Download the process of selection of erythromycin

J. Cell Sci. 14, 475-497 (i974)
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475
THE PROCESS OF SELECTION OF
ERYTHROMYCIN-RESISTANT MITOCHONDRIA
BY ERYTHROMYCIN IN PARAMECIUM
R. PERASSO
Laboratoire de Biologie Cellulaire IV
AND A. ADOUTTE*
Laboratoire de Ge'ne'tique, Universite Paris XI,
Centre d'Orsay, 91405, France
SUMMARY
Mitochondrial mutations conferring erythromycin resistance (E E ) 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 (E s ) 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 E s genomes can be
demonstrated.
The complete genetical and cytological transformation from erythromycin sensitivity to
erythromycin resistance can occur after less than three fissions in erythromycin-containing
medium.
The results indicate that intensive selective multiplication of E R mitochondria occurs, under
the pressure of erythromycin, virtually in the absence of cellular division. The possibility of
dissociating mitochondrial division from cell division emphasizes the extent of mitochondrial
autonomy.
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
E s mitochondria and a minority of E R ones. When such a cell is placed in erythromycin-containing medium (ERY) it first behaves like a sensitive cell: its multiplication
* Present address: Centre de Gen6tique Moleculaire, CNRS, 91190 Gif-sur-Yvette, France.
476
R. Perasso and A. Adoutte
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
E R 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 E R 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 E R and E s mitochondria within a mixed cell placed in erythromycin.
(2) When a mixed cell (containing E R and E s mitochondria) is grown in the absence
of antibiotic, E s mitochondria show a selective advantage over E R ones. Even if the
initial number of E s mitochondria is low in the original cell, after a number of cell
generations, no E R 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% E R mitochondria and 50 % E s mitochondria produces progeny in which no E R mitochondria
can be detected, whereas 35-40 generations are needed in E]^,2 + E s 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 E R 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
Selection of mitochondria in Paramecium
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
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
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" mitochondria and hence were able to grow and 3 cells were subcloned individually
in normal medium. The 3 subclones were grown for 10 new fissions, tested and subcloned
again, only one cell being subcloned for each of the 3 clones, etc. . .. For each mixed cell
studied, starting from the 10th fission, 2 sub-clones were thus maintained for a large number
of generations in normal medium and tested every 10 fissions.
Definition of the various types of cells. It is possible to estimate the number of EB mitochondria
in a mixed cell using a formula reported by Preer (1948). In the present paper we do not attempt
a precise estimation of the composition of mixed cells but only distinguish the following 5 convenient classes, defined experimentally: (1) pure EB cells, with 100% EH mitochondria. These
never give rise to sensitive cells. In some cases the tests have been carried out up to the 140th
generation in normal medium. (2) Highly enriched cells. These start giving rise to cells lacking
E" mitochondria only after at least 20 generations in normal medium, for the EJ1 mitochondria,
or 30-40 generations, for the E1'O2 mitochondria. (3) Moderately enriched cells. These give rise
to 100 % E" cells after 10 generations in normal medium and start giving rise to cells lacking EB
mitochondria after about 15 generations in normal medium (for EB mitochondria), or 25 generations (for the EB02 mitochondria). (4) Weakly enriched cells. Sensitive cells appear as early as the
10th generation in normal medium. And (5) purely sensitive cells, with 0% ER mitochondria.
No resistant cells appear, whatever the stage at which the cells are tested.
By comparison with the results obtained for cells containing 50 % EB and 50 % Es mitochondria (Adoutte & Beisson, 1972) it can be very roughly estimated that the proportion of E B
mitochondria is 75-90 % in highly enriched cells, 25-75 % m moderately enriched cells, and
1-25 % in weakly enriched cells.
Electron microscopy
All studies have been carried out on single cells. Each cell was fixed in 2 % glutaraldehyde in
0-05 M cacodylate buffer, pH 7^3, at 4 °C for 30 min, washed rapidly several times in the same
buffer and postfixed for 1 h in 2 % osmium tetroxide in the same buffer. After a brief washing
R. Perasso and A. Adoutte
47 8
the cell was embedded in a i-mm cube of i % agar. The cube thus obtained was dehydrated in
ethanol and propylene oxide and embedded in Epon. Sections were cut on an OmU2 ultramicrotome equipped with a diamond knife. They were recovered on grids coated with a
Parlodion membrane and contrasted with uranyl acetate and lead citrate. Observations were
carried out in a Siemens Elmiskop IA electron microscope.
