FEMS MicrobiologyLetters 25 (1984) 271-274
Published by Elsevier
271
FEM 01949
At low monovalent cation concentrations K + can have a specific
effect on the amoebo-flagellate transformation in Naegleria gruberi
(Amoeba; differentiation; membrane potential)
M. Burbidge, W.T. C o a k l e y , B. D e N y e u r t a n d A.J. G r i f f i t h s
Department of Microbiology, UniversityCollege, Cardiff, CF2 1TA, Wales, U.K.
Received 6 July, 1984
Revision receivedand accepted 5 September 1984
1. S U M M A R Y
The amoebo-flagellate transformation that occurs when Naegleria gruberi is transferred from
growth medium to distilled water can be suppressed by the addition of ions. At concentrations
of 3.2 mM and above the percentage of amoeba
enflagellating decreases as the concentration of
KC1, NaC1, LiC1, CaC12 and MgC12 is increased.
KC1, alone, shows a marked trough, with a minim u m at 1.2 mM, in enflagellation over the concentration range 0-3.2 mM.
2. I N T R O D U C T I O N
Vegetative, amoeboid N. gruberi transform to
flagellates on transfer from growth medium to
distilled water. The transformation is inhibited by
low temperatures, mechanical agitation and the
presence of ions in the suspending medium [1].
The transformation process has been studied as a
system in which to examine the controls on differentiation which involve changes in phenotype
without change in genotype [2,3]. The mechanisms
by which changes in extracellular ionic concentration influence the transformation are not well understood [4]. In previous work the ionic control on
enflagellation did not appear to have a strong
cation specificity [5]. We report here a specific
influence of K + ions, at low concentrations, on
the transformation.
3. M A T E R I A L S A N D M E T H O D S
N. gruberi (strain 1518/le, from The Culture
Centre of Algae and Protozoa, Cambridge, U.K.)
were grown as monoxenic cultures with Klebsiella
aerogenes on non-nutrient agar (NNA; 15 g Bacto
Agar per litre of Page's amoeba saline [6]). Overnight cultures (25 ml) of K. aerogenes, grown on
Nutrient Broth (Oxoid) and incubated at 30°C,
were washed twice in amoeba saline and were
resuspended in 5.0 ml of amoeba saline. N N A
plates were spread with 0.5 ml of the bacterial
suspension and 0.1 ml of amoeba saline containing
10 5 cysts. Cultures were incubated at 25°C for
about 36 h at which time the amoebae were approaching the late log phase of growth. The
amoebae were harvested with a glass spreader in 4
ml of distilled water per plate. A pooled cell
suspension obtained from three N N A plates was
centrifuged at 400 g for 4 rain and the pellet was
washed three times in distilled water before resuspending in 1 ml of distilled water. Distilled water
0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies
272
or NaC1, KC1, CaCI 2, MgC12 or LiCI solutions (2
ml) were placed in test tubes. About 4 drops of
amoeba suspension (to give 5 • 105 cells/ml) were
added to each test tube. The test tubes were placed
in a shaking water-bath at 25°C and shaken at an
amplitude of 2.5 cm at 100 rev./min. At 15-min
time intervals 3 drops of cell suspension were
removed and mixed with a drop of Lugol's iodine
(4 g 12, 6 g KI in 100 ml of distilled water). The
percentage flagellates was determined from phase
contrast microscopy of the fixed cells on a
haemocytometer.
loo I
,,J
4. RESULTS
Control cells in distilled water showed some
transformation within 30 min of the first resuspension of the amoeba pellet in distilled water. Transformation reached an average plateau value of
97% within 140 min. The average time, T0.5, to
reach half of the plateau enflagellation percentage
was 73 min. These values compare reasonably well
with recent data on transformation in low ionic
strength media [7,8].
Fig. 1 shows the dependence of the maximum
transformation percentage on NaC1, KC1 and LiC1
over a concentration range of 0-25 mM. In each
of two series of experiments four data points were
obtained for each concentration shown. For each
0
'
10
'
210
SALT CONC. ImM)
Fig. 1. The average percentage of flagellates observed in KCI
(I), NaCI (e) and LiCI (A) over the concentration range of
0 25 mM. The mean percentage for cells in water (D) is
included.
series the experimental results were obtained by
examining the enflagellation response of a batch
of amoebae at each concentration. The experiment
was repeated four times with different batches of
amoebae. The agreement within each series of
experiments was good but the agreement between
Table 1
A comparison of results from the present work and from a number of other studies, for the salt concentration necessary to give 50%
flagellates
Strain
1518/1
KC1
(mM)
12.5
NaCl
(mM)
LiC1
(mM)
CaC12
(mM)
3
1
6
6
12.5
MgCl 2
(mM)
0.8
1518/le
= 30
= 30
10
1518/la
75
60
< 30
45
NEG-M
65
65
< 50
-
Food
Bacteria
Ref.
'Unknown
bacterial
flora'
[2]
Klebsiella
aerogenes
Klebsiella
aerogenes
present
work
[7]
Axenic
[5]
Media (g/l) used in these studies were: present work, Bacto agar (15), NaC1 (0.12), M g S O 4 . 7 H 2 0 (0.004), CaC12.2H20 (0.004),
N a 2 H P O 4 (0.142) and KHEPO 4 (0.136); [2] powdered agar (15), Lemco meat extract (1.0), glucose (2.0); [5] Difco yeast extract (5.0),
L-methionine (0.044), dextrose (5.4), phosphate buffer (1 mM) and calf serum (100); [7] Difco Bacto agar (20), Difco Bacto Peptone
(2), dextrose (2), K 2 H P O a (1.5), KHEPO 4 (1).
