence for polyphyletic origins of plastids.

The nucleotide sequences of the plastidal psbD genes of Bumilleriopsis filiformis and Porphyridium purpureum (encoding the D2 protein of photosystem II) are reported in this paper. The Bumilleriopsis sequence clusters together with Porphyridium when a most parsimonious protein tree of D2 sequences is constructed. A composite D1/D2 protein-similarity network reveals that neither the three red algal sequences nor the two heterokontophyte sequences (Bumilleriopsis, xanthophytes and Ectocarpus, phaeophytes) group together. Therefore, the Heterokontophyta and Rhodophyta may be heterogeneous groups. Instead, it emerges that the D1/D2 proteins of Porphyridium and Bumilleriopsis clearly form a tight cluster. D1 and D2 proteins apparently do not provide a reliable molecular clock. These results fit into hypotheses proposing a polyphyletic origin for complex plastids, even among the supposedly "natural" group of heterokontophytes.

Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads

In cryptomonads, unicellular phototrophic flagellates, the plastid(s) is (are) located in a special narrow compartment which is bordered by two membranes; it harbours neither mitochondria nor Golgi dictyosomes but comprises eukaryotic ribosomes and starch grains together with a small organelle called the nucleomorph. The nucleomorph contains DNA and is surrounded by a double membrane with pores. It is thought to be the vestigial nucleus of a phototrophic eukaryotic endosymbiont. Cryptomonads are therefore supposed to represent an intermediate state in the evolution of complex plastids from endosymbionts. We have succeeded in isolating pure nucleomorph fractions, and can thus provide, using pulsed field gel electrophoresis, polymerase chain reaction and sequence analysis, definitive proof for the eukaryotic nature of the symbiont and its phylogenetic origin.