RESULTS
Altogether 9 pairs have been studied both genetically and cytologically (3 from the
Ef x E s cross, 6 from E ^ x E s cross) and 14 pairs genetically only (all from the
Ef x E s cross). After conjugation and cytoplasmic exchange had occurred and the pairs
had separated the ex-conjugants were isolated individually and placed in ERY either
immediately (EJ^2 * Es) or after having undergone one fission in normal medium
(E? x Es).
1 day
2 days
3 days
5 days
in ERY
4 days
14a
>100 cells
oo0ooo
0
QQ Q0
Er, x E s
pair 14
"\>=#
14b
Fig. 1. The development of pair 14. The exact number of cells has been drawn
except when stated. Heavy arrows indicate that a cell has been removed for EM study,
the cell being referred to, in the text and on figures, by a number (14a, 14b, . . .).
Light arrows indicate that a cell has been removed and placed back in normal medium
to study the extent of its genetic purification.
In all cases, the E R ex-conjugant grew normally in the presence of erythromycin
whereas the E s ex-conjugant underwent 2 or 3 residual fissions before fissions were
blocked for 2-4 days. During this period the E s cells first displayed the E s phenotype
(dark, slow swimming), then progressively acquired an E R phenotype (clear, rapid
swimming), and finally resumed growth. In the course of this development, some cells
were taken for EM observation, others for study of the extent of genetic purification
reached, that is the proportions of E R and E s mitochondria they contained.
All the pairs showed a roughly similar development. That of pair No. 14 from the
E R x E s cross (Fig. 1) can be considered as representative. The cytological results on
20
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PureR
PureR
High
PureR
PureR
Pure R
High
High
PureR
Nil
Weak
Moderate
High
Moderate
Moderate
Moderate
Moderate
Moderate
High
High (or
pure R)
High
chondria
Enrichment
, in ER mito-
indicates that all tested cells are ER, -, that
tions (see Material and methods).
1 - , that the
all are E"; 1 - , that approximately half were ER and half ES;
majority were ER, 1 - - that the majority were ES. When the different subclones
yielded different results they have been fully represented (e.g. No. 67). I n pair
No. 4, 6 cells (out of 13)in process of transformation were analysed.
+
+
+
+
+
+
+
++I++I-
+I-
-
-
-
+
40
Results of the genetical purification tests, fissions
T h e column ' Generations in ERY' indicates the stage at which cells in process of
transformation in ERY medium have been removed and placed back in normal
medium. Number of generations such as 2-3 indicate that some cells of the clone
have not yet gone through the 3rd fission (e.g. j,6 or 7 cells present).
For the tests of genetical purification 3-30 cells are tested in ERY every 1 0genera-
EP~,x E~
E: x E~
Cross
No. of
pair
Duration
of cytoplasmic
bridge,
min
Table r . The enrichment of ES cells in ER mitochondria as a function of time and number of generations in erythromycin-containing medium
\O
P
h
1.
2
2
2
'
s.
%
%
s
%
3
2
$.
Fd
2
480
R. Perasso and A. Adoutte
this pair are presented in Figs. 4-10. The genetical results are summarized in Table 1.
The method of numbering the cells is indicated on Fig. 1.
1st day. Cell 14a (Fig. 4). After 24 h in ERY the first mitochondrial alterations
appear: beginning with loss of cristae, reduction of mitochondrial diameter and increase in mitochondrial length, irregularity in the contours of some mitochondria.
These alterations are characteristic effects of ERY on sensitive mitochondria (Adoutte
et al. 1972).
A sister cell of 14 a was placed in normal medium and yielded a clone (1 o generations)
from which 15 cells were tested and were found to be sensitive. This result indicates
that the cell did not contain significant amounts of E R mitochondria (<^ 25%). No
significant increase in E R mitochondria has thus occurred in the cells derived from the
sensitive exconjugant of this pair after 24 h in ERY.
As a control, cell 14b (Fig. 5), derived from the E R ex-conjugant of the same pair,
contains a relatively 'healthy' mitochondrial population. The slight mitochondrial
alterations detectable (slight decrease in cristae, irregular contours) are typical of the
E R mutant which is only moderately resistant (Adoutte et al. 1972).
