The Process of Selection of Erythromycin-Resistant Mitochondria by Erythromycin in Paramecium

A. ADOUTTE 0 0 Laboratoire de Ge'ne'tique, Universite Paris XI, Centre d'Orsay , 91405 , France R. PERASSO Laboratoire de Biologie Cellulaire IV Mitochondrial mutations conferring erythromycin resistance (EE) are available in Paramecium and it is possible to obtain (by conjugation and cytoplasmic exchange) exconjugant cells containing a majority of wild-type erythromycin-sensitive (Es) mitochondria and a minority of E11 ones. In the presence of erythromycin, such 'mixed' cells progressively become resistant. This process of acquisition of resistance has been studied cytologically (on thin sections of single cells) and genetically (by evaluating, on the basis of previous data, the proportion of E " / E s mitochondrial genomes at various times). While at early stages of the process of transformation the whole mitochondrial population appears rather homogeneous, at later stages, (i.e. when the cell has resumed growth in the antibiotic-containing medium) one finds, side by side, both resistant-looking mitochondria (structurally normal) and sensitive-looking ones, showing the typical alterations induced in E s cells by erythromycin. Conversely, a progressive decrease in the number of Es genomes can be demonstrated. ERYTHROMYCIN-RESISTANT MITOCHONDRIA BY ERYTHROMYCIN IN PARAMECIUM INTRODUCTION Mutations conferring resistance to erythromycin have been isolated in Paramecium aurelia and shown to be located in mitochondrial DNA (Beale, 1969; Adoutte & Beisson, 1970; Beale, Knowles & Tait, 1972). In the course of the genetical analysis of these mutations the following observations have been made. After conjugation between an erythromycin-sensitive (Es) and an erythromycin-resistant cell (ER), if cytoplasmic exchange has occurred, the sensitive ex-conjugant harbours a majority of Es mitochondria and a minority of ER ones. When such a cell is placed in erythromycin-containing medium (ERY) it first behaves like a sensitive cell: its multiplication is blocked; but after a lag of 2-4 days, it becomes progressively resistant and resumes growth. This lag has been interpreted as reflecting the selective multiplication of the ER mitochondria until their number has become sufficient to allow the cells to resume growth (Adoutte & Beisson, 1970). A detailed genetical and cytological analysis of this process of transformation from erythromycin-sensitivity to erythromycin-resistance was carried out on the basis of the two following facts, previously established: (1) Erythromycin exerts profound effects on the structure of mitochondria in E s strains of Paramecium (disappearance of cristae, appearance of rigid plates, etc.) whereas the mitochondria of ER strains remain little or not at all affected (Adoutte, Balmefrezol, Beisson & Andre, 1972). It could thus be hoped that a distinction would be possible between ER and Es mitochondria within a mixed cell placed in erythromycin. (2) When a mixed cell (containing ER and E s mitochondria) is grown in the absence of antibiotic, Es mitochondria show a selective advantage over ER ones. Even if the initial number of Es mitochondria is low in the original cell, after a number of cell generations, no ER mitochondria can be detected: all the cells in the clone become pure sensitives. This evolution towards sensitivity follows reproducible kinetics which are a function of the relative proportion of the 2 types of mitochondria in the original mixed cell and of the genotype of the mitochondria confronted. We have established, for instance that after 20-25 fissions in non-selective medium, a cell containing 50% ER mitochondria and 50 % Es mitochondria produces progeny in which no ER mitochondria can be detected, whereas 35-40 generations are needed in E]^,2 + Es combinations (Adoutte & Beisson, 1972). We thus have a means of roughly evaluating the proportion of resistant mitochondria in a mixed cell, at any chosen time, by placing the cell in nonselective medium and determining the stage at which the clone obtained gives rise to pure sensitive cells. This paper describes in parallel the genetical and cytological evolution in ERYof the progeny of mixed cells obtained after conjugation and cytoplasmic exchange between a wild-type (Es) strain and either a moderately (ER) or a strongly (E}^2) resistant strain. An abstract of this work has been published in J. Microscopie, 1972, 14, 78 a. An analogous situation has been studied cytologically by Knowles (1972); in this case the ER mitochondria were introduced into sensitive cells by a microinjection technique. MATERIAL AND METHODS Strains The strains described below have been used. Es is the wild-type strain of Paramecium aurelia, stock d 4-2, syngen 4, sensitive to erythromycin. The cells of this strain are unable to multiply in the presence of 100 /<g/ml of erythromycin. E? and Ef02, are two erythromycin-resistant strains isolated from the wild-type strain. Strain Ef is moderately resistant to erythromycin: the cells undergo approximately 3 fissions/ day in the presence of 100 /tg/ml erythromycin. Strain EJ'O2 is highly resistant: the cells undergo 4-5 fissions/day in 100 fig/ml erythromycin, which is equivalent to the growth rate of all 3 strains in the absence of antibiotic. Mitochondria in Ef cells placed in ERY show slight ultrastructural alterations, whereas mitochondria in EK02 cells placed in ERY are not at all affected (Adoutte et al. 1972). Media and culture conditions have already been described (Sonneborn, 1970; Adoutte & Beisson, 1970); cells were cultured in a 'Scotch Grass' infusion bacterized with Aerobacter aerogenes. Suitable volumes of a concentrated erythromycin stock solution were added to this bacterized medium to reach a concentration of 100 fig/m\ of erythromycin. This concentration of erythromycin in the culture medium was used as the selective medium (ERY) throughout the experiments. Crosses have been carried out in the usual way (Sonneborn, 1970; Adoutte & Beisson, 1970). From a great number of pairs isolated, those that remained united by a cytoplasmic bridge after fertilization were selected for study. The presence of this bridge indicates that cytoplasm, including mitochondria, has been exchanged between the mates. The bridges are usually transient (5 to 20 min in the cases studied). It is known that the amount of mitochondria exchanged between conjugants is, roughly, a function of the persistence of the cytoplasmic bridge (Adoutte & Beisson, 1970). Tests for genetic purification Protocol. Starting with a mixed cell and growing it in normal medium, the number of cell generations necessary to reach the stage where all the cells will be Es is an indication of the initial proportion of resistant mitochondria present in the mixed cell. In order to ascertain when the progeny of a mixed cell no longer contained ER mitochondria the cell was placed in normal medium; after 10 fissions a sample of 3-30 cells was tested in ERY to discover whether they contained E" mitoch (...truncated)