FEMS Microbiology Letters 49 (1988) 75-79
Published by Elsevier
75
FEM 03017
Thermoadaptation of methanogenic bacteria
by intracellular ion concentration
R e i n h a r d H e n s e l 1 a n d H e l m u t K/Snig
2
1 Max-Planck-Institut fftr Biochemie, 8033 Martinsried and 2 Institut ]'fir Mikrobiologie der Universitdt't, 8400 Regensburg, lq R. G.
Received 31 August 1987
Accepted 2 September1987
Key words: Thermoadaptation; Thermostability; Intracellular ion concentration;
Cyclic 2,3-diphosphoglycerate; Methanogens
1. SUMMARY
An inter- and intra-species correlation was
found between the intracellular potassium concentration and growth temperature within the
Methanobacteriales, comprising mesophiles as well
as moderate ( Methanobacteriurn therrnoautotrophicum) and extreme thermophiles (Methanothermus fervidus, Mt. sociabilis). Potassium concentrations in different species were determined at
optimal growth temperatures and for the same
species cultured at different temperatures. The
main anionic component was found to be the
unusual trianionic cyclic 2,3-diphosphoglycerate.
In vitro experiments with the thermolabile enzymes glyceraldehyde-3-phosphate dehydrogenase
and malate dehydrogenase from Mr. fervidus indicated that the potassium salt of the cyclic diphosphoglycerate acts as potent thermostabilizer. Thus
it appears that, for the methanogens, changes in
the intracellular ion concentration are the basis of
thermoadaptation.
major branches of the urkingdom of archaebacteria [1-3].
To investigate the biochemical adaptations enabling these organisms to live in extremely hot
environments the members of the Methanobacteriales are suitable subjects because closely related
meso- and extremely thermophilic organisms can
be compared. Analyses of enzymes from the extreme thermophile Methanothermus fervidus (maximal growth temperature 90-97 ° C) showed unexpectedly low thermostabilities [4,5], suggesting the
presence of stabilizing factors in vivo. The in vitro
thermostabilization of the glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) of Mr. fervidus by
salts (with potassium phosphate being most effective, [5]) and the relatively high intracellular potassium concentration found in methanogens ( > 200
mM [6]) indicated that the intracellular ion concentrations may be responsible for thermoadaptation in these organisms.
3. MATERIALS A N D M E T H O D S
2. I N T R O D U C T I O N
Extreme thermophiles with optimal growth
temperatures above 80 ° C occur within almost all
Correspondence to: Dr. R. Hensel, Max-Planck-Institut fiJr
Biochemie, 8033 Martinsried, F.R.G.
3.1. Bacteria and growth conditions
Following strains were used: Methanobacterium
bryantii (DSM 863), Mb. thermoautotrophicum
(DSM 1053), Methanothermus fervidus (DSM
2088), Mt. sociabilis (DSM 3496). Mb. strain To
was obtained from K.O. Stetter, University of
Regensburg. All cultures were grown in medium 1
0378-1097/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
76
[7], gassed with H 2 / C O 2 (80/20) in 10-1 fermentors and harvested at an absorbance (578 nm) of
0.7 0.8.
3.2. Determination of intracellular contents of potassium and cyclic 2,3-diphosphoglycerate (cDPG 3 - )
For calculating the intracellular ion concentration the intracellular volume was determined by
the centrifugation method [8] using 3H20 and 14C
sucrose to label total and extracellular water, respectively. Usually the intracellular volume of the
pellet ranged from 10 to 20 #l. In equally treated
samples (however, without labelling) potassium
and c D P G 3 were quantified. Determination of
potassium was done by flame photometry.
c D P G 3 was determined according to [9]. The
determination of the intracellular ion content immediately after harvesting and after 2-fold washing the cells in 0.2 M sucrose, yielded virtually the
same results indicating that the cells were not
susceptible to leaching within an appropriate time.
pH 7.0) were incubated anaerobically in heatsealed glass capillaries. After incubation at the
respective temperature the samples were cooled in
an ice bath, centrifuged briefly and immediately
tested for residual activity.