2nd day. Cell 14a! (Fig. 6). After 2 days in ERY, the mitochondrial alterations are
increased in cells derived from the sensitive ex-conjugant just as in purely sensitive
cells. Cristae disappear in most mitochondria, but when still present they display a
wavy configuration. The matrix becomes denser and some plates begin to appear.
Mitochondria are more elongated and more slender, some even collapse. No 'resistantlooking' mitochondria are yet observable.
Although this cell looks cytologically like a pure sensitive one, one of its sister cells is
already enriched in E R mitochondria; indeed at this stage, after 10fissionsin normal
medium, the sister cell yielded 20 resistant cells out of 27 tested cells, but all 30 cells
tested after 20 fissions were sensitive. Increase of E R mitochondria has thus begun
between stages 14a and i4a x (see Fig. 1); the proportion of E R mitochondria must
have been small because they were overgrown by E s ones after only 20 fissions in
normal medium.
Cells from the clone derived from the resistant conjugant still retain nearly normal
mitochondria (Fig. 7). Sensitive-looking mitochondria coming from the E s partner
have never been observed in these cells.
\th day. Cell 1483 (Fig. 8). The sensitive ex-conjugant has now undergone 3-4 fissions in ERY and the cells are slowly resuming a normal growth rate. The mitochondrial population assumes a much ' healthier' aspect, in contrast with what can be
observed in a purely sensitive cell, taken as a control, in which alterations have become
quite dramatic (Fig. 9). Three types of mitochondria can be observed in cell i4a 3 : (1)
Clearly sensitive looking mitochondria with a dense matrix and very few or no cristae.
(2) Normal, resistant-looking mitochondria (clear matrix, abundant cristae and mitochondrial ribosomes). The shape of these mitochondria is, however, irregular and some
pictures suggest a budding process (Fig. 11). And (3) Intermediate-type mitochondria.
These resemble resistant mitochondria by their clear matrix and sensitive mitochondria by the wavy arrangement of their cristae.
Three sister cells of cell 1483 were cloned in normal medium. Two of them were
Selection of mitochondria in Paramecium
K
481
shown to be moderately enriched in E mitochondria and the third highly enriched.
Thus, the genetical enrichment in E K mitochondria has progressed since stage 14 a! but
it is clear that the cells are not yet pure E R (Table 1).
At the same stage the cells issued from the resistant ex-conjugant retain normal
mitochondria.
In summary, the cytological as well as genetical evolution in ERY of the ex-sensitive
conjugant of pair 14 displays 3 stages: (1) slight enrichment in E R genetic determinants
without notable modification of the sensitive aspect of the mitochondrial population;
(2) considerable genetical enrichment in E R determinants correlated with the appearance of numerous resistant-looking mitochondria; and (3) rapid genetical purification
associated with the acquisition of a homogeneous resistant mitochondrial population,
after cells have resumed growth.
With some variations, the process described for pair No. 14 has been observed for all
the pairs of the Ef x E s cross. Genetical results for some other pairs are reported in
Table 1. It can be seen that, as the number of fissions (or number of days) in ERY
increases, cells of sensitive origin are increasingly enriched in ERmitochondria, but they
are not yet purely E R after 4 fissions in ERY since they can still yield sensitive cells if
grown for numerous fissions in normal medium.
At late stages (5-6 fissions), some sensitive mitochondria have exceptionally been
observed among a vast majority of resistant mitochondria (Fig. 12, part of a cell from
pair No. 36, that has resumed normal growth in ERY slightly earlier than 14a). The
distinction between the 2 types of mitochondria is very striking under these conditions.
At this stage most cells behave as genetically pure E R (pair No. 3, Table 1).
In the E{^2 x E s combination, the important fact emerging is the greater rapidity of
transformation from E s to E R . This is observable genetically as well as cytologically.
The development of pairs 69 and 72 is shown schematically in Figs. 2 and 3. The
genetic data for these 2 representative pairs and some others are recorded in Table i,
and the cytological data in Figs. 12 and 13.
