Nicholas E. Curtis

Horizontal Transfer of Functional Nuclear Genes Between Multicellular Organisms

SIDNEY K. PIERCE*, STEVEN E. MASSEY, JEFFREY J. HANTEN, ANDNICHOLAS E. CURTISDepartment of Biology, University of South Florida, SCA 110, 4202 E. Fowler Ave.,Tampa, Florida 33620The horizontal transfer of functional genes between or-ganisms is the theoretical foundation of the endosymbioticorigin of cellular organelles, as well as the basis of genetictherapies and the technology of genetic modification. With-out doubt, transfer of functional genes is routine betweenprokaryotes (1), has occurred between both mitochondria(2) and chloroplasts (3), and the cell nucleus. In addition,DNA has been transferred from endosymbiotic bacteria intoinsect host cell nuclei (4). However, no direct evidenceexists for the natural transfer of nuclear genes betweenmulticellular organisms. We have recently presented cir-cumstantial and pharmacological evidence that nucleargenes encoding for chloroplast proteins are transferredfrom an alga to an ascoglossan sea slug (5, 6). We nowdemonstrate, using molecular techniques, that such a geneis present in the genomic DNA of the slug.Elysia ( Tridachia) crispata is one of a few species ofelysiid sea slugs that has an intracellular symbiosis of sev-eral months’ duration with chloroplasts acquired from spe-cific, siphonaceous algal food. The slug slits open the algalfilament with its radula and sucks the contents into itsdigestive system. As digestion proceeds, certain cells liningthe digestive diverticula phagocytize the plastids into intra-cellular vacuoles. In some species, the chloroplasts reside intheir vacuole for as long as 8–9 months (5, 6, 7), 3–4months in E. crispata (8, 9). In several elysiid slugs, includ-ing E. crispata, the plastids remain photosynthetically ac-tive, and photosynthetic carbon fixation contributes to avariety of molecules that participate in the slug’s energymetabolism and mucus production (9, 10, 11).Maintenance of a chloroplast’s photosynthetic functionsrequires that a variety of proteins associated with the pho-tosystems turn over, but chloroplast genomes only code fora small fraction of the proteins needed for plastid function(e.g., 11, 12). For example, in chromophytic algae—thefood source of some species of elysiids—the chloroplastgenome encodes only 13% of the plastid proteins (13). Inhigher plants, the genes for as many as 90% of plastidproteins, including many of the photosystem components,are located in the cell nucleus (3). Therefore, the persistenceof photosynthesis in the endosymbiotic plastids indicatesthat protein turnover must be occurring, and that supportfrom the nuclear genome of the slug must be necessary.The algal species providing the plastids in E. crispata(and many other species of elysiid slugs) is unknown andcontroversial. Some reports indicate that E. crispata eats,primarily, species of Caulerpa, especially C. verticellata(14). Others (9) report that E. crispata does not consumeCaulerpa spp. at all, but rather eats other genera, such asBatophora, Bryopsis, Halimeda, and Penicillus. These con-flicting results suggest that E. crispata eats a variety ofulvophytic, coenocytic algae; but whether it retains chloro-plasts from multiple algal species is unknown and is amatter that we are currently investigating. Regardless oftheir origin, the endosymbiotic chloroplasts in E. crispataare unexceptional in that they require substantial proteinsynthesis support from the nucleus. When slugs are incu-bated in

Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica.

Analysis of the transcriptome of the kleptoplastic sea slug, Elysia chlorotica, has revealed the presence of at least 101 chloroplast-encoded gene sequences and 111 transcripts matching 52 nuclear-encoded genes from the chloroplast donor, Vaucheria litorea. These data clearly show that the symbiotic chloroplasts are translationally active and, of even more interest, that a variety of functional algal genes have been transferred into the slug genome, as has been suggested by earlier indirect experiments. Both the chloroplast- and nuclear-encoded sequences were rare within the E. chlorotica transcriptome, suggesting that their copy numbers and synthesis rates are low, and required both a large amount of sequence data and native algal sequences to find. These results show that the symbiotic chloroplasts residing inside the host molluscan cell are maintained by an interaction of both organellar and host biochemistry directed by the presence of transferred genes.