3.4. Preparation of the cyclic 2, 3-diphosphoglycerate
(cDPG ~ )
The c D P G 3 was isolated from 10 g Mt. fert,idus cells and purified by chromatography on
QAE-Sephadex [10]. The potassium salt was prepared by treatment with Dowex 50W and subsequent neutralization with KOH.
3.5. Determination of the 7",, of the DNA from Mt.
fervidus
The Tm was determined using the Gilford spectrophotometer system 2600; the thermocuvettes
were linearly heated from 7 0 ° C to 9 7 ° C within
27 min, the basic solvent buffer contained 10 mM
K 2 H P O 4 and 1 mM EDTA, pH 7.0; the A260 was
adjusted to 0.4.
3.3. Enzyme preparations and stability tests
G A P D H from Mt. fervidus was purified as
previously described [5]. The M D H purification
follows the same procedure with minor deviations
in the hydroxylapatite chromatography [Hensel et
al., to be published]. G A D P H from rabbit muscle
was obtained from Sigma.
The half-lives of enzyme inactivation were assessed from the sere±logarithmic plots of first-order
denaturation kinetics. The homogeneous enzymes
(30 ~ g . m l - 1 ; standard solvent buffer: 10 mM
potassium phosphate, 15 mM 2-mercaptoethanol,
4. RESULTS A N D D I S C U S S I O N
In mesophilic, moderately and extremely thermophilic members of the Methanobacteriales,
grown at optimal temperature, the respective intracellular potassium concentrations increased
from about 0.3 to 1.0 M (Table 1). The unusual
trivalent anionic cyclic 2,3-diphosphoglycerate
( c D P G 3-) recently described as the dominant
organic phosphate in Methanobacterium thermoau-
Table 1
lntracellular potassium and cyclic 2,3-diphosphoglycerate (cDPG3 ) concentrations of meso- and thermophilic methanogens
measured at their optimal growth temperatures
Organism
Growth temperature
(°C)
Intracellularconcentration ~'
K+
cDPG 3
(raM)
(mM)
Ratio
K* :cDPG ~
Methanobacteriumbryantii
Mb. strain To (isolated by K.O. Stetter)
Mb. thermoautotrophicum
Methanothermusfervidus
Mt. sociabilis
37
37
64
84
88
370+ 20
400 +_100
710 + 40
985± 30
1060+ 30
3.9
5.5
10.9
3.3
3.3
Mean values±SD (n =4 6);
95± 6
72 ± 15
65 __+ 4
300± 5
320+_10
77
Table 2
Half lile of
inactivation
Intracellular potassium and cDPG concentration of Mt. fercidus and Mb. thermoautotrophicum grown at different temperatures
Organism
Growth
temperature
( ° C)
Intracellular concentration
K+
cDPG
(mM)
(mM)
Mt. fervidus
70
78
84 a
88
720 _+20
920_+ 80
985_+30
750+20
195 _+25
280_+ 8
300_ 5
220___15
Mb. thermoauto- 55
trophicum
64 a
530 +_15
710 ± 40
535 _+10
78 _+10
65 _ 5
42 +_ 5
-/:-° /
100
50
70
a Optimal temperature.
tO
totrophicum [10,11] p l a y s an i m p o r t a n t role in
b a l a n c i n g the positive charge of K +. In the extreme t h e r m o p h i l e s (Mt. fervidus a n d Mt. sociabilis) c D P G 3- is the p r i m a r y p o t a s s i u m counterion,
w h e r e a s in the m o d e r a t e t h e r m o p h i l e a n d
m e s o p h i l e s ( Mb. bryantii, Mb. strain To) there are
a p p a r e n t l y a d d i t i o n a l counterions.