In the case of pair No. 72, the progeny of the sensitive ex-conjugant behaved
genetically as purely resistant after fewer than 3fissionsin ERY. The population of
mitochondria in a cell of the same clone observed in the EM after 2fissionsin ERY
already is nearly pure for resistant characteristics (Fig. 12); only 1 out of 20 mitochondria shows sensitive characteristics. In addition some mitochondria show a
curious 'mosaic' condition: one part having resistant characteristics while another
shows sensitive ones (collapsed extremities and engulfed glycogen).
In the case of pair No. 69, the cell tested genetically after 1 or 2 fissions (3 cells
present) in ERY is already enriched in resistant mitochondria (Table 1). At this stage
it is difficult to classify mitochondria cytologically as sensitive or resistant. At the next
test (after 3fissionsin ERY), of the 2 cells studied genetically, one is purely resistant,
the other is very highly enriched for E K mitochondria. In good agreement with these
observations we observe a large majority of resistant-looking mitochondria and a
minority of sensitive-looking ones, the discrimination between the 2 types being now
very clear (Fig. 13).
The steps of acquisition of erythromycin-resistance thus appear to be analogous in
R. Perasso and A. Adoutte
482
1 day
3 days
2 days
72b,
ERY
00
72b,
00
2S-30 cells
72a
5 days in ERY
4 days
00
0...16-20
"fy/V >100 cells
cells
'"'[•' >100
cells
pair 72
72a,
Fig. 2. The development of pair No. 72 (same conventions as in Fig. 1).
1 day
2 days
tlost
3 days
69b,
4 days
5 days in ERY
69b,, 69b,2
-On— 00 00
•••"•.••v-";- > i o o
cells
Er x E !
pair 69
c
>100 cells
102
x
c
Fig. 3. The development of pair No. 69 (same conventions as in Fig. 1).
the 2 combinations studied, the events being accelerated in the case of the highly
resistant mutant Ef-02. This is so even when the ex-conjugants in the E}Q2 X E S cross
are allowed to undergo one fission in normal medium before being placed in ERY as
was the case in the Ej1 x E s combination. The differences between the 2 types of crosses
are therefore due only to the different levels of resistance of the 2 types of E R mitochondria studied.
Selection of mitochondria in Paramecium
483
DISCUSSION
Validity of the results
This work has consisted of analysing both genetically and cytologically the acquisition of erythromycin-resistance by sensitive cells. Of course, it is not the identical
cell which is studied in the EM and analysed genetically. Furthermore, can one be
sure that the two cells which are taken for testing from the clone in the process of
transformation, are representative of all the cells of the clone? These questions raise
the same problem: are all cells of a clone in the process of transformation identical
and thus can the results obtained on one of them be extended to the others? Several
lines of evidence, cited below, indicate that this is indeed the case.
When several cells have been isolated from a clone in process of transformation and
the extent of genetic purification determined the results are fairly uniform (e.g. pairs
No. 3 and 69 in Table 1).
When two cells from the same clone are observed in the electron microscope, they
are found to be strikingly similar.
In the E' 1 x E s cross, the sensitive ex-conjugant had undergone onefissionin normal
medium and the two resulting cells were then transferred independently into ERY.
It was observed that these two cells followed exactly the same evolution in ERY,
becoming resistant after the same lag. This means that they contain approximately
the same proportion of E R mitochondria, since the lag in ERY is closely linked to the
proportion of E R mitochondria harboured by the mixed cell (Adoutte & Beisson,
1970). Thus the mitochondria derived from those transferred from the E R conjugant
have been distributed in approximately equal numbers to the two cells derived from
the E s conjugant.
Finally, it was already known that, in normal medium, mixed cells yield clones the
cells of which are very similar in their mitochondrial population (Adoutte & Beisson,
I972).
All these observations suggest that there is an intense mixing of mitochondria in
paramecium; added to the fact that the cells divide by binary fission, this leads to a
great cytoplasmic similarity between the cells of a clone.
The facts
A number of clear cut facts emerge from this study. The first concerns the rapidity
of the process of transformation, especially when the E{Q2 strain is used. Sensitive
cells, into which a minority of E$,2 mitochondria are introduced (less than 10%),
become purely erythromycin-resistant, genetically and cytologically, after 3 or fewer
generations in ERY. (It must be noted that the number of generations necessary to
reach genetic purification is probably overestimated since the first 2 fissions in ERY
medium are residual fissions, carried out even by purely sensitive cells.) Thus an
active multiplication of E H mitochondrial genomes must have occurred along with an
active synthesis of the products of these genomes conferring the resistant phenotype to
mitochondria, while practically no cell divisions were occurring.
