Crystal structure of the pheromone Er-13 from the ciliate Euplotes raikovi, with implications for a protein-protein association model in pheromone/receptor interactions.

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.

Bioactive molecules from ciliates: Structure, activity, and applicative potential.

Ciliates are a rich source of molecules synthesized to socialize, compete ecologically, and interact with prey and predators. Their isolation from laboratory cultures is often straightforward, permitting the study of their mechanisms of action and their assessment for applied research. This review focuses on three classes of these bioactive molecules: (i) water-borne, cysteine-rich proteins that are used as signaling pheromones in self/nonself recognition phenomena; (ii) cell membrane-associated lipophilic terpenoids that are used in interspecies competitions for habitat colonization; (iii) cortical granule-associated molecules of various chemical nature that primarily serve offence/defense functions.

Two new species of Euplotes with cirrotype-9, Euplotes foissneri sp. nov. and Euplotes warreni sp. nov. (Ciliophora, Spirotrichea, Euplotida), from the coasts of Patagonia: implications from their distant, early and late branching in the Euplotes phylogenetic tree.

Two new Euplotes species have been isolated from cold shallow sandy sediments of the extreme Southern Chilean coasts: Euplotes foissneri sp. nov., from a low-salinity site at Puerto Natales on the Pacific coast, and Euplotes warreni sp. nov., from a marine site at Punta Arenas on the Atlantic coast. Euplotes foissneri has a medium body size (53×36 µm in vivo), a dorsal surface marked by six prominent ridges, a double dargyrome, six dorsal and two ventrolateral kineties, a buccal field extending to about 3/4 of the body length, an adoral zone composed of 28-32 membranelles, and nine fronto-ventral, five transverse and two or three caudal cirri. The bulky, hook-, horseshoe- or 3-shaped macronucleus is associated with one sub-spherical micronucleus. The central body region hosts taxonomically unidentified endosymbiotic eubacteria. Euplotes warreni has a small body size (39×27 µm in vivo), a smooth dorsal surface marked by three deep grooves, a double dargyrome, four dorsal and two ventrolateral kineties, a buccal field extending to about 2/3 of the body length, an adoral zone composed of 23-25 adoral membranelles, and nine fronto-ventral, five transverse and three caudal cirri. The macronucleus is hook- or C-shaped and associated with one spherical micronucleus. Endosymbiotic bacteria belonging to the genus Francisella reside preferentially in the anterior cell region. Both species lack the fronto-ventral cirrus numbered 'V/2', whereby their cirrotype-9 conforms to the so-called 'pattern I', which is the basic distinctive trait of the genus Euplotopsis Borror and Hill, 1995. Phylogenetic analyses of small subunit rRNA gene sequences, however, classify E. warreni into its own early branching clade and E. foissneri into a late branching clade. This indicates a polyphyletic nature and taxonomic inconsistency of the genus Euplotopsis, which was erected to include Euplotes species with cirrotype-9 pattern I.

The Sub-Chromosomic Macronuclear Pheromone Genes of the Ciliate Euplotes raikovi: Comparative Structural Analysis and Insights into the Mechanism of Expression.

In Euplotes raikovi, we have determined the full-length sequences of a family of macronuclear genes that are the transcriptionally active versions of codominant alleles inherited at the mating-type (mat) locus of the micronuclear genome, and encode cell type-distinctive signaling pheromones. These genes include a 225-231-bp coding region flanked by a conserved 544-bp 5'-leader region and a more variable 3'-trailer region. Two transcription initiation start sites and two polyadenylation sites associated with nonconventional signals cooperate with a splicing phenomenon of a 326-bp intron residing in the 5'-leader region in the generation of multiple transcripts from the same gene. In two of them, the synthesis of functional products depends on the reassignment to a sense codon, or readthrough of a strictly conserved leaky UAG stop codon. That this reassignment may take place is suggested by the position this codon occupies in the transcripts, close to the transcript extremity and far from the poly(A) tail. In such a case, one product is a 69-amino acid protein in search of function and the second product is a 126-amino acid protein that represents a membrane-bound pheromone isoform candidate to function as a cell type-specific binding site (receptor) of the soluble pheromones.

