MicrObiology (1994), 140, 3241-3247
Printed in Great Britain
Induction of rnyxospores in Stigmatella
aurantiaca (myxobacteria) : analysis of
inducer-inducer and inducer-inhibitor
interactions by dose-response curves
Klaus Gertht and Hans Reichenbacht
Author for correspondence: Klaus Gerth. Tel: +49 531 6181 433. Fax: +49 531 6181 482.
lnstitut fur Biologie II der
Universitat Freiburg,
LehrstuhI fur Mikrobiologie,
Freiburg, Germany
Theories and methods developed in molecular pharmacology for drug receptor
interactions were used to explain artificially induced myxospore formation.
The correlation between inducer concentrations and the yield of myxospores,
i.e. the experimentally obtained dose-response curves, were much steeper
than expected for an interaction based on mass action law. We postulate that
the interaction of an inducer with one of the different receptors of the
bacterial cell causes the production of a common stimulus which programmes
the cells to turn into myxospores, if a threshold value is reached. Simultaneous
addition of inducers of the same or of different inducer groups showed
synergistic interactions. Inducer concentrations that by themselves were not
inducing gave maximum myxospore formation when combined. Addition of
pyrrole, a specific inhibitor, caused a concentration-dependent parallel shift of
the glycerol dose-response curve. Compounds, inducers or inhibitors, with
identical receptor specificities competed for the common receptor according to
their affinity. Interactions of inducers with different receptor specificities
could be predicted from calculations with a mathematical model of functional
synergism.
Keywords : myxobacteria, Stigmutellu uuruntiucu, dose-response curves, inducers and
inhibitors, myxospore
INTRODUCTION
Myxospore formation in Stigmatella aurantiaca Sg a1 can be
induced by a wide variety of chemicals which appear to
interact with at least three different inducer-specific,
independent receptors of the bacterial cell (Gerth e t al.,
1993). Receptor-defective mutants M 13 (defective in
receptor I), M 50 (defective in receptor 11) and M 16
(defective in receptors I1 and 111)were used to classify the
40 known inducers into four groups. The receptor
hypothesis was further supported by the discovery of
inhibitors of myxospore formation specific for glycerol
receptor I, viz. pyrrole, oxindole and tert-butanol. The
increased sensitivity of mutant M 20 specifically to
inducers of group I, and the difference in the kinetics of
myxospore formation between group I and I1 inducers,
also support the hypothesis. The objective of this study
t Present address: GBF,
Gesellschaft fur Biotechnologische Forschung,
Naturstoffbiologie, Mascheroder Weg 1, D-38124 Braunschweig, Federal
Republic of Germany.
0001-9002 0 1994 SGM
was to gain insights into the molecular basis of the
interaction between inducers and receptors, into the
competition and synergism between inducers of the same
and of different inducer groups, and into the interference
of inhibitors with the induction mechanism. Methods and
theories developed in molecular pharmacology for drugreceptor interactions (Ariens, 1964) were applied to
analyse the inducer-receptor interactions in 5’. awantzaca
and the resulting signals leading to myxospore formation.
METHODS
Experimental organism and culture conditions. Stigmatellu
uuruntiucu strain Sg a1 has been characterized, and the general
conditions for its cultivation have been described in detail in our
previous papers (Gerth & Reichenbach, 1978; Gerth e t ul.,
1993).
Dose-response curves. The bacteria were cultivated in 50 ml
Casitone liquid medium (CLM) for 3 d, after which time the
ODcz3of the culture was adjusted with fresh medium to 0.5
(1 cm cuvette, Eppendorf filter photometer). T o obtain the
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3241
K. G E R T H a n d H. R E I C H E N B A C H
desired inducer or inhibitor concentrations, different volumes of
the respective stock solutions were pipetted into 100 ml sterile
Erlenmeyer flasks and adjusted to a constant volume of 1 ml
with distilled water. Then 9 ml of the diluted bacterial culture
was added, and the flasks were incubated for 2 h at 32 “C on a
rotary shaker.
T o separate vegetative cells from myxospores, the cultures were
treated with ultrasound at 1.6 A for 30 s (MSE Ultrasonic
Power Unit). While vegetative cells were completely destroyed,
the myxospores remained intact. Then the suspension was
centrifuged at 6000 r.p.m., and the pellet was suspended in 1 ml
cold medium and carefully pipetted on top of 2 ml of a 30%
(w/v) sucrose solution layered over 2 ml of a 40 O
h solution in :i
centrifugation tube. T o prevent clumping of the myxosporea;
during sedimentation, 2-5YO(w/v) NaCl was added to the sugar
solutions. After centrifugation at 5000 r.p.m. for 15 min, the
sedimented myxospores were separated from all debris. The
pellet was resuspended in fresh medium, the OD,,, read, and
the number of myxospores determined from a calibration curve.