Secondly, there is a very good parallelism between the extent of purification of the
%2
C E L 14
484
R- Perasso and A. Adoutte
R
cells in E determinants, as determined genetically, and their cytological aspect. In
particular, when a cell is denned as purely resistant cytologically it is also purely
resistant genetically and vice versa. This indicates that mitochondria that are genetically E s but phenotypically E R do not exist, at least in notable amounts.
These two comments apply quite well to cells 72b, which have undergone only
2 fissions in ERY since conjugation. However, they already are purely resistant cytologically and genetically. It must be noted that if a simple dilution of the E s mitochondria had occurred one would expect to find one quarter of them in the 72 b cell,
while practically none are found. This result suggests that, in addition to the active
multiplication of E R genomes, active elimination of the sensitive mitochondria has
occurred. This elimination is observed only during the process of acquisition of
resistance since it is known that purely sensitive cells can remain in erythromycin for as
long as 7 days and still be able to resume growth when put back into normal medium.
In this case no irreversible destruction of all the sensitive mitochondria has occurred.
The complete elimination of E s mitochondria then appears to be linked to active
processes occurring during the acquisition of resistance.
Thirdly, cytological discrimination between sensitive and resistant mitochondria is
difficult at early stages of the acquisition of resistance. At intermediate stages (2-3 days)
a range of mitochondrial phenotypes is observed. It is only when cells have become
practically pure E R that one can distinguish clearly sensitive from resistant mitochondria, lying sometimes side by side.
Finally, it appears that cells of sensitive origin resume growth in ERY at a normal
rate only when they have reconstituted the totality of their mitochondrial population
(both cytologically and genetically).
Interpretations
The central question in this study is the mechanism of acquisition of erythromycinresistance. At least two mechanisms can be proposed, as already suggested by Knowles
(1972).
1. Multiplication of the resistant mitochondria that have initially penetrated into
the sensitive cell, along with the degeneration of the sensitive mitochondria (with
possible reutilization of some elements recovered from the sensitive mitochondria
for the building of the resistant ones).
2. Multiplication of the initially resistant mitochondria along with genetic transformation of E s mitochondria by E R DNA, a possibility raised by the discovery of
mitochondrial recombination in yeast (Thomas & Wilkie, 1968; Bolotin et al. 1971)
and the preliminary evidence for analogous events obtained in Paramecium (Adoutte,
1973)The EM pictures do not allow discrimination between these 2 possibilities. Although
we have noticed in several instances images suggesting division of resistant mitochondria by elongation and constriction as appears to be the case in normal cells
(J. Beisson & R. Perasso, in preparation), the cytological situation is not simple,
since during the intermediate phase, 3 types of mitochondria can be recognized: (1)
Selection of mitochondria in Paramecium
485
clearly resistant-looking; (2) clearly sensitive-looking and (3) a range difficult to classify, often closer to sensitive types (for instance with wavy or few cristae) but not
as altered as expected for sensitive mitochondria (i.e. without dense matrix or
reduced diameter, etc... .). These mitochondria could correspond to sensitive mitochondria in the process of transformation towards resistance as postulated by the
second hypothesis. It is important to note, however, that we are not dealing with
purely sensitive cells, and that sensitive mitochondria may appear less affected precisely because they are in the vicinity of resistant mitochondria. One can imagine
either direct interactions between mitochondria (exchange of products) bringing some
'relief to sensitive mitochondria, or, more likely, indirect interactions resulting from
the fact that the intracellular environment is more 'normal' in a cell in the course of
transformation then in a purely sensitive cell.
The great rapidity of purification observed with mutant E^ 2 could appear to favour
the hypothesis of complete transformation of sensitive mitochondria into resistant ones.
Sensitive mitochondria would be disappearing rapidly not because they are degenerating rapidly but because they are rapidly transformed into resistant ones. It must be
noted however that we are dealing with a rapid phenomenon in terms of cell generations; it is not rapid in time. Cells do not become purely resistant in less than 48 h.
Assuming that a few tens resistant mitochondria pass from the resistant to the sensitive conjugant and that they multiply exponentially at the rate of one fission every 6 h
(generation time of Paramecium), 48 h are enough to produce from them the several
thousand mitochondria per cell.