A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.

Molecular Structures and Coding Genes of the Water-Borne Protein Pheromones of Euplotes petzi, an Early Diverging Polar Species of Euplotes.

Euplotes is diversified into dozens of widely distributed species that produce structurally homologous families of water-borne protein pheromones governing self-/nonself-recognition phenomena. Structures of pheromones and pheromone coding genes have so far been studied from species lying in different positions of the Euplotes phylogenetic tree. We have now cloned the coding genes and determined the NMR molecular structure of four pheromones isolated from Euplotes petzi, a polar species which is phylogenetically distant from previously studied species and forms the deepest branching clade in the tree. The E. petzi pheromone genes have significantly shorter sequences than in other congeners, lack introns, and encode products of only 32 amino acids. Likewise, the three-dimensional structure of the E. petzi pheromones is markedly simpler than the three-helix up-down-up architecture previously determined in another polar species, Euplotes nobilii, and in a temperate-water species, Euplotes raikovi. Although sharing the same up-down-up architecture, it includes only two short α-helices that find their topological counterparts with the second and third helices of the E. raikovi and E. nobilii pheromones. The overall picture that emerges is that the evolution of Euplotes pheromones involves progressive increases in the gene sequence length and in the complexity of the three-dimensional molecular structure.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi.

BACKGROUND: Deleterious phenomena of protein oxidation affect every aerobic organism and methionine residues are their elective targets. The reduction of methionine sulfoxides back to methionines is catalyzed by methionine-sulfoxide reductases (Msrs), enzymes which are particularly active in microorganisms because of their unique nature of individual cells directly exposed to environmental oxidation. RESULTS: From the transcriptionally active somatic genome of a common free-living marine protist ciliate, Euplotes raikovi, we cloned multiple gene isoforms encoding Msr of type A (MsrA) committed to repair methionine-S-sulfoxides. One of these isoforms, in addition to including a MsrA-specific nucleotide sequence, included also a sequence specific for a Msr of type B (MsrB) committed to repair methionine-R-sulfoxides. Analyzed for its structural relationships with MsrA and MsrB coding sequences of other organisms, the coding region of this gene (named msrAB) showed much more significant relationships with Msr gene coding sequences of Rhodobacterales and Rhizobiales (Alphaproteobacteria), than of other eukaryotic organisms. CONCLUSIONS: Based on the fact that the msrAB gene is delimited by Euplotes-specific regulatory 5' and 3' regions and telomeric C4A4/G4T4 repeats, it was concluded that E. raikovi inherited the coding region of this gene through a phenomenon of horizontal gene transfer from species of Alphaproteobacteria with which it coexists in nature and on which it likely feeds.

Evidence for gene duplication and allelic codominance (not hierarchical dominance) at the mating-type locus of the ciliate, Euplotes crassus.

The high-multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating-type substance (pheromone). In strain L-2D, now known to be homozygous at the mating-type locus, we previously identified two pheromones (Ec-α and Ec-1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR-73 characterized by a heterozygous genotype and strong mating compatibility with L-2D strain. It was found that POR-73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec-α pheromone of L-2D cells. The other two pheromones were shown to be new and were designated Ec-2 and Ec-3 to denote their structural homology with the Ec-1 pheromone of L-2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating-type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating.

Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii, based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the three-dimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.

Homo- and hetero-oligomeric protein-protein associations explain autocrine and heterologous pheromone-cell interactions in Euplotes.

