DNA-binding and protein structure of nuclear factors likely acting in genetic information processing in the Paulinella chromatophore.

The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.

A neo-functionalized homolog of host transmembrane protein controls localization of bacterial endosymbionts in the trypanosomatid Novymonas esmeraldas.

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

Host-symbiont interactions in Angomonas deanei include the evolution of a host-derived dynamin ring around the endosymbiont division site.

The trypanosomatid Angomonas deanei is a model to study endosymbiosis. Each cell contains a single ß-proteobacterial endosymbiont that divides at a defined point in the host cell cycle and contributes essential metabolites to the host metabolism. Additionally, one endosymbiont gene, encoding an ornithine cyclodeaminase (OCD), was transferred by endosymbiotic gene transfer (EGT) to the nucleus. However, the molecular mechanisms mediating the intricate host/symbiont interactions are largely unexplored. Here, we used protein mass spectrometry to identify nucleus-encoded proteins that co-purify with the endosymbiont. Expression of fluorescent fusion constructs of these proteins in A. deanei confirmed seven host proteins to be recruited to specific sites within the endosymbiont. These endosymbiont-targeted proteins (ETPs) include two proteins annotated as dynamin-like protein and peptidoglycan hydrolase that form a ring-shaped structure around the endosymbiont division site that remarkably resembles organellar division machineries. The EGT-derived OCD was not among the ETPs, but instead localizes to the glycosome, likely enabling proline production in the glycosome. We hypothesize that recalibration of the metabolic capacity of the glycosomes that are closely associated with the endosymbiont helps to supply the endosymbiont with metabolites it is auxotrophic for and thus supports the integration of host and endosymbiont metabolic networks. Hence, scrutiny of endosymbiosis-induced protein re-localization patterns in A. deanei yielded profound insights into how an endosymbiotic relationship can stabilize and deepen over time far beyond the level of metabolite exchange.

A bipartite chromatophore transit peptide and N-terminal protein processing in the Paulinella chromatophore.

The amoeba Paulinella chromatophora contains photosynthetic organelles, termed chromatophores, which evolved independently from plastids in plants and algae. At least one-third of the chromatophore proteome consists of nucleus-encoded (NE) proteins that are imported across the chromatophore double envelope membranes. Chromatophore-targeted proteins exceeding 250 amino acids (aa) carry a conserved N-terminal extension presumably involved in protein targeting, termed the chromatophore transit peptide (crTP). Short imported proteins do not carry discernable targeting signals. To explore whether the import of proteins is accompanied by their N-terminal processing, here we identified N-termini of 208 chromatophore-localized proteins by a mass spectrometry-based approach. Our study revealed extensive N-terminal acetylation and proteolytic processing in both NE and chromatophore-encoded (CE) fractions of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Surprisingly, only the N-terminal ∼50 aa (part 1) become cleaved upon import. This part contains a conserved adaptor protein-1 complex-binding motif known to mediate protein sorting at the trans-Golgi network followed by a predicted transmembrane helix, implying that part 1 anchors the protein co-translationally in the endoplasmic reticulum and mediates trafficking to the chromatophore via the Golgi. The C-terminal part 2 contains conserved secondary structural elements, remains attached to the mature proteins, and might mediate translocation across the chromatophore inner membrane. Short imported proteins remain largely unprocessed. Finally, this work illuminates N-terminal processing of proteins encoded in an evolutionary-early-stage organelle and suggests host-derived posttranslationally acting factors involved in regulation of the CE chromatophore proteome.

Paulinella chromatophora.

Macorano and Nowack provide an overview of Paulinella chromatophora, a filose amoeba that harbors an organelle called a chromatophore and only the second known case of a eukaryote forming a primary endosymbiosis with a photosynthetic bacterium. Studying this relatively young relationship offers the chance to study the early stages of endosymbiosis.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

The Puzzle of Metabolite Exchange and Identification of Putative Octotrico Peptide Repeat Expression Regulators in the Nascent Photosynthetic Organelles of Paulinella chromatophora.

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles-termed chromatophores-that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in P. chromatophora nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

Putative sponge biomarkers in unicellular Rhizaria question an early rise of animals.