Finally a process of reutilization of elements, at least, from the sensitive mitochondria,
may occur, to account for their very rapid cytological disappearance (in the case of
mutant EJ*02). The details of this process are unknown. In particular we never
observed organelles analogous to the cytolysomes seen engulfing and digesting mitochondria when cells of Tetrahymena are starved (Nillson, 1970).
On the whole, then, the process of acquisition of resistance appears to involve
multiplication of resistant mitochondria, but the occurrence, in addition, of transformation of sensitive mitochondria into resistant ones remains a possibility.
CONCLUSIONS
Whatever mechanism one favours for the interpretation of the acquisition of resistance, one thing is clear. There must occur an active multiplication of resistant genomes,
while the cells are dividing slowly or not at all. This appears to be one important conclusion of this study since it implies that a separation of the mechanisms ensuring the
coordination between mitochondrial multiplication and cell division has occurred. This
fact is interesting in the light of the current hypotheses concerning the mechanisms
regulating mitochondrial division (Williamson, 1970; Lloyd et al. 1971; Barath &
Kuntzel, 1972). It does not contradict any of the models proposed by these authors.
The situation analysed here after conjugation also accounts for the initial isolation
of the E R strains which implied an intense selective multiplication under the pressure of
erythromycin of 'the' initially mutated E R mitochondria. Furthermore, replication of
mitochondrial DNA without cell multiplication is also accompanied by the expression
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of the functions carried by this DNA, since the mitochondria become phenotypically
resistant. (This is most probably the necessary condition for their multiplication.)
On the whole, we have studied a very peculiar system of mitochondrial biogenesis,
which reveals the great capacity of these organelles to respond to a selective pressure,
pointing out, therefore, their important extent of autonomy in replication and in
expression of their functions.
We thank Drs J. Beisson and J. Andre for support and encouragement throughout this work
and Dr J. Beisson for many helpful discussions on the manuscript. We are specially grateful to
Dr T. M. Sonneborn for critical reading of the manuscript and numerous suggestions.
This study was supported in part by the Centre National de la Recherche Scientifique
(ERA 174, LA 86, and R.C.P. No. 284) and by the Direction des Recherches et Moyens
d'Essais (contract 72/778).
REFERENCES
ADOUTTE, A. (1973). Mitochondrial mutations in Paramecium: phenotypical characterization and recombination. In Int. Conf. on The Biogenesis of Mitochondria (ed. A. Kroon & C.
Saccone). London and New York: Academic Press (in Press).
ADOUTTE, A. & BEISSON, J. (1970). Cytoplasmic inheritance of erythromycin-resistant mutations
in Paramecium. Mol. gen. Genet. 108, 70-77.
ADOUTTE, A. & BEISSON, J. (1972). Evolution of mixed populations of genetically different
mitochondria in Paramecium aurelia. Nature, Lond. 235, 393-395.
ADOUTTE, A., BALMEFREZOL, M., BEISSON, J. & ANDRE, J. (1972). The effects of erythromycin
and chloramphenicol on the ultrastructure of mitochondria in sensitive and resistant strains
of Paramecium. J. Cell Biol. 54, 8-19.
BARATH, Z. & KUNTZEL, H. (1972). Cooperation of mitochondrial and nuclear genes in specifying the mitochondrial genetic apparatus in Neurospora crassa. Proc. natn. Acad. Sci. U.S.A.
69, 1371-1374.
BEALE, G. H. (1969). A note on the inheritance of erythromycin-resistance in Paramecium
aurelia. Genet. Res. 14, 341-342.
BEALE, G. H., KNOWLES, J. K. & TAIT, A. (1972). Mitochondrial genetics in Paramecium.
Nature, Lond. 235, 396-397BOLOTIN, M., COEN, D., DEUTSCH, J., DUJON, B., NETTER, P., PETROCHILO, E. & SLONIMSKI, P.
(1971). La recombinaison des mitochondries chez Saccharomyces cerevisiae. Bull. Inst.
Pasteur 69, 215-239.
KNOWLES, J. K. (1972). Observations on two mitochondrial phenotypes in single paramecium
cells. Expl Cell Res. 70, 223-226.