In Euplotes, protein pheromones regulate cell reproduction and mating by binding cells in autocrine or heterologous fashion, respectively. Pheromone binding sites (receptors) are identified with membrane-bound pheromone isoforms determined by the same genes specifying the soluble forms, establishing a structural equivalence in each cell type between the two twin proteins. Based on this equivalence, autocrine and heterologous pheromone/receptor interactions were investigated analyzing how native molecules of pheromones Er-1 and Er-13, distinctive of mating compatible E. raikovi cell types, associate into crystals. Er-1 and Er-13 crystals are equally formed by molecules that associate cooperatively into oligomeric chains rigorously taking a mutually opposite orientation, and each burying two interfaces. A minor interface is pheromone-specific, while a major one is common in Er-1 and Er-13 crystals. A close structural inspection of this interface suggests that it may be used by Er-1 and Er-13 to associate into heterodimers, yet inapt to further associate into higher complexes. Pheromone-molecule homo-oligomerization into chains accounts for clustering and internalization of autocrine pheromone/receptor complexes in growing cells, while the heterodimer unsuitability to oligomerize may explain why heterologous pheromone/receptor complexes fail clustering and internalization. Remaining on the cell surface, they are credited with a key role in cell-cell mating adhesion.

Phylogeographical pattern of Euplotes nobilii, a protist ciliate with a bipolar biogeographical distribution.

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild-type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.

Cell aging-induced methionine oxidation causes an autocrine to paracrine shift of the pheromone activity in the protozoan ciliate, Euplotes raikovi.

Ciliates of the genus Euplotes rely on the autocrine (self) and paracrine (non-self) activities of their water-borne protein pheromones to control the two fundamental phenomena of their life cycle, i.e. vegetative (mitotic) growth and sex manifested as cell union in mating pairs. We observed that cell aging determines the synthesis of increasing concentrations of pheromones that are oxidized at the level of methionine residues which are more exposed on the molecular surface. The oxidized form of the E. raikovi pheromone Er-1 was purified and its interactions with its source cells were shown no longer to be of autocrine type directed to promote cell growth, but changed to interactions of the paracrine type directed to induce cell unions in mating pairs of the selfing type (i.e. involving genetically identical cells). These pairs generate viable offspring, like pairs formed between genetically different cells. It was therefore concluded that aging cells may paradoxically gain beneficial effects from the synthesis of oxidized forms of their pheromones. By undergoing mating in response to the interactions with these forms, they can re-initiate a new life cycle and, in fact, rejuvenate.

Characterization and expression of the gene encoding En-MAPK1, an intestinal cell kinase (ICK)-like kinase activated by the autocrine pheromone-signaling loop in the Polar Ciliate, Euplotes nobilii.

In the protozoan ciliate Euplotes, a transduction pathway resulting in a mitogenic cell growth response is activated by autocrine receptor binding of cell type-specific, water-borne signaling protein pheromones. In Euplotes raikovi, a marine species of temperate waters, this transduction pathway was previously shown to involve the phosphorylation of a nuclear protein kinase structurally similar to the intestinal-cell and male germ cell-associated kinases described in mammals. In E. nobilii, which is phylogenetically closely related to E. raikovi but inhabits Antarctic and Arctic waters, we have now characterized a gene encoding a structurally homologous kinase. The expression of this gene requires +1 translational frameshifting and a process of intron splicing for the production of the active protein, designated En-MAPK1, which contains amino acid substitutions of potential significance for cold-adaptation.

A new dataset of pheromone and pheromone-gene structures from the ciliate, Euplotes crassus.