The percentage of myxospores was calculated from the number
of vegetative cells before induction. About 10-15% of the
myxospores were lost during the procedure.
Definition and calculation of intrinsic activities. In order to
produce an effect, an inducer must interact with a specific
receptor, i.e. it must have an affinity (defined as l/K,) to the
receptor. Also, the inducer must interact in an effective way, i.e.
it must have what is called an intrinsic activity. The intrinsic
activity a of a drug is expressed as a fraction of the intrinsic
activity of some reference compound, the intrinsic activity of
which is taken as unity. T h s reference compound is able to
produce the maximum effect Em(Ariens, 1964). To measure the
intrinsic activity of an inducer, the maximum yield of myxospores was determined for the compound as the mean value of
several parallel measurements performed under optimal nduction conditions. The reference compound for induced
myxospore formation was indoline, with a myxospore yield of
81 Yo (Em= 1).
Constant of inducer-receptor association. This constant, K ,
(for compound A), is defined as the molar concentration of an
inducer that gives the half-maximum response ( K , = [A] for
E,: Em= 0.5). T o determine the constants for inducers acting
simultaneously on more than one receptor, mutants defective in
one of the receptors (M 13 or M 50) were used (Gerth e t al.,
1993).
Calculation of dose-response curves. If the values for K, and
for intrinsic activity (a) of an inducer are known, the dose
response curve based on mass action law can be calculated
E, =
‘A
Em
+(KA/[AI)
(1)
Determination of Hill coefficients. The Hill coefficient is an
often-used measure of deviations from hyperbolic behaviour in
binding systems (Ariens, 1964; Jard e t al., 1968). log (E, [YO]/
Em[%]-E, [%I) is plotted against the log of inducer concentrations on the abscissa. The slope of the resulting
straight line, which is defined as the Hill coefficient, n, is 1 jf the
dose-response curve obeys mass action law.
Calculation of competitive interactions. O n the basis of mass
action law, the effect, EAB,of the combined inducers A and B (or
inducer A and inhibitor B) competing for the same receptor may
be expressed as a fraction of the maximum effect. For our
calculations, we used E,, = 50Y0, because at this point the
effector concentration of the theoretical curve, based on mass
action law, and that of the sigmoidal experimental curves
3242
coincide. The contributions of compound A and compound B
to their common effect are determined by their concentrations
[A] and [B], the constants K , and KB, and their intrinsic
activities a, and aB (Ariens, 1964). If five of the parameters are
known, the remaining one, e.g. the association constant of an
inhibitor, can be calculated from equation 2.
Calculation of functional synergism. In the case of a functional
interaction, two inducers, A and Byeach interact with their own
specific receptor system, R, and RB, respectively, but produce
their effect by means of a common stimulus. The resulting effect
can be expressed by
Definition of trigger value and threshold value. An all-ornone relation between stimulus and effect is assumed, because
the cell only can or cannot form a myxospore. This means that
an effect is produced only if the stimulus reaches a critical value,
the trigger value z. As a consequence of biological variation
within a large population of bacterial cells, no sharp threshold
value will be observed for the culture. Rather, a ‘graded’ log
dose-response curve is obtained, the slope of which is determined by statistical variation of the individual trigger values
of the single cells due to biological variance (Ariens, 1964).
RESULTS
Dose-response curves
The effect of increasing glycerol concentrations on the
yield of ultrasound-resistant myxospores is shown in Fig.
1. The slope of the experimentally determined doseresponse curve was much steeper than that of the
theoretical curve calculated on the basis of mass action
law (equation 1). This was also corroborated by a Hill
coefficient of n = 8-4 rather than n = 1.
For the very efficient inducer indole (Gerth e t a/., 1993),
we obtained three different curves for the wild strain and
two receptor-defective mutants (Fig. 2). The half-maximum yield of myxospores was obtained at a concentration
of 0.062 mM with the wild strain Sg a l , and at 0.088 with
mutant M 50. Mutant M13 was much less sensitive: the
required concentration was 4.5 times higher than that for
the wild strain.