LLOYD, D., TURNER, G., POOLE, R. K., NICHOLL, W. G. & ROACH, G. I. (1971). An hypothesis
of nuclear mitochondrial interactions for the control of mitochondrial biogenesis based on
experiments with Tetrahymena. Subcellular Biochem. 1, 91-95.
NILSSON, J. (1970). Cytolysomes in Tetrahymena pyriformis GL. C.r. Trav. Lab. Carlsberg 38,
87-121.
PREER, J. R.,
Jr. (1948). A study of some properties of the cytoplasmic factor 'Kappa' in
P. aurelia, variety 2. Genetics, Princeton 33, 349-404.
SONNEBORN, T. M. (1970). Methods in Paramecium research. In Methods in Cell Physiology,
vol. 4 (ed. D. Prescott), pp. 241-339. London and New York: Academic Press.
THOMAS, D. Y. & WILKIE, D. (1968). Recombination of mitochondrial drug-resistance factors
in Saccharomyces cerevisiae. Biochem. biophys. Res. Comnmn. 30, 368-372.
WILLIAMSON, D. H. (1970). The effect of environmental and genetic factors on the replication
of mitochondrial DNA in yeast. In Control of Organelle Development, Symp. Soc. exp. Biol.
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(Received 15 August 1973)
Selection of mitochondria in Paramecium
Figs 4 and 5. For legend see p. 488.
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All micrographs are magnified 30000 times.
Fig. 4. Cell 14a (see Fig. 1), that is, a cell derived from a sensitive ex-conjugant after
24 h in erythromycin-containing medium (ERY). Some mitochondrial profiles (arrows)
are devoid of cristae; most show a marked decrease in the amount of cristae. A trichocyst (t), is seen in transverse section.
Fig. 5. Cell 14b (see Fig. i), that is, a cell derived from the resistant ex-conjugant of
pair 14 after 24 h in ERY; most mitochondria appear normal; some (arrows) show a
slight decrease in the amount of cristae. Peroxysomes (p) mimick mitochondria devoid
of cristae; they cannot be confused, however, since their envelope is a single membrane.
Fig. 6. Cell 14a!, i.e. a cell derived from the sensitive ex-conjugant after 48 h in ERY.
Cristae have almost completely disappeared, p, peroxysome.
Fig. 7. Cell I4b1; i.e. a cell derived from the resistant ex-conjugant after 48 h in ERY.
Mitochondria are normal except for some slight decrease in diameter and in amount
of cristae. tb, trichocyst body; tt, trichocyst tip.
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Fig. 8. Cell 14 a3, i.e. a cell derived from the sensitive ex-conjugant that has just started
to resume growth after 4 days in ERY. r, resistant-looking mitochondria; s, sensitivelooking mitochondrion; tb, trichocyst body; tt, trichocyst tip.
Selection of mitochondria in Paramecium
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Fig. 9. A purely Es cell, taken as a control, after 4 days in ERY. Note the considerable
differences when compared with cell 14a3 (Fig. 8). Mitochondria are very elongated
and cristae have disappeared from most of them, but when they do remain they have
a wavy configuration (w); plates (pi) of rigid appearance occur within mitochondria.
Fig. 10. A typical field of cell i4a3, like other cells of the E[! x E,s cross when resuming
growth, r, resistant-looking mitochondria, one of which is apparently undergoing a
budding process (b); s, sensitive-looking mitochondrion.
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Fig. 11. A late stage of the process of transformation: this cell has gone through 5 or 6
fissions in ERY and is resuming growth at a nearly normal rate. A typically sensitivelooking mitochondrion can be seen (s) surrounded by resistant-looking mitochondria
(r). p, peroxysome; tb, trichocyst body.
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Fig. 12. A cell derived from the sensitive ex-conjugant of pair No. 72, from the E[l02 x Es
combination, after 48 h in ERY (cell 72 bx). A large majority of mitochondria have the
characteristics of resistant mitochondria; 2 'mosaic' mitochondria can be seen (in).
Glycogen is indicated by arrows.
Fig. 13. A field from cell 69b 1]; derived from the sensitive ex-conjugant of pair 69,
after 3 fissions and 4 days in ERY. The distinction between sensitive-looking mitochondria (s) and resistant-looking ones (r) is very clear.
Selection of mitochondria in Paramecium