Steven E. Massey

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

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

A Comparative Genomics Analysis of Codon Reassignments Reveals a Link with Mitochondrial Proteome Size and a Mechanism of Genetic Code Change Via Suppressor tRNAs

Using a comparative genomics approach we demonstrate a negative correlation between the number of codon reassignments undergone by 222 mitochondrial genomes and the mitochondrial genome size, the number of mitochondrial ORFs, and the sizes of the large and small subunit mitochondrial rRNAs. In addition, we show that the TGA-to-tryptophan codon reassignment, which has occurred 11 times in mitochondrial genomes, is found in mitochondrial genomes smaller than those which have not undergone the reassignment. We therefore propose that mitochondrial codon reassignments occur in a wide range of phyla, particularly in Metazoa, due to a reduced “proteomic constraint” on the mitochondrial genetic code, compared to the nuclear genetic code. The reduced proteomic constraint reflects the small size of the mitochondrial-encoded proteome and allows codon reassignments to occur with less likelihood of lethality. In addition, we demonstrate a striking link between nonsense codon reassignments and the decoding properties of naturally occurring nonsense suppressor tRNAs. This suggests that natural preexisting nonsense suppression facilitated nonsense codon reassignments and constitutes a novel mechanism of genetic code change. These findings explain for the first time the identity of the stop codons and amino acids reassigned in mitochondrial and nuclear genomes. Nonsense suppressor tRNAs provided the raw material for nonsense codon reassignments, implying that the properties of the tRNA anticodon have dictated the identity of nonsense codon reassignments.

A Sequential “2-1-3” Model of Genetic Code Evolution That Explains Codon Constraints

With regard to the recently published paper by Wu, Bagby, and den van Elsen, ‘‘Evolution of the Genetic Triplet Code via Two Types of Doublet Codons’’ (J Mol Evol 61:54–64, 2005), I should like to make some constructive comments, outlined below. First, the authors appear to make the a priori assumption of an initial singlet code, with little supporting evidence. This aspect needs development, as there is some evidence available, not discussed by the authors, supporting the assumption. Second, the observation that several aminoacyltRNA synthetases (aaRSs) only recognize two nucleotides of the tRNA anticodon (e.g., thrRS, proRS, valRS) is not surprising, given the degeneracy of the standard genetic code. The observation does not support an ancestral doublet code, any more than it simply reflects the fact that there is no requirement for these aaRSs to recognize the anticodon wobble base (given that the cognate amino acids are coded for by fourfold degenerate codons, there is no coding information at the wobble base). Third, the authors suggest that the model explains the number of amino acids in the standard genetic code (20), based on an implied requirement for leu, ser, and arg to be encoded by six codons (sections ‘‘Why Are There 64 Codons and Only 20 Amino Acids’’ and ‘‘Codon Patterns’’). The authors do not make clear what the requirement is. In addition, the model does not explain why there are two additional amino acids, coded for by stop codons in some proteins: selenocysteine and pyrrolysine. Clearly, the code is not restricted to 20 amino acids and could feasibly accommodate 21 or 22 amino acids. The model does not explain why the standard genetic code does not have more amino acids. Fourth, lack of anticodon recognition by an aaRS does not necessarily imply that its cognate amino acid was one of the first amino acids added to the code. The authors suggest that this is the case for ser, leu, and ala, in the section ‘‘An Ancestral Doublet Code.’’ An alternate possibility is that the aaRSs have simply evolved identity determinants other than the anticodon. This is especially clear for serRS, which recognizes the tRNA extra arm as a major identity determinant (Wu and Gross 1993). In addition, leuRS is known to recognize the anticodon in yeast but not in bacteria (Soma et al. 1996) and contacts the anticodon in mitochondria (Sohm et al. 2004). Fifth, the authors assume throughout that the present-day aaRSs coevolved with the code. At the most extreme this implies that, given an initial singlet code of four codons, the four respective aaRSs would have been composed of just those four amino acids. A more likely scenario is that ribozyme aaRSs were replaced with protein aaRSs after the majority of the modern code was established. Finally, as the authors acknowledge, the idea of a singlet–doublet–triplet transition, and that of an ancestral triplet reading frame, is not a new one. The authors provide strong evidence for an ancestral triplet reading frame from modern structural studies. The novelty of their model derives from the idea of a mixture of prefix and suffix doublet codons. However, the model does not account for a fundamental property of the genetic code, namely, the order of constraint on the individual codon positions. The second codon position is the most constrained codon position, followed by the first codon position and, Correspondence to: Steven E. Massey; email: semassey@ chuma1.cas.usf.edu J Mol Evol (2006) 62:809–810 DOI: 10.1007/s00239-005-0222-0

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.

A Preliminary Molecular and Phylogenetic Analysis of the Genome of a Novel Endogenous Retrovirus in the Sea Slug Elysia chlorotica

An endogenous retrovirus that is present in the sea slug Elysia chlorotica is expressed in all individuals at the end of the annual life cycle. But the precise role of the virus, if any, in slug senescence or death is unknown. We have determined the genomic sequence of the virus and performed a phylogenetic analysis of the data. The 6060-base pair genome of the virus possesses a reverse transcriptase-domain-containing protein that shows similarity to retrotransposon sequences found in Aplysia californica and Strongylocentrotus purpuratus. However, nucleotide BLAST analysis of the whole genome resulted in hits to only a few portions of the genome, indicating that the Elysia chlorotica retrovirus is novel, has not been previously sequenced, and does not have great genetic similarity to other known viral species. When more invertebrate retroviral genomes are examined, a more precise phylogenetic placement of the Elysia chlorotica retrovirus can be determined.