Molecular cold-adaptation of protein function and gene regulation: The case for comparative genomic analyses in marine ciliated protozoa.

Euplotes focardii is a marine ciliated protozoan discovered in the Ross Sea near Terra Nova Bay, Antarctica. This organism is strictly psychrophilic, survives and reproduces optimally at 4-5 °C, and has a genome rich in A/T base pairs. Like other ciliated protozoans, Euplotes spp. are characterized by nuclear dimorphism: 1) the germline micronucleus contains the entire genome as large chromosomes; and 2) the somatic macronucleus (∼50 megabases, or 5% of the micronuclear genome) contains small linear DNA nanochromosomes [1-12 kilobases], each of which constitutes a single genetic unit. These characteristics make E. focardii an ideal model for genome-level analysis to understand the evolutionary mechanisms that determine the adaptation of organisms to cold environments. Here we describe two examples that are controlled by phylogenetically appropriate comparison with mesophilic and psychrotolerant Euplotes species: 1) the genes and encoded proteins of the E. focardii tubulin superfamily, including α-, ß-, and γ-tubulins; and 2) the genes of the heat-shock protein (Hsp) 70 family. The tubulins provide particular insight into protein-level structural changes that are likely to facilitate microtubule nucleation and polymerization in an energy poor environment. By contrast, the hsp70 genes of E. focardii and of its psychrotolerant relative E. nobilii reveal adaptive alterations in the regulation of gene expression in the cold. The unique characteristics of the E. focardii genome and the results that we present here argue strongly for a concerted effort to characterize the relatively low complexity macronuclear genome of this psychrophilic organism.

Different roles of two gamma-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature.

Gamma-tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of gamma-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, gamma-tubulin is encoded by two genes, gamma-T1 and gamma-T2, that produce distinct isotypes. Comparison of the gamma-T1 and gamma-T2 primary sequences to a Euplotesgamma-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in gamma-T2. Structural models of gamma-T1 and gamma-T2, obtained using the 3D structure of human gamma-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal 'plus end' contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to gamma-T1, the gamma-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the gamma-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only gamma-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the gamma-T1 isotype associates only with basal bodies. Our results suggest that gamma-T1 and gamma-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that gamma-T1 and gamma-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the gamma-tubulin ring complex at cold temperatures.

Ribosomal cold-adaptation: characterization of the genes encoding the acidic ribosomal P0 and P2 proteins from the Antarctic ciliate Euplotes focardii.

Molecular adaptation at low temperature requires specificities represented mainly by modifications in the gene sequence and consequently in the protein primary structure. To characterize the molecular mechanisms responsible for ribosome cold-adaptation, we compared the ribosomal P0 and P2 genes from the Antarctic ciliate Euplotes focardii with homologous genes from mesophilic organisms, including the ciliates Tetrahymena thermophila and non cold-adapted Euplotes species. This analysis revealed the presence of non synonymous mutations unique to E. focardii. In the P0 protein the mutations produced amino acid substitutions that increased the molecular flexibility that may facilitate a conformational adjustment associated with the interaction with the GTPase center of the large subunit rRNA, and increased the hydrophobicity of the region involved in the interaction with P1/P2 heterodimer, probably to keep associated the ribosomal stalk in the cold. In the P2 protein the mutations produced amino acid substitutions that increased the N-terminus flexibility, which may facilitate interactions with P1 protein in the formation of the heterodimer, and reduced the mobility of the C-terminus, to stabilize the stalk during ribosomal activity. Finally, P proteins appeared to be valid markers for investigating the phylogenetic origin of early eukaryotes.