The dawn of animals remains one of the most mysterious milestones in the evolution of life. The fossil lipids 24-isopropylcholestane and 26-methylstigmastane are considered diagnostic for demosponges-arguably the oldest group of living animals. The widespread occurrence and high relative abundance of these biomarkers in Ediacaran sediments from 635-541 million years (Myr) ago have been viewed as evidence for the rise of animals to ecological importance approximately 100 Myr before their rapid Cambrian radiation. Here we show that the biosynthesis of 24-isopropylcholestane and 26-methylstigmastane precursors is common among early-branching unicellular Rhizaria-heterotrophic protists that play an important role in trophic cycling and carbon export in the modern ocean. Negating these hydrocarbons as sponge biomarkers, our study places the oldest evidence for animals closer to the Cambrian Explosion. Cambrian silica hexactine spicules that are approximately 535 Myr old now represent the oldest diagnostic sponge remains, whereas approximately 558-Myr-old Dickinsonia and Kimberella (Ediacara biota) provide the most reliable evidence for the emergence of animals. The proliferation of predatory protists may have been responsible for much of the ecological changes during the late Neoproterozoic, including the rise of algae, the establishment of complex trophic relationships and the oxygenation of shallow-water habitats required for the subsequent ascent of macroscopic animals.

Genomics-Informed Insights into Endosymbiotic Organelle Evolution in Photosynthetic Eukaryotes.

The conversion of free-living cyanobacteria to photosynthetic organelles of eukaryotic cells through endosymbiosis transformed the biosphere and eventually provided the basis for life on land. Despite the presumable advantage conferred by the acquisition of photoautotrophy through endosymbiosis, only two independent cases of primary endosymbiosis have been documented: one that gave rise to the Archaeplastida, and the other to photosynthetic species of the thecate, filose amoeba Paulinella. Here, we review recent genomics-informed insights into the primary endosymbiotic origins of cyanobacteria-derived organelles. Furthermore, we discuss the preconditions for the evolution of nitrogen-fixing organelles. Recent genomic data on previously undersampled cyanobacterial and protist taxa provide new clues to the origins of the host cell and endosymbiont, and proteomic approaches allow insights into the rearrangement of the endosymbiont proteome during organellogenesis. We conclude that in addition to endosymbiotic gene transfers, horizontal gene acquisitions from a broad variety of prokaryotic taxa were crucial to organelle evolution.

Massive Protein Import into the Early-Evolutionary-Stage Photosynthetic Organelle of the Amoeba Paulinella chromatophora.

The endosymbiotic acquisition of mitochondria and plastids more than 1 Ga ago profoundly impacted eukaryote evolution. At the heart of understanding organelle evolution is the re-arrangement of the endosymbiont proteome into a host-controlled organellar proteome. However, early stages in this process as well as the timing of events that underlie organelle integration remain poorly understood. The amoeba Paulinella chromatophora contains cyanobacterium-derived photosynthetic organelles, termed "chromatophores," that were acquired more recently (around 100 Ma ago). To explore the re-arrangement of an organellar proteome during its integration into a eukaryotic host cell, here we characterized the chromatophore proteome by protein mass spectrometry. Apparently, genetic control over the chromatophore has shifted substantially to the nucleus. Two classes of nuclear-encoded proteins-which differ in protein length-are imported into the chromatophore, most likely through independent pathways. Long imported proteins carry a putative, conserved N-terminal targeting signal, and many specifically fill gaps in chromatophore-encoded metabolic pathways or processes. Surprisingly, upon heterologous expression in a plant cell, the putative chromatophore targeting signal conferred chloroplast localization. This finding suggests common features in the protein import pathways of chromatophores and plastids, two organelles that evolved independently and more than 1 Ga apart from each other. By combining experimental data with in silico predictions, we provide a comprehensive catalog of almost 450 nuclear-encoded, chromatophore-targeted proteins. Interestingly, most imported proteins seem to derive from ancestral host genes, suggesting that the re-targeting of nuclear-encoded proteins that resulted from endosymbiotic gene transfers plays only a minor role at the onset of chromatophore integration.

Metabolic Integration of Bacterial Endosymbionts through Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are massively produced by eukaryotic hosts during symbiotic interactions with bacteria. Among other roles, these symbiotic AMPs have the capacity to permeabilize symbiont membranes and facilitate metabolite flow across the host-symbiont interface. We propose that an ancestral role of these peptides is to facilitate metabolic exchange between the symbiotic partners through membrane permeabilization. This function may be particularly critical for integration of endosymbiont and host metabolism in interactions involving bacteria with strongly reduced genomes lacking most small metabolite transporters. Moreover, AMPs could have acted in a similar way at the onset of plastid and mitochondrion evolution, after a host cell took up a bacterium and needed to extract nutrients from it in the absence of dedicated solute transporters.