Using Algal Transcriptome Sequences to Identify Transferred Genes in the Sea Slug, Elysia chlorotica

The first molecular evidence of horizontal gene transfer between multicellular eukaryotes was our discovery of the presence of three Vaucheria litorea nuclear-encoded genes [fucoxanthin chlorophyll a/c-binding protein (fcp) and light-harvesting complex 1 and 2 (Lhcv1 and 2)] in the genomic DNA of the sea slug, Elysia chlorotica, which are used to support the chloroplast endosymbiosis in the slug. These genes are translated and transcribed in the host cell, and vertically transmitted to subsequent generations of the host species. In order to provide a database of native V. litorea sequences to facilitate the search for additional transferred genes between these two species, we have partially sequenced and annotated the transcriptome of V. litorea, using 454 Life Science’s next generation pyrosequencing technology. Preliminary analysis of the sequence data has led to the discovery of six additional algal nuclear genes in E. chlorotica cDNA and genomic DNA, which encode enzymes in the chlorophyll synthesis pathway as well as additional light-harvesting and metabolic enzymes. Furthermore, we confirm the recent discovery of the Calvin-Benson cycle gene, prk.

Cell biology of the chloroplast symbiosis in sacoglossan sea slugs.

Chloroplasts removed from their species of origin may survive for various periods and even photosynthesize in foreign cells. One of the best studied and impressively long, naturally occurring examples of chloroplast persistence, and function inside foreign cells are the algal chloroplasts taken up by specialized cells of certain sacoglossan sea slugs, a phenomenon called chloroplast symbiosis or kleptoplasty. Among sacoglossan species, kleptoplastic associations vary widely in length and function, with some animals immediately digesting chloroplasts, while others maintain functional plastids for over 10 months. Kleptoplasty is a complex process in long-term associations, and research on this topic has focused on a variety of aspects including plastid uptake and digestive physiology of the sea slugs, the longevity and maintenance of symbiotic associations, biochemical interactions between captured algal plastids and slug cells, and the role of horizontal gene transfers between the sea slug and algal food sources. Although the biochemistry underlying chloroplast symbiosis has been extensively examined in only a few slug species, it is obvious that the mechanisms vary from species to species. In this chapter, we examine those mechanisms from early discoveries to the most current research.

Chlorophyll a synthesis by an animal using transferred algal nuclear genes

Chlorophyll synthesis is an ongoing requirement for photosynthesis and a ubiquitous, diagnostic characteristic of plants and algae amongst eukaryotes. However, we have discovered that chlorophyll a (Chla) is synthesized in the symbiotic chloroplasts of the sea slug, Elysia chlorotica, for at least 6 months after the slugs have been deprived of the algal source of the plastids, Vaucheria litorea. In addition, using transcriptome analysis and PCR with genomic DNA, we found 4 expressed genes for nuclear-encoded enzymes of the Chla synthesis pathway that have been horizontally transferred from the alga to the genomic DNA of the sea slug. These findings demonstrate the first discovery of Chla production in an animal using transferred nuclear genes from its algal food.

PHYLOGENETIC ANALYSIS OF THE LARGE SUBUNIT RUBISCO GENE SUPPORTS THE EXCLUSION OF AVRAINVILLEA AND CLADOCEPHALUS FROM THE UDOTEACEAE (BRYOPSIDALES, CHLOROPHYTA)(1).

