Reversal of Ethionine Inhibition by Methionine during 
Slime Mold Development 
Hans R. Hohl and Susan T. Hamamoto 1 
Dictyostelium discoideum , a cellular slime mold, 
has been shown by Filosa (I960) to produce 
abnormal fruiting bodies when grown on Es- 
cherichia coli in the presence of 1.2 x 10- 3 * * * 
M ethionine. The fruiting bodies obtained un- 
der those conditions were described as short, 
with thick stalks and elongated sori. At a con- 
centration of 4.8 X 10 -3 M no growth oc- 
curred. Starting from this initial observation 
we have attempted to answer the following 
questions : Which elements of development, 
such as morphogenesis or differentiation of 
spores and stalk cells, are affected at various 
concentrations of ethionine? Which phases of 
the life cycle are sensitive to ethionine? What 
is the mechanism of ethionine inhibition ? 
Answers to these questions might possibly pro- 
vide clues to the mechanism of development in 
these organisms, where an integrated mass of 
myxamoebae produces, in the absence of an 
external source of energy, a well-proportioned 
fruiting body composed of a cellulose-ensheathed 
stalk bearing a lobose sorus of spores. 
MATERIALS AND METHODS 
Myxamoebae of Dictyostelium discoideum 
NC-4(S2), a haploid strain, were grown at 
24°C in the presence of Escherichia coli B/r 
on a medium containing 1% lactose, 1% pep- 
tone, and 1.5% Difco agar. After 44-46 hours, 
shortly before the cells reached the stationary 
growth phase, they were harvested with ice- 
cold Sorensen’s phosphate buffer (0.016 M, 
pH 6.0), washed, and their concentration ad- 
justed to 1. 5-2.0 X !0 8 cells/ml. A 0.5-ml 
sample of cell suspension containing 0.75- 
1.0 X 10 8 cells was spread onto millipore filters 
1 Pacific Biomedical Research Center and Depart- 
ment of Microbiology, University of Hawaii, Hono- 
lulu, Hawaii. Manuscript received September 26, 1966. 
This work was supported by a grant from the 
National Institutes of Health (GM-1 1758-03), 
United States Public Health Service. 
according to the method of Sussman and Lov- 
gren (1965). For this the millipore filters (48 
mm diameter, black), resting on absorbent filter 
pads, were placed in plastic petri dishes (60 mm 
diameter) . Prior to the addition of myxamoebae 
the pads were soaked with 1.5 ml of phosphate 
buffer containing per ml 0.67 mg of strepto- 
mycin and 0.13 mg of sodiumlaurylsulfate to- 
gether with appropriate concentrations of the 
test solution. The sodiumlaurylsulfate appears 
to contribute to a more uniform development 
of the population and to enhanced effectiveness 
of at least some compounds, probably by in- 
creasing the cell permeability without having 
any obvious effect on normal development. All 
cultures were incubated at 24 °C in the dark; 
in some experiments when the plates were 
kept at room temperature overnight, the tem- 
perature varied between 21° and 25 °C. The 
plates were scored every 4 hours until no fur- 
ther developmental changes were observed. To 
test the effect of ethionine on growth and ag- 
glutination, the cells were grown on E. coli in 
shake cultures in the presence of the compound 
and the growth curves determined (Hohl and 
Raper, 1963), or washed cells were incubated 
in roller tubes and their pattern of agglutina- 
tion was observed (Hohl and Raper, 1964). 
For the growth in shake cultures, D. discoideum 
V-12 was used. 
RESULTS 
Ethionine completely inhibits growth of 
Dictyostelium at a concentration of 3.0 X 
IQ - 2 M and shows no inhibitory effect at 
1.0 X 10 -3 M (Fig. 1). In no concentration, 
however, does it interfere with agglutination of 
myxamoebae as judged from the behavior of 
cells in roller tubes. Even at a concentration of 
3.0 X 10 _2 M there was no sign of inhibition 
either in the rate of agglutination or in the size 
of the agglutinates formed. This indicates that 
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