I n the case of Mt. feruidus, grown at different
t e m p e r a t u r e s , a p a r a l l e l b e t w e e n the i n t r a c e l l u l a r
p o t a s s i u m a n d c D P G 3 c o n c e n t r a t i o n a n d temp e r a t u r e was f o u n d up to the o p t i m a l g r o w t h
t e m p e r a t u r e ( T a b l e 2). A b o v e the o p t i m u m the ion
c o n c e n t r a t i o n decrease was p r o b a b l y due to a
decrease in the viable cells in the culture.
Similarly for Mb. thermoautotrophicum the
p o t a s s i u m c o n c e n t r a t i o n increased to the t e m p e r a ture o p t i m u m a n d decreased at higher t e m p e r a tures, b u t the c o n c e n t r a t i o n of c D P G 3- d i d n o t
follow this tendency. In this organism, as in the
mesophiles, b o t h ions ( K + a n d c D P G 3 - ) , are not
as strongly c o r r e l a t e d as in the e x t r e m e t h e r m o philes.
I n d i c a t i o n s t h a t K + a n d c D P G ~- p l a y an imp o r t a n t role in the b i o c h e m i c a l t h e r m o a d a p t a t i o n
for the e x t r e m e l y t h e r m o p h i l i c m e t h a n o g e n s do
n o t only c o m e f r o m the almost ideal K + : c D P G 3r a t i o of 3 . 3 : 1 within the cell, b u t also f r o m the
t h e r m o s t a b i l i z i n g effect of the t r i p o t a s s i u m salt of
the c D P G 3- on the cell p r o t e i n s from Mt. fervidus
in vitro. U p o n a d d i t i o n of this salt (300 m M ) the
half-lives of i n a c t i v a t i o n of the g l y c e r a l d e h y d e - 3 -
5
2.8
2.7
100
HaH ~ile ot
inactivation
1/3" X l 0 -3
90
°C
80
(b)
rnin
100
5O
J
o
5
j°
j1
3.02
57.5
3.04
55
3,06
3.08
52.5
3.1
50
1/T x 10"3
°C
Fig. l. Heat inactivation of GAPDH and MDH from Mt.
fervidus (a) and GAPDH from rabbit muscle (b) in the presence (filled symbols) and absence (open symbols) of K3cDPG.
78
phosphate dehydrogenase (GAPDH) and malate
dehydrogenase (MDH) increased from 1.5 min
(GAPDH) and 8 min (MDH) to 3.5 h and 16 h,
respectively, at 90 °C, the upper growth limit of
Mt. fervidus under the experimental conditions
(Fig. la). Comparing the stabilizing efficiency of
K3cDPG with several other salts, the stabilization
effect is obviously based on both the nature of the
cation and the phosphate moiety of the anion
(Fig. 2a, b).
Thermostabilization experiments with an arbitrarily chosen enzyme, the G A P D H from rabbit
(Fig. lb) indicated that the thermostabilization by
K3cDPG requires specific prerequisites of the protein structure.
Residual
activity
(a)
%
1O0
l ~ /
•--•
80
6O
4O
2O
. . . .
~-~.~.
....
100
.
200
.
,
.
" ~ I .
300
mM
Residual
activity
Considering the low G + C content of the DNA
of Mt. fervidus and Mr. sociabilis (33 mol% [12,13],
an extrinsic stabilization of the DNA duplex seems
to be unavoidable at high growth temperatures.
However, as shown for the D N A of Mt. fervidus
K3cDPG can not stabilize the duplex more efficiently than would be expected from KCI solutions equimolar in [K +] (Fig. 3). Even in the
presence of the maximal concentration of 300 mM
the DNA is thermally denatured for the most part
at the upper growth limit of 90°C. Thus additional stabilizing factors like DNA-binding proteins must be assumed to ensure a native DNA
structure at elevated growth temperatures.
While we are far from understanding the subtile intracellular interactions required for growth
at elevated temperatures, the data presented here
strongly suggest that at least in the thermophilic
methanogens the intracellular ion concentration
plays an important role in the biochemical thermoadaptation