In contrast to the results with indole, the dose-response
curves of 2-propanol-induced myxospore formation were
identical for the wild strain and mutant M 13. In this case
the concentration for half-maximum myxospore induction was increased with mutant M 50 by a factor of two
(Fig. 3).
Intrinsic activities
While the efficiency of myxospore induction was similar
for the compounds mentioned so far, with an intrinsic
activity close to 1,there were some inducers that possessed
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Induction of myxospores in Stigmatella aurantiaca
80 I
100
4Y
n
S 80
v
60
.-5
601
6
Y-
$
40-
2
-
s
r" 20 -
20
f
0.01
. . . . . . . .'
.
' " * . * * I
0.1
1
Glycerol (M)
. . . . . . . .I
YI
I
5
10
1
10
20
30
2-Propanol (mM)
40 50
........................................................................................................................................................
..........................................................................................................................................................
Fig, 1. Dose-response curves of myxospore induction with
glycerol with the wild strain: 0 , experimentally determined
calculated on the basis of mass
(Hill coefficient, n = 8.4); 0,
action law with the values taken from the experimental curve
(Hill coefficient, n = 1).
Fig. 3. Dose-response curves of myxospore induction with 2propanol: 0, wild strain and M 13; 0, mutant M 50.
.-
+.'
rn
I
40
loo
S 80 v
n
C
.-0
c,
60 -
b
100
"1 0
Y-
$P 4 0 -
Inducer (mM)
VI
2
r" 20 -
t
01
0.01
Fig- 4. Dose-response curves of inducers with low intrinsic
activity: 0,KCI (intrinsic activity = 0-38); 0 , proline (intrinsic
activity = 0.63).
d
I
0.1
lndole (mM)
1
........................................................................................................................................................
Fig. 2. Dose-response curves of myxospore induction with
mutant M 50 defective
indole: 0 , wild strain ( K A = 0.062); 0,
at receptor II ( K A = 0.088); V, mutant M 13 defective a t
receptor I ( K A = 0.28).
a much lower intrinsic activity. Among those were 2methylindole, with an intrinsic activity of a = 0.75;
proline, with a = 0.63; and KC1, with 01 = 0.38, the
lowest intrinsic activity observed (Fig. 4). The intrinsic
activities and association constants of selected inducers
and inhibitors are summarized in Table 1.
that concentration ethylene glycol alone could induce
only 10-15% of the cells to convert into myxospores,
simultaneous addition of glycerol at concentrations between 10 and 70 mM, which were not inducing by
themselves, resulted in a typical dose-response curve.
Maximum yield could be obtained. The interaction can be
calculated for half-maximum myxospore formation using
equation 2. Experimentally, we found a glycerol concentration of 37 mM, which was in good agreement with
the calculated 31.5 mM.
Interaction of inducers of myxospore formation
Inducers which differed with respect to their receptors,
their intrinsic activities and their inducing concentrations,
e.g. 2-phenylethanol and NaC1, were also synergistic (Fig.
6). The maximum effect of an 82% yield of myxospores
was reached with 4 mM 2-phenylethanol and 30mM
NaCl.
Ethylene glycol when applied at a constant concentration
of 136.5 mM showed a strong synergistic effect with
increasing concentrations of glycerol (Fig. 5). While at
The synergism between low concentrations of tertbutanol, a compound that at high concentrations induces
via receptors I1 and 111, and increasing concentrations of
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I<. G E R T H a n d H. REICHENBACH
Table 1. Summary of intrinsic activities (a) and
association constants (K,) of selected inducers and
inhibitors of myxospore formation
Compound
loo
I
Intrinsic Interaction Association
activity
with
constant
receptor
(mM)
Ethylene glycol
Dimethyl sulfoxide
Glycerol
P yrrole
Oxindole
2,3-Benzofuran
Indole
2-Phen ylet hanol
Indoline
2-Methylindole
I
I
I
I
I
I
I
I1
I1
I1
I1
0914
0.97
0.984
0
0
0.956
0.984
0.984
0.898
1.0
0.75
166
118
100
2.35'
0.273*
0.08
0.088
0.28
4.8
2.4
0.4
* Values calculated as described in Methods.