The placement of Avrainvillea and Cladocephalus in the family Udoteaceae (order Bryopsidales) has been questioned on the basis of nuclear, plastid, and other ultrastructural characteristics unique to these genera. Bayesian analysis of the chloroplast‐encoded LSU RUBISCO (rbcL) gene showed that the Udoteaceae is paraphyletic. Cladocephalus luteofuscus (P. Crouan et H. Crouan) Børgesen, Avrainvillea nigricans f. floridana D. Littler et Littler, and A. mazei G. Murray et Boodle form a clade with the freshwater alga Dichotomosiphon tuberosus (A. Braun ex Kütz.) A. Ernst that is basal to a clade that includes other members of the Udoteaceae, the Halimedaceae, and the Caulerpaceae. The noncalcified species Boodleopsis pusilla (Collins) W. R. Taylor, A. B. Joly et Bernat. groups with species of the calcified Udoteacean genera Penicillus, Rhipocephalus, Udotea, and Halimeda.

The Intracellular, Functional Chloroplasts in Adult Sea Slugs ( Elysia crispata ) Come from Several Algal Species, and are Also Different from those in Juvenile Slugs.

The sacoglossan sea slug, Elysia crispata, sequesters chloroplasts from its algal food source within specialized cells lining the digestive diverticulum. These stolen chloroplasts photosynthesize within the slug cell cytoplasm as long as four months--one of the longest kleptoplastic associations known [1]. While many other sacoglossan species feed on and sequester chloroplasts from only one species of algae, adult E. crispata sequester plastids from three different species of algae; Penicillus capitatus, Halimeda incrassata, and Halimeda monile [2]. We have now done feeding experiments testing the ability of newlymetamorphosed, juvenile E. crispata, raised from egg masses in the lab, to sequester chloroplasts from multiple algal species using a large range of potential algal food sources. Surprisingly, juvenile E. crispata fed on different algal species (Bryopsis plumosa and Derbesia tenuissima) from those utilized for sources of symbiotic plastids in the adults. Transmission electron microscopy (TEM) verified that the B. plumosa and D. tenuissima chloroplasts were sequestered intracellularly in the juvenile slugs. In addition, juvenile E. crispata fed exclusively on B. plumosa could be grown to adult size, and, as adults, they would switch to feeding on Penicillus capitatus if presented with it. Since the fine structure of B. plumosa and P. capitatus chloroplasts are easily distinguishable, TEM indicated that both types of chloroplasts are sequestered simultaneously inside the same cell in animals fed on both species of algae (Fig. 1). Finally, a newly discovered population of E. crispata which lives in an area where only B. plumosa is present showed the presence of B. plumosa chloroplasts sequestered in adult slug digestive cells using TEM analysis and using molecular markers. Adult slugs fed on B. plumosa in the lab maintained chloroplasts for approximately as long as the field-collected animals. These results indicate that E. crispata not only eats several species of algae, but also is capable of maintaining symbiotic plastids concurrently from those species for long periods.

Microscopic, Biochemical, and Molecular Characteristics of the Chilean Blob and a Comparison With the Remains of Other Sea Monsters: Nothing but Whales

We have employed electron microscopic, biochemical, and molecular techniques to clarify the species of origin of the “Chilean Blob,” the remains of a large sea creature that beached on the Chilean coast in July 2003. Electron microscopy revealed that the remains are largely composed of an acellular, fibrous network reminiscent of the collagen fiber network in whale blubber. Amino acid analyses of an acid hydrolysate indicated that the fibers are composed of 31% glycine residues and also contain hydroxyproline and hydroxylysine, all diagnostic of collagen. Using primers designed to the mitochondrial gene nad2, an 800-bp product of the polymerase chain reaction (PCR) was amplified from DNA that had been purified from the carcass. The DNA sequence of the PCR product was 100% identical to nad2 of sperm whale (Physeter catadon). These results unequivocally demonstrate that the Chilean Blob is the almost completely decomposed remains of the blubber layer of a sperm whale. This identification is the same as those we have obtained before from other relics such as the so-called giant octopus of St. Augustine (Florida), the Tasmanian West Coast Monster, two Bermuda Blobs, and the Nantucket Blob. It is clear now that all of these blobs of popular and cryptozoological interest are, in fact, the decomposed remains of large cetaceans.