Klaus Heckmann

Interspecific Morphogens Regulating Prey-Predator Relationships in Protozoa

The ciliate Euplotes octocarinatus and some close relatives of it are triggered by predator-released substances to undergo morphogenetic changes that inhibit their engulfment. The changes occur within a few hours and do not require cell division. They are perpetuated during reproduction so long as the concentration of the morphogen is maintained. The ability of Euplotes to respond to predator-produced signals by a defensive change in cell architecture probably provides an effective mechanism for damping population oscillations ofboth prey and predators andfosters coexistence. The signal-induced cell transformation merits study for its own sake because of its developmental implications.

Predation risk of typical ovoid and ‘winged’ morphs of Euplotes (Protozoa, Ciliophora)

Freshwater species of the genus Euplotes (Protozoa, Ciliophora) change their morphology in the presence of some of their predators. The ciliates develop extended lateral ‘wings’ as well as dorsal and ventral projections which make engulfment by predators more difficult. In a series of laboratory experiments ingestion rates of four protozoan predators, the ciliates Lembadion bullinum, Dileptus anser, Stylonychia mytilus and Urostyla grandis, and one metazoan predator, the turbellarian Stenostomum sphagnetorum, on three species of Euplotes (E. octocarinatus, E. patella and E. aediculatus) were determined. It was calculated that the probability of rejection by a predator changed from 1:1 for ovoid morphs of Euplotes to about 2:1–20:1 for ‘winged’ morphs of Euplotes, dependent on the prey and predator species that were combined. The nutritional condition of the prey also had some influence. In mixed-species cultures of prey and predators, transformed cells of E. octocarinatus survived for several months.

Prokaryotic Symbionts of Ciliates

Prokaryotes living in ciliates were first noticed over a century ago by J. Muller (1856). Rod-shaped structures were observed in the macro-nuclei and micronuclei of a number of ciliates, and less commonly, in their cytoplasm. In the beginning, it was not clear whether they were parasites or spermatozoa because the micronucleus was considered to be a testis and the macronucleus an ovary, while chromosome filaments and endonuclear symbionts were mistaken for spermatozoa. This view was corrected by Butschli (1876), who also wrote the first review on parasites in ciliates (Butschli, 1889). Accounts of early observations of bacteria in protozoa that followed this initial period were reviewed by Kirby (1941), Wichterman (1953), and Ball (1969).

Nuclear processes in Euplotes octocarinatus during conjugation

The pregamic and metagamic nuclear divisions during conjugation of Euplotes octocarinatus and the development of new micro- and macronuclei were investigated with the help of phase contrast microscopy and a staining of the cells by aceto carmine. The most important stages are illustrated with microphotographs and a time table of the events based on experiments carried out at 26°C is presented. It is shown that E. octocarinatus has n = 35 chromosomes and that all of them enter the macronuclear anlage and undergo polytenisation before they break down into the short DNA-pieces typical of the adult macronucleus of hypotrich ciliates. The nuclear events suggest that, similar to what has been reported for E. patella, frequently sister nuclei become the pronuclei which should result in the formation of a high proportion of isogenic co-conjugant lines. A comparison of the nuclear events in heterotypic pairs (co-conjugants of different mating types) with those in homotypic pairs (co-conjugants of the same mating type) revealed no differences apart from a tendency of homotypic pairs to become arrested at the premeiotic division and then to separate. Although hundreds of cells were investigated, regeneration of pieces of the old macronucleus or fusion of fragments with a newly formed anlage, as is reported for certain other Euplotes species, has not been observed.

The two γ-tubulin-encoding genes of the ciliate Euplotes crassus differ in their sequences, codon usage, transcription initiation sites and poly(A) addition sites

We have isolated and sequenced two gamma-tubulin (gamma-Tub)-encoding macronuclear genes of the ciliate Euplotes crassus (Ec), as well as their corresponding cDNAs. Our results reveal that the two genes (gamma-tub 1 and gamma-tub 2) have introns in homologous positions, but differ in their sequences, codon usage, transcription initiation sites and poly(A) addition sites. They both consist of three exons, two introns and two short non-coding sequences on both ends, and they both code for polypeptides of 462 amino acids (aa). The two genes share 76% identity at the nucleotide (nt) level, 86% at the deduced aa level and show 61-92% aa homology to the gamma-Tubs of other organisms. The gamma-tub 2 gene contains two in-frame UGA codons which, like UGA codons in other Euplotes genes, probably code for cysteines. No UGA triplet was found in the gamma-tub 1 gene. Further studies on the cDNA ends indicate that gamma-tub 1 uses at least three transcription initiation sites and two poly(A) addition sites. In contrast, only one transcription initiation site and one poly(A) addition site were identified in gamma-tub 2.

EXPRESSION OF THE PHEROMONE 3-ENCODING GENE OF EUPLOTES OCTOCARINATUS USING A NOVEL BACTERIAL SECRETION VECTOR

The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.

A comparative study of RNAs from four ciliate groups and a mollusc and data on their poly(A)+ RNA in vitro translation

RNAs from the ciliates Euplotes octocarinatus, Paramecium tetraurelia, Stylonychia lemnae, Tetrahymena thermophila and the snail Lymnaea stagnalis were isolated and compared for structural differences. The organisms were found to differ considerably in the size and structure of their rRNAs, the extent of polyadenylation and the length of the respective poly A tails of their mRNAs. The mRNAs of Euplotes, Paramecium, Stylonychia and Lymnaea were also compared for in vitro translatability in the rabbit reticulocyte lysate and the wheat germ translation systems. In accordance with earlier observations for Paramecium [15] and the finding that Paramecium, Stylonychia, Oxytricha and Tetrahymena deviate from the universal genetic code by using the stop codons UAA and UAG to encode glutamine [4, 8,11,12,21] it was found that Paramecium and Stylonychia poly(A)(+) RNAs were poor templates for in vitro translation. Unexpectedly, however, poly(A)(+) RNA of the ciliate Euplotes was readily translated in vitro and yielded a great number of polypeptides with different molecular weights comparable to the data obtained with poly(A)(+) RNA of Lymnaea.