"1
10
Inducer (mM)
100
Fig. 6. Synergistic effects of inducers of groups I and II: 0,
dose-response curve with constant 2-phenylethanol (4 mM) and
increasing NaCl concentrations; V, induction with 2phenylethanol; 0,
induction with NaCI.
induction was only marginal: at low concentrations a
slight synergistic effect was observed ;at higher concentrations maximum yield was reduced to 45 %.
n
S 80-
Interaction of inducers and inhibitors of myxospore
formation
v
C
.-0
z
+,
L
60
-
The effect of pyrrole on glycerol-induced myxospore
formation is shown in Fig. 9. The resulting dose-response
curves were shifted to higher inducer concentrations as
the pyrrole concentration applied was increased. The
yield of myxospores remained unaffected.
0
Y-
e
40-
.
s
r" 20 I
O'-10
'
100
Inducer (mM)
*
'
"""
.................................................................................. ........................................................................
Fig. 5. Synergistic effects of inducers of inducer group I: 0,
dose-response curve with constant ethylene glycol (136.5 mM)
and increasing glycerol concentrations; 0,induction with
glycerol; V, induction with ethylene glycol.
NaCl is shown in Fig. 7 . With the receptor 11-deficient
mutant M 50, a maximum effect of 65 % myxospores was
obtained with 23 mM NaCl and 16 mM tert-butanol,
concentrations that are not inducing by themselves. A
further increase in NaCl concentration reduced the
efficiency of myxospore formation.
Glycerol and NaCl showed totally different patterns of
interaction with tert-butanol (Fig. 8). While glycerolinduced myxospore formation declined sharply with
increasing tert-butanol concentrations, the effect on NaCl
3244
Calculation of the association constant KAof
oxindole
The dose-response curves of glycerol induction in the
presence of two different concentrations of the inhibitor
oxindole were experimentally determined (not shown),
and the glycerol concentrations that were required for
half-maximum myxospore formation were taken from
these curves. We used the following values for our
calculations :
Association constant of glycerol: Kglycerol= 0.1 M
(Table 1)
Intrinsic activity of glycerol :
(Table 1)
aG = 0.984
Intrinsic activity of oxindole :
a, = 0
Oxindole concentrations used:
B, = 0.2 mM
B, = 0.3 mM
The glycerol concentrations required for half-maximum
yields of myxospores in the presence of the given inhibitor
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Induction of myxospores in Stigmatella aurantiaca
loo
i
I
loo
0'
'
'
Inducer (mM)
,,
.....,...
. . .
.
I
1000
100
Glycerol (mM)
....,...,.....,......,,,...................................................
. ...,.,.. ..... ...... ............ ...........................,
Fig. 7. Synergistic effects of inducers of group I and Ill. Mutant
M 50, defective a t receptor II, was used to investigate the
interaction of inducers acting simultaneously on receptors I and
111: 0 , dose-response curve with constant tert-butanol (16 mM)
and increasing NaCl concentrations; 0, induction with NaCl;
induction with tert-butanol.
................................................................................................................................................
.........
Fig. 9. Glycerol-induced myxospore formation in the presence
in the presence of
of pyrrole: 0 , control without pyrrole; 0,
1.82 mM pyrrole; V,in the presence of 3.64 mM pyrrole.
v,
100 I
n
S 80-
W
C
.-0
i 60-
c,
b
'c
40-
s f . .
r"s 20-
(
t
"
0.1
O
4
10
100
tert-Butanol (mM)
Fig. 8. Effect of increasing concentrations of tert-butanol on
glycerol- and NaCI-induced myxospore formation : 0 , induction
with NaCl (55 mM); 0,
induction with glycerol (136 mM).
lndole (mM)
1
Fig. 10. Effect of oxindole (0.4 mM) on indole-induced
myxospore formation. A dissociation constant of K, = 0.17 was
determined from this curve.
response curve (Fig. 10). The experimental value of
0.17 mM was in good agreement with the calculated one.
DISCUSSION
concentrations were: A, = 182 mM; A, = 215 mM. The
association constants of oxindole were calculated using
equation 2. The mean value was Koxindole= 0.273 mM
(s = 0-007).
The reliability of this value was corroborated with a
different set of experimental conditions. From the experimentally determined value of KA = 0.062 mM (Fig.
2) for the wild strain Sg a1 we calculated the indole
concentration which would be necessary for half-maximum spore formation in the presence of 0.4 mM oxindole.
This calculated value of 0.1 58 mM was compared with the
concentration determined experimentally from a dose-
In order to interact with a receptor, the inducer must have
an affinity to it, which is defined as the reciprocal of the
association constant KA. The lower the affinity, the higher
is the concentration required for myxospore induction.
