The water-borne protein signals (pheromones) of the Antarctic ciliated protozoan Euplotes nobilii : structure of the gene coding for the En-6 pheromone 1 Antonietta La Terza, Nicoleta Dobri, Claudio Alimenti, Adriana Vallesi, and Pierangelo Luporini Abstract: The marine Antarctic ciliate, Euplotes nobilii, secretes a family of water-borne signal proteins, denoted as phero- mones, which control vegetative proliferation and mating in the cell. Based on the knowledge of the amino acid sequences of a set of these pheromones isolated from the culture supernatant of wild-type strains, we designed probes to identify their encoding genes in the cell somatic nucleus (macronucleus). The full-length gene of the pheromone En-6 was determined and found to contain an open-reading frame specific for the synthesis of the En-6 cytoplasmic precursor (pre-pro-En-6), which requires 2 proteolytic cleavages to remove the signal peptide (pre) and the prosegment before secretion of the mature protein. In contrast to the sequence variability that distinguishes the secreted pheromones, the pre- and pro-sequences appear to be tightly conserved and useful for the construction of probes to clone every other E. nobilii pheromone gene. Potential intron sequences in the coding region of the En-6 gene imply the synthesis of more En-6 isoforms. Key words: Antarctic ciliates, pheromone genes, cold adaptation, Antarctic biology. Re ´sume ´: Euplotes nobilii, un cilie ´ marin de l’Antarctique, secre `te une famille de prote ´ines signale ´tiques transmises par l’eau appele ´es phe ´romones, qui contro ˆlent la prolife ´ration et la conjugaison des cellules ve ´ge ´tatives. Nous avons e ´labore ´ des sondes a ` partir des informations tire ´es de la se ´quence d’acides amine ´s d’un groupe de ces phe ´romones isole ´es du sur- nageant de culture de souches sauvages, afin d’identifier les ge `nes qui les codent dans le noyau somatique des cellules (macronoyau). Le ge `ne de pleine longueur de la phe ´romone En-6 a e ´te ´ caracte ´rise ´ et il contient un cadre de lecture ouvert spe ´cifique a ` la synthe `se du pre ´curseur cytoplasmique de En-6 (pre ´-pro-En-6), qui doit e ˆtre clive ´ par prote ´olyse a ` 2 reprises afin d’enlever le peptide signal (« pre ´ ») et le segment « pro », avant la se ´cre ´tion de la prote ´ine mature. A ` la diffe ´rence des se ´quences qui distinguent les phe ´romones se ´cre ´te ´es qui sont variables, les se ´quences « pre ´ » et « pro » semblent haute- ment conserve ´es et pourraient e ˆtre utiles pour e ´laborer des sondes permettant de cloner les autres ge `nes des phe ´romones de E. nobilii. La pre ´sence de se ´quences d’introns pre ´sume ´s dans la re ´gion codante du ge `ne En-6 implique que plusieurs isoformes de En-6 sont synthe ´tise ´es. Mots-cle ´s : cilie ´s de l’Antarctique, ge `nes de phe ´romones, adaptation au froid, biologie de l’Antarctique. [Traduit par la Re ´daction] Introduction A major component of the microbial community of the Antarctic coastal waters is represented by protozoan ciliates (Petz et al. 1995; Vincent 2000; Petz 2005). Some species, of Euplotes in particular, are easy to identify, isolate, and expand into massive laboratory cultures capable of repro- ducing true-to-type for long (practically indefinite) times (Valbonesi and Luporini 1990). Therefore, they represent excellent (as well as inexpensive) research material for the investigation of various relevant aspects of the biology and adaptive mechanisms and strategies that organisms evolved to thrive successfully in the polar environment. We have recently focused the most interest on 1 of these Antarctic (psychrophilic) Euplotes species, Euplotes nobilii, in relation to the capacity that it shares with a few other spe- cies of Euplotes, Euplotes raikovi and Euplotes octocarina- tus in particular, to constitutively secrete signal proteins (Vallesi et al. 2008). These water-borne molecules, desig- nated as pheromones, are known to promote cell growth (mitotic reproduction) and mating (sex) activities through binding to specific cell membrane receptors (Vallesi et al. 2005). The structural characterization by NMR spectroscopy analysis of 3 of these E. nobilii pheromones, purified from the culture supernatant of wild-type strains and designated En-1, En-2, and En-6 (Alimenti et al. 2003; Placzek et al. 2007; Pedrini et al. 2007), has permitted us to investigate cold-adaptive specificities that distinguish these proteins from their mesophilic counterparts previously characterized from E. raikovi (for a review see Luporini et al. (2005)). Received 5 September 2008. Accepted 6 October 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 31 January 2009. A. La Terza, N. Dobri, C. Alimenti, A. Vallesi, and P. Luporini. 2 Dipartimento di Biologia Molecolare Cellulare Animale, University of Camerino, 62032 Camerino (MC), Italy. 1 This article is one of a selection of papers in the Special Issue on Polar and Alpine Microbiology. 2 Corresponding author (e-mail: piero.luporini@unicam.it). 57 Can. J. Microbiol. 55: 57–62 (2009) doi:10.1139/W08-122 Published by NRC Research Press