273
at the concentrations shown in Fig. 1.
Table 1 shows the concentration of the chloride
salts of the different cations required to reduce the
% flagellates from values in excess of 90% in
distilled water to 50%. Data from other sources
[2,5,7] are also included in Table 1 for comparison.
100
E
(/) 5(
5. D I S C U S S I O N
I,LI
,_1
I
I
I
1
2
3
SALT CONC. (mM)
Fig. 2. The average percentage of flagellates observed in KCI
(ll L NaC1 (e) and LiC1 (A) over the concentration range 0-3.2
raM. The error bars represent _+1 standard error of the mean.
The data points for the KCI and NaCI cases are means of 8
measurements. The LiC1 data are based on 2 - 4 measurements
at each concentration. The mean percentage for cells in water
(D) is included.
the means of the two sets of data decreased at the
higher ion concentrations. These quantitative differences between the means at high concentrations
did not alter the general shapes of the curves or
alter the relative potency of the different salts. The
data points shown in Fig. 1 are the means of the
results at each concentration. In each of the two
series of results there was a significant trough (at
0.8 mM KC1) in the dependence of the transformation on KC1 concentration. The presence of a
trough was confirmed in the experiments shown in
Fig. 2. The position of the minimum enflagellation
for cells in KC1 is close to 1.2 mM. There is no
evidence of a minimum in the response to NaC1 or
LiCI over the concentration range 0-3.2 mM. The
flagellate dependence on KCI concentration increased again at concentrations in excess of 1.2
m M to reach percentages higher than those observed in NaC1 or LiC1 at 3.2 mM.
There was no indication of a minimum in the
response of the transformation to CaC12 or MgC12
Table 1 shows that the chloride salt concentration for 50% flagellation is higher for all cations in
our work compared with that of Willmer [2]. There
is good agreement between the results of Jeffery
and Hawkins [7] and those of Fulton [5]. Their
values are closer to our results than they are to
Willmer's [2]. Willmer's study of the ionic concentration dependence of the amoeboflagellate
transformation did not reveal the specific K + effect at 0.8 mM KC1 reported here and he did not
examine the concentration range between 0.8 mM
and 3.2 mM. Fulton [5] and Jeffery and Hawkins
[7] did not report results at concentrations below
10 and 30 mM KC1, respectively. Table 1 shows
widely differing results for transformation of
strains 1518/1 and 1 5 1 8 / l a (which were claimed
to be the same strain [7]). Table 1 also shows
agreement between the results for the monoxenically grown 1 5 1 8 / l a and the axenically grown
NEM-M. This indicates that strain differences do
not necessarily account for the observed variation
in results. This suggestion is supported by the
failure of Jeffery and Hawkins [7] to detect any
strain-specific effect on exposing three different
strains of N. gruberi 1518/1 to a transformation
medium. Differences in the growth media employed in the four studies are also shown in Table
1. Fulton [5] has shown that the yeast extract, and
only the yeast extract, contained a component
which, when added to transformation medium,
could inhibit enflagellation. This component was
isolated and was found to act synergistically with
ions in inhibiting enflagellation. Amoebae grown
in medium enriched with the active component are
much less sensitive than cells grown in normal
medium to prevention of transformation by NaC1
[5]. The presence of bacteria on transfer of amoebae to transformation medium can also have a
274
marked inhibitory effect on transformation [7].
The differences in nutrient and in experimental
procedures may then explain the wide range of
results shown in Table 1 and may also explain the
fact that Willmer [2] did not report a low value for
enflagellation in 0.8 mM KC1.
The mechanism of the specific effect of K ÷ in
the present study has not been established. If the
effect of the ion is totally electrical then it could
operate both by shielding the negative charges on
the cell surface (non-specific cation effect on the
surface potential) and by influencing membrane
potential (potassium has been shown to have a
much greater influence on membrane potential in
the amoeba Chaos chaos than does sodium [9]).
Gingell has already suggested that surface
potential changes may be the transducer which
makes N. gruberi sensitive to the relatively nonspecific effects of high electrolyte concentrations,
as shown in Fig. 1 and Table 1 [10]. Some physical
properties (e.g. fluidity and bending stresses) of a
variety of membranes can be altered by surface
potential change [11-13] and by membrane potential change [13-16].
In addition to the above electrostatic effects of
changes in potassium ion concentration the consequences of ion flow across the membrane need to
be considered. It has been shown in Chaos chaos
and Amoeba proteus that ion currents, which are
intimately involved in cell movement, leave the
amoeba at one region and return at another [17].
The currents decrease when an increase in potassium ion concentration depolarises the membrane
[17]. These ion currents also give rise to electric
fields with components which are tangential to the
cell membrane and can lead to movement of
charged particles on the cell surface [18,19]. The
forces responsible for these movements are them-
selves dependent on the ionic shielding of the
surface charges.
The above results indicate that a study of the
response of enflagellation to low potassium ion
concentrations may give information on the influence of membrane potential on phenotypic
change.
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