The association constants of the myxospore inducers
varied between 166 mM for ethylene glycol and 0.088 mM
for indole (Table 1).
For myxospore induction the inducer-receptor complex
must produce a stimulus, measured as the inducer's
intrinsic activity. Some inducers, such as proline, induced
only a certain proportion of the cells in the culture to form
myxospores (Fig. 4). A further concentration increase
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3245
K. G E R T H a n d H. R E I C H E N B A C H
from 100 to 200 mM had no effect on the yield. Thus,
proline is an inducer with a low intrinsic activity in spite
of its fairly low K A of 65 mM. NaC1, which also had a low
intrinsic activity, behaved differently. The dose-response
curve had the shape of an optimum curve (Fig. 6),
suggesting that at higher salt concentrations toxic effects
may interfere with metabolism and prevent myxospore
formation.
While a typical dose-response curve was obtained for
glycerol-induced myxospore formation (Fig. l), the
experimental curve (with a Hill coefficient of 8.4) was
much steeper than the calculated one (Hill coefficient 1).
The result is not in agreement with the mass action law.
However, the result can be explained by postulating that
the conversion of a vegetatively growing cell into a
myxospore is induced only if a ‘trigger value’ for the
induction is surpassed. The ‘graded ’ log dose-response
curves obtained with bacterial cultures do not exclude
such an all-or-none response. The slope could be due to
biological variance of the individual trigger values of a
large number of single cells.
As shown previously (Gerth e t al., 1993) the inducers fall
into distinct groups. Glycerol and ethylene glycol are
both inducers of the group which interacts with the
hypothetical (glycerol) receptor I. Inducers of the same
group would compete for the common receptor according
to their affinity; the resulting dose-response curves run
parallel to one another. It is not the concentration of the
individual inducer, but the common stimulus produced
by the inducer-receptor complexes, that triggers myxospore formation (Fig. 5). The experimentally determined
and the calculated glycerol concentrations required for
half-maximum yield are in good agreement with the
theory of competitive agonists.
A compound with low intrinsic activity but high affinity
would be a competitive inhibitor of myxospore formation.
Pyrrole caused a parallel shift of the log dose-response
curve for glycerol to higher inducer concentrations (Fig.
9). But the effect of the inhibitor was overcome by a
higher glycerol concentration, showing competition between glycerol and pyrrole. A non-competitive inhibitor
would decrease the maximum yield of myxospores, and
this inhibition would be insurmountable. The association
constant of another inhibitor of the glycerol receptor,
oxindole, was calculated using equation 2 and COTroborated experimentally (Fig. 10).
Myxospore induction as a result of inducer-inducer as
well as inducer-inhibitor interactions at (glycerol) receptor I can be explained with a simplified model of
competitive interaction. Synergism between compounds
which induce myxospore formation via independent
receptors, e.g. NaCl (receptor I) and 2-phenylethanol
(receptor II), is also possible (Fig. 6). Completely different
and physiologically independent pathways for the conversion of vegetative cells into myxospores are not likely,
and indeed no numerically additive effect, as would be
expected under such circumstances, was observed in our
experiments. Thus, the simultaneous addition of 4 mM 2phenylethanol and 33 mM NaCl, each weakly inducing by
3246
itself, caused 82 % of the cells to convert into myxospores.
We therefore postulate a functional synergism, in which
the inducers interact with their own receptors R, and R,,,
each producing its effect by means of the same type of
stimulus. The above considerations were also true for the
synergistic effects between NaCl and tert-butanol, a
compound which induces via receptors I1 and 111. This
could be seen when mutant M 50, defective in receptor 11,
was used. The concentrations of tert-butanol and NaCl
used by themselves did not induce myxospores at all.
Toxic effects of increasing salt concentrations explain the
decline in the overall yield of myxospores (Fig. 7).
Some compounds induced sporulation by acting simultaneously on two different, specific receptors. The
dose-response curves of 2-propanol, an inducer of group
I11 (see below), were identical for the wild strain Sg a1 and
mutant M 13, defective in receptor I. Thus, 2-propanol
did not use receptor I for induction. With M 50, mutated
in receptor 11, an increased concentration of 2-propanol
was required for induction (Fig. 3) demonstrating that
receptor I1 and receptor I11 are both used for induction by
this compound. Indole, one of the most potent inducers
discovered so far, induced myxospores with all types of
receptor mutants, e.g. M 13, M 50 and M 16. A higher
indole concentration was required to induce the receptor
I mutant M 13 than for the receptor I1 mutant M 50 (Fig.
