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

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

Evolution of the intracellular transport mechanisms in eukaryotes: ciliates and mammals use the same translocation and nuclear localization signals

In the ciliate E. raikovi, self/non-self recognition phenomena are controlled by cell type-specific, water-borne signal proteins (pheromones) by their binding to target cell-surface receptors. The downstream signal transduction pathway activated by the pheromone-receptor interactions of self type (that promote the vegetative, mitogenic cell growth) involves the phosphorylation of a nuclear protein kinase, designated Er-MAPK1, which is structurally similar to the "intestinal-cell kinase" and "male germ cell-associated kinase" described in mammals. To identify the sequence segments responsible for Er-MAPK1 nuclear localization, mouse fibroblasts were transfected with plasmids containing the reporter gene for the "Green-Fluorescent Protein" (GFP) associated to different fragments of the Er-MAPK1 coding sequence. By expressing GFP-tagged protein constructs in mammalian cells, in the C-terminal domain of Er-MAPK1 it was effectively possible to identify an Arg/Lys-rich motif that is required for the nuclear entry of GFP-fused constructs. These results provide evidence that distant related organisms such as ciliates and mammals use the same molecular language for the nuclear translocation and localization of proteins, thus suggesting that this language arose early in the evolution of the eukaryotic cell

Bipolar distribution of the symbiotic association between the ciliate Euplotes nobilii and the γ-proteobacterium Francisella adeliensis.

Ciliates host a huge variety of bacteria, carried either as epibionts onto the cell body surface or as endosymbionts in the cell cytoplasm. Endosymbionts include the gamma-proteobacterium Francisella, which may colonize and harm a variety of hosts, mammals included. Our interest is focused on a new species of Francisella, F. adeliensis, originally isolated from a population of an Antarctic marine species of Euplotes, E. petzi, and subsequently identified also in sympatric populations of three other congeneric Antarctic species, E. euryhalinus, E. focardii and E. nobilii. In consideration of the bipolar distribution of E. nobilii, we inquired whether the F. adeliensis/E. nobilii symbiotic association is limited to the Antarctica or radiates to the Arctic, by screening E. nobilii strains isolated from the coasts of southern Patagonia, Svalbard, Greenland and northern Alaska with a F. adeliensis-specific16S gene amplification and in-situ hybridization. All E. nobilii strains resulted positive to F. adeliensis endosymbionts. By gene and genome sequence comparisons, we are currently analyzing how the E. nobilii/F. adeliensis symbiotic association evolved in time and space

EUPLOTES PHEROMONE GENES: STRUCTURALLY SIMPLE STRUCTURE AND COMPLEX EXPRESSION MECHANISMS

Ciliates are unique in possessing two genomes within the same cytoplasm: a chromosomic transcriptionally silent germinal genome residing inside the cell micronucleus and a sub-chromosomic transcriptionally active somatic genome residing inside the cell macronucleus. The latter is anew generated from the former at every sexual event through short-term dramatic phenomena of chromosome fragmentation, DNA elimination and gene amplification. In spirotrichous ciliates such as Euplotes, the macronuclear genome comprises only gene-size DNA molecules characterized by telomere-capped extremities and a single coding region flanked by 5’ and 3’ un-translated regions of variable lengths. We analyzed the expression of these macronuclear genes in two gene families encoding water-borne signaling pheromones responsible for cell-cell communication and recognition phenomena in two common marine species of Euplotes, E. crassus and E. raikovi. Although structurally simple, these genes regulate their expression through mechanisms that are much more complex than previously thought. They result in the synthesis of multiple transcripts generated by the use of different transcription start sites and the removal of intron sequences from the conserved 5’ regions. However, the presence of several in-frame UAA and UAG stop codons positioned far from the gene poly(A) tail raises the question on whether every transcript is actually able to specify a functional product. We are currently verifying the hypothesis that these stop codons are translated, as it occurs in other ciliates, as sense codons specific for the amino acid glutamine

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

THE BIPOLAR CILIATE EUPLOTES NOBILII IS A TRANS-TROPICAL VECTOR OF POTENTIAL PATHOGENIC PARAFRANCISELLA γ-PROTEOBACTERIA

Ciliates are common hosts of a huge variety of bacteria, which are either carried as epibionts on the cell body surface, or endosymbionts in the cell cytoplasm as is the case of Euplotes species. Francisella γ-proteobacteria (which may colonize and harm a variety of hosts, mammals included) were originally isolated as endosymbionts from an Antarctic population of E. petzi, assigned to F. adeliensis sp. nov. (now re-named Parafrancisella adeliensis), and later identified in other Antarctic E. focardii and E. nobilii populations. In consideration of the bipolar distribution of E. nobilii, we inquired whether and to which extent non-Antarctic conspecific populations host P. adeliensis endosymbionts. Screened in PCR amplifications and in-situ hybridizations with a P. adeliensis-specific 16S rRNA gene probe, E. nobilii populations from coastal sites of southern Patagonia, Svalbard Islands, eastern and western Greenland and northern Alaska all proved to be positive to P. adeliensis, thus supporting the conclusion that E. nobilii may act as a trans-tropical P. adeliensis vector. Insights on the E. nobilii/P. adeliensis symbiotic relationships were next obtained by sequencing and analyzing the genomes of the two species. While the P. adeliensis genome lacks genes encoding enzymes for the synthesis of essential amino acids such as lysine, cysteine, methionine and tyrosine, the E. nobilii genome lacks genes encoding methionine sulfoxide reductase of type A which is an essential enzyme to repair diastereomeric S-form of methionine sulfoxide in oxidized proteins. It thus likely that P. adeliensis relies on E. nobilii cytoplasm to recover essential amino acids, and that E. nobilii relies on P. adeliensis antioxidant enzymes released from the Type-6 Secretion System to face damage from oxidative stress imposed by the high (saturated) oxygen concentrations of the polar sea waters

Protein transport into the nucleus: characterization of nuclear localization signals in the protozoan ciliate Euplotes

In E. raikovi, a nuclear protein kinase, designated Er-MAPK1, appears to be phosphorylated in association with the mechanism of signal transduction which promotes cell proliferation through autocrine interactions between cell type-specific signaling protein pheromones and their membrane receptors. This kinase shows significant structural matching to mammalian kinases that are localized in the nucleus of specialized cell types, such as the "Male germ cell-Associated Kinases" and the "Intestinal Cell Kinases". Two Arg/Lys-rich motifs were identified in the Er-MAPK1 C-terminal domain as putative “nuclear localization signals” and their effective function in directing this protein into the nucleus was studied by expressing GFP-tagged protein constructs in mammalian fibroblasts. The results obtained provide evidence that distant related organisms such as ciliates and mammals use the same molecular language for the nuclear translocation and localization of proteins, suggesting that this language arose early in the evolution of the eukaryotic cell

Primary Structure and Coding Genes of Two Pheromones from the Antarctic Psychrophilic Ciliate, <i>Euplotes focardii</i>

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