Impact of light intensity and quality on chromatophore and nuclear gene expression in Paulinella chromatophora, an amoeba with nascent photosynthetic organelles.

Plastid evolution has been attributed to a single primary endosymbiotic event that occurred about 1.6 billion years ago (BYA) in which a cyanobacterium was engulfed and retained by a eukaryotic cell, although early steps in plastid integration are poorly understood. The photosynthetic amoeba Paulinella chromatophora represents a unique model for the study of plastid evolution because it contains cyanobacterium-derived photosynthetic organelles termed 'chromatophores' that originated relatively recently (0.09-0.14 BYA). The chromatophore genome is about a third the size of the genome of closely related cyanobacteria, but 10-fold larger than most plastid genomes. Several genes have been transferred from the chromatophore genome to the host nuclear genome through endosymbiotic gene transfer (EGT). Some EGT-derived proteins could be imported into chromatophores for function. Two photosynthesis-related genes (psaI and csos4A) are encoded by both the nuclear and chromatophore genomes, suggesting that EGT in Paulinella chromatophora is ongoing. Many EGT-derived genes encode proteins that function in photosynthesis and photoprotection, including an expanded family of high-light-inducible (ncHLI) proteins. Cyanobacterial hli genes are high-light induced and required for cell viability under excess light. We examined the impact of light on Paulinella chromatophora and found that this organism is light sensitive and lacks light-induced transcriptional regulation of chromatophore genes and most EGT-derived nuclear genes. However, several ncHLI genes have reestablished light-dependent regulation, which appears analogous to what is observed in cyanobacteria. We postulate that expansion of the ncHLI gene family and its regulation may reflect the light/oxidative stress experienced by Paulinella chromatophora as a consequence of the as yet incomplete integration of host and chromatophore metabolisms.

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei.

BACKGROUND: Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems. RESULTS: Here we show that the trypanosomatid Angomonas deanei, which harbors a ß-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei. CONCLUSIONS: After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.

Gene transfers from diverse bacteria compensate for reductive genome evolution in the chromatophore of Paulinella chromatophora.

Plastids, the photosynthetic organelles, originated >1 billion y ago via the endosymbiosis of a cyanobacterium. The resulting proliferation of primary producers fundamentally changed global ecology. Endosymbiotic gene transfer (EGT) from the intracellular cyanobacterium to the nucleus is widely recognized as a critical factor in the evolution of photosynthetic eukaryotes. The contribution of horizontal gene transfers (HGTs) from other bacteria to plastid establishment remains more controversial. A novel perspective on this issue is provided by the amoeba Paulinella chromatophora, which contains photosynthetic organelles (chromatophores) that are only 60-200 million years old. Chromatophore genome reduction entailed the loss of many biosynthetic pathways including those for numerous amino acids and cofactors. How the host cell compensates for these losses remains unknown, because the presence of bacteria in all available P. chromatophora cultures excluded elucidation of the full metabolic capacity and occurrence of HGT in this species. Here we generated a high-quality transcriptome and draft genome assembly from the first bacteria-free P. chromatophora culture to deduce rules that govern organelle integration into cellular metabolism. Our analyses revealed that nuclear and chromatophore gene inventories provide highly complementary functions. At least 229 nuclear genes were acquired via HGT from various bacteria, of which only 25% putatively arose through EGT from the chromatophore genome. Many HGT-derived bacterial genes encode proteins that fill gaps in critical chromatophore pathways/processes. Our results demonstrate a dominant role for HGT in compensating for organelle genome reduction and suggest that phagotrophy may be a major driver of HGT.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora.

Endosymbiotic acquisition of bacteria by a protist, with subsequent evolution of the bacteria into mitochondria and plastids, had a transformative impact on eukaryotic biology. Reconstructing events that created a stable association between endosymbiont and host during the process of organellogenesis--including establishment of regulated protein import into nascent organelles--is difficult because they date back more than 1 billion years. The amoeba Paulinella chromatophora contains nascent photosynthetic organelles of more recent evolutionary origin (∼60 Mya) termed chromatophores (CRs). After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome. Three transferred genes--psaE, psaK1, and psaK2--encode subunits of photosystem I. Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes. Additionally, our data suggest that proteins routed to CRs pass through the Golgi apparatus. Whereas genome reduction and transfer of genes from bacterial to host genome have been reported to occur in other obligate bacterial endosymbioses, this report outlines the import of proteins encoded by such transferred genes into the compartment derived from the bacterial endosymbiont. Our study showcases P. chromatophora as an exceptional model in which to study early events in organellogenesis, and suggests that protein import into bacterial endosymbionts might be a phenomenon much more widespread than currently assumed.