2), indicating that receptor I is used preferentially. The
response of the wild strain represents a synergism of
receptor I and receptor I1 effects. We can use equation 2 to
calculate the combined effect of the receptors. The
dissociation constants of the receptors are taken from Fig.
2, the intrinsic activities from Table 1. The indole
concentration required for Sg a1 to give E,/2 was
experimentally determined to be 0.062 mM and calculated
to be 0.059 mM, which supports the hypothesis of
functional synergism.
As described previously (Gerth e t al., 1993), the group I11
inducers tert-butanol and 2-propanol are not only
inducers at receptors I1 and 111, but at concentrations
below those causing induction also inhibit receptor I. tertButanol caused a decline in the efficiency of glycerol
induction to almost zero, but induction with NaCl
remained more or less unaffected (Fig. 8). Since glycerol
and NaCl interact with the same receptor I, we must
assume a fundamental difference between the interactions
of the two molecules with the receptor, such as hydrogen
bonding in one case and ionic interaction in the other.
In molecular pharmacology, a molecule is regarded
formally as possessing two different domains, one conferring affinity for the receptor, and another determining
intrinsic activity (Ariens, 1964). We suggest that the
situation is similar with inducers of myxospore formation.
tert-Butanol has only the moiety responsible for the
affinity for receptor I, but no intrinsic activity. It thus
becomes an inhibitor for receptor I. In contrast, the small,
positively charged sodium ion may interact only with that
region of the receptor which is responsible for the
production of the internal signal or stimulus, and so the
ion possesses only intrinsic activity. An inhibitor that
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Induction of myxospores in Stigmatella awantiaca
Table 2. Correlation between chemical structure and receptor specifity
Inducer
tert-Butanol
y
receptor I11
KA receptor I
K
A receptor I1
(MI
(MI
(MI
1o*
72
91
12.8
0
0
3.1
0
KA
3
HO-C-CH,
I
CH3
tert-Butylamine
CH3
I
NH,’-C-CH,
I
CH3
2-Phenylpro panol
0
* Inhibitor at receptor I
occupies a certain binding region of the receptor would
not necessarily prevent induction by a compound that
acted directly on the active centre of the receptor.
If a positive charge was required for intrinsic activity at
receptor I, it might be possible to convert by chemical
modification a non-inducing compound that can bind to
the receptor (i.e. a competitive inhibitor) into an inducer.
Replacement of the hydroxyl group of tert-butanol by a
positively charged amino group, forming tert-butylamine,
produced an inducer at receptor I. The two compounds
had approximately the same affinity for receptor I, but the
amine had lost its affinity for receptors I1 and I11 (Table 2).
2-Phenylpropanol can be regarded as a tert-butanol
molecule with one methyl group in position 2 replaced by
an aromatic ring. By this substitution, tert-butanol, which
induces via receptor I11 at high concentrations, has
become more lipophilic, a quality which is important for
interactions with receptor I1 (Gerth etal., 1993). In fact, 2phenylpropanol has a high affinity for receptor I1 and at
the same time has lost all affinity for receptors I and 111.
Thus, even with these small inducer molecules it is
possible to differentiate between a moiety responsible for
affinity and a moiety responsible for intrinsic activity.
ACKNOWLEDGEMENTS
We want t o thank the Deutsche Forschungsgemeinschaft and
the Sonderforschungsbereich 46 ‘ Molekulare Grundlagen der
Entwicklung ’ for their financial support.
REFERENCES
Ariens, E. J. (editor) (1964). Mofectlfar Pharmacology, vol. 1. New
York & London: Academic Press.
Getth, K. & Reichenbach, H. (1978). Induction of myxospore
formation in Stigmatefla atlrantiaca (Myxobacterales). Arch Microbiof
117, 173-182.
Getth, K., Metzger, R. & Reichenbach, H. (1993). Induction of
myxospores in Stigmatella atlrantiaca (myxobacteria) : inducers and
inhibitors of myxospore formation, and mutants with a changed
sporulation behaviour. J Gen Microbiof 139, 865-871.
Jard, S., Bastide, F. & Morel, F. (1968). Analyse de la relation ‘doseeffect biologique ’ pour l’action de l’ocytocine et de la noradrenaline
sur la peau et la vessie de la grenouille. Biochim Biopbys A c t a 150,
124-130.
Received 17 February 1994; revised 22 July 1994; accepted 5 August
1994.
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