Like many other organisms, ciliates communicate and interact socially via diffusible chemical signals, named pheromones, that are functionally associated with a genetic mating-type mechanism of cell self/not-self recognition. In Euplotes species, pheromones form species-specific families of small, globular, and disulfide-rich proteins folding into exclusively helical secondary structures. Each is specified by one of a series of high-multiple alleles that are inherited in Mendelian fashion with relationships of co-dominance at the so-called mat genetic locus of the cell transcriptionally inert micronuclear genome, and expressed in the transcriptionally active macronuclear genome as individual DNA molecules in which the central coding region is flanked by 5'-leader and 3'-trailer noncoding regions ending with C4A4/T4G4 telomeric repeats. In E. crassus, a cosmopolitan marine species with a long tradition in the study of ciliate mating systems and breeding patterns, oligonucleotides specific to amino acid sequences of pheromones Ec-1 and Ec-α were previously used to clone and sequence a first set of four structurally distinct macronuclear (mac) pheromone coding genes, mac-ec-α, mac-ec-1, mac-ec-2 and mac-ec-3, from two interbreeding strains, L-2D and POR-73. The use of these oligonucleotides in PCR amplifications of macronuclear DNA preparations from three other E. crassus interbreeding strains, ES10, Fava4 and MN4, has now resulted in the characterization of a second set of eight new pheromone coding genes, mac-ec-ß, mac-ec-γ, mac-ec-δ, mac-ec-ε, mac-ec-µ, mac-ec-4, mac-ec-5 and mac-ec-6. Multiple alignment between previously and newly determined pheromone-gene sequences reinforces the concept that the E. crassus pheromone-gene family includes two sub-families, which likely reflect a duplication of the micronuclear mat gene locus and represent an apomorphic trait of the E. crassus clade. Members of one sub-family (each identified with a Greek letter) show a 500-bp 5'-leader noncoding region rich in AGGA/AGGGA repetitions, and encode 56-amino acid pheromones with eight conserved Cys residues. Members of the other sub-family (each identified with an Arabic numeral) show an 800-bp 5'-leader noncoding region without AGGA/AGGGA repetitions, and encode 45-amino acid pheromones with ten conserved Cys residues.

Primary Structure and Coding Genes of Two Pheromones from the Antarctic Psychrophilic Ciliate, Euplotes focardii.

In ciliates, diffusible cell type-specific pheromones regulate cell growth and mating phenomena acting competitively in both autocrine and heterologous fashion. In Euplotes species, these signaling molecules are represented by species-specific families of structurally homologous small, disulfide-rich proteins, each specified by one of a series of multiple alleles that are inherited without relationships of dominance at the mat-genetic locus of the germinal micronuclear genome, and expressed as individual gene-sized molecules in the somatic macronuclear genome. Here we report the 85-amino acid sequences and the full-length macronuclear nucleotide coding sequences of two pheromones, designated Ef-1 and Ef-2, isolated from the supernatant of a wild-type strain of a psychrophilic species of Euplotes, E. focardii, endemic to Antarctic coastal waters. An overall comparison of the determined E. focardii pheromone and pheromone-gene structures with their homologs from congeneric species provides an initial picture of how an evolutionary increase in the complexity of these structures accompanies Euplotes speciation.

Analysis of autapomorphic point mutations provides a key for the tangled taxonomic distinction of the closely related species, Euplotes crassus, E. minuta and E. vannus (Ciliophora, Euplotida).

A well-defined clade of the Euplotes phylogenetic tree is represented by marine species characterized by a single-type dargyrome and ten fronto-ventral cirri. Three of them, namely Euplotes crassus, E. minuta and E. vannus, form a complex of closely related species of large use in experimental ciliatology. Despite morphometric and genetic analyses having substantiated their taxonomic separation, ambiguities still persist in strain assignments to one or another species. In addition to objective reasons intrinsic to significant overlapping of most morphological parameters, ambiguities also result from divergences (inherited from past literature) in deciding which of the two morphotypes, E. crassus or E. vannus, is characterized by a larger or a medium cell body size (E. minuta being clearly distinct by a smaller morphotype). By analysing nuclear SSU-rRNA gene and ITS region sequences from 37 strains, previously assigned to E. crassus, E. minuta and E. vannus based on conventional taxonomic parameters, we identified and used ITS autapomorphic point mutations to design three species-specific primers. In combination with an Euplotes-generic primer, they proved to be very effective in running polymerase chain reactions that produce amplicons of species-specific size that reliably resolve ambiguities in assigning strains to E. crassus, E. minuta or E. vannus.

Isolation and structural characterization of two water-borne pheromones from Euplotes crassus, a ciliate commonly known to carry membrane-bound pheromones.

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-α and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-α and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-α and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome.

Functional chimeric genes in ciliates: An instructive case from Euplotes raikovi.

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.

Ancient Immunity. Phylogenetic Emergence of Recognition-Defense Mechanisms.

Although still scarcely considered by the majority of the biomedical world, invertebrates have greatly contributed to the elucidation of fundamental biological problems [...].