Endosymbiotic gene transfer and transcriptional regulation of transferred genes in Paulinella chromatophora.

Paulinella chromatophora is a cercozoan amoeba that contains "chromatophores," which are photosynthetic inclusions of cyanobacterial origin. The recent discovery that chromatophores evolved independently of plastids, underwent major genome reduction, and transferred at least two genes to the host nucleus has highlighted P. chromatophora as a model to infer early steps in the evolution of photosynthetic organelles. However, owing to the paucity of nuclear genome sequence data, the extent of endosymbiotic gene transfer (EGT) and host symbiont regulation are currently unknown. A combination of 454 and Illumina next generation sequencing enabled us to generate a comprehensive reference transcriptome data set for P. chromatophora on which we mapped short Illumina cDNA reads generated from cultures from the dark and light phases of a diel cycle. Combined with extensive phylogenetic analyses of the deduced protein sequences, these data revealed that 1) about 0.3-0.8% of the nuclear genes were obtained by EGT compared with 11-14% in the Plantae, 2) transferred genes show a distinct bias in that many encode small proteins involved in photosynthesis and photoacclimation, 3) host cells established control over expression of transferred genes, and 4) not only EGT, but to a minor extent also horizontal gene transfer from organisms that presumably served as food sources, helped to shape the nuclear genome of P. chromatophora. The identification of a significant number of transferred genes involved in photosynthesis and photoacclimation of thylakoid membranes as well as the observed transcriptional regulation of these genes strongly implies import of the encoded gene products into chromatophores, a feature previously thought to be restricted to canonical organelles. Thus, a possible mechanism by which P. chromatophora exerts control over the performance of its newly acquired photosynthetic organelle may involve controlling the expression of nuclear-encoded chromatophore-targeted regulatory components of the thylakoid membranes.

Endosymbiotic associations within protists.

The establishment of an endosymbiotic relationship typically seems to be driven through complementation of the host's limited metabolic capabilities by the biochemical versatility of the endosymbiont. The most significant examples of endosymbiosis are represented by the endosymbiotic acquisition of plastids and mitochondria, introducing photosynthesis and respiration to eukaryotes. However, there are numerous other endosymbioses that evolved more recently and repeatedly across the tree of life. Recent advances in genome sequencing technology have led to a better understanding of the physiological basis of many endosymbiotic associations. This review focuses on endosymbionts in protists (unicellular eukaryotes). Selected examples illustrate the incorporation of various new biochemical functions, such as photosynthesis, nitrogen fixation and recycling, and methanogenesis, into protist hosts by prokaryotic endosymbionts. Furthermore, photosynthetic eukaryotic endosymbionts display a great diversity of modes of integration into different protist hosts. In conclusion, endosymbiosis seems to represent a general evolutionary strategy of protists to acquire novel biochemical functions and is thus an important source of genetic innovation.

Chromatophore genome sequence of Paulinella sheds light on acquisition of photosynthesis by eukaryotes.

BACKGROUND: It is commonly accepted that a single primary endosymbiosis gave rise to the photosynthetic organelles of plants, the plastids. Recently, we presented evidence that photosynthetic inclusions, termed "chromatophores," present in the filose thecamoeba Paulinella chromatophora originated from an independent, more recent primary endosymbiotic event. To clarify metabolic capabilities of the chromatophore and its state of integration into the host, we present here the complete genome sequence of the chromatophore. RESULTS: Our data reveal a fundamental reduction of the chromatophore genome. The single, circular chromosome of 1.02 Mb encodes 867 protein-coding genes and is, therewith, the smallest cyanobacterial genome reported to date. Compared to Synechococcus WH5701, a free-living relative of the chromatophore, only 26% of the genes were retained. Eleven putative pseudogenes were identified, indicating that reductive genome evolution is ongoing. Although the chromatophore genome contains a complete set of photosynthesis genes, it lacks not only genes thought to be dispensable for an intracellular lifestyle but also genes of essential pathways for amino acid and cofactor synthesis. CONCLUSIONS: Our data characterize the chromatophore as a photosynthetic entity that is absolutely dependent on its host for growth and survival. Thus, the chromatophores of P. chromatophora are the only known cyanobacterial descendants besides plastids with a significantly reduced genome that confer photosynthesis to their eukaryotic host. Their comparison with plastids and bacterial endosymbionts of invertebrates sheds light on early steps of the integration of a photosynthetic prokaryote into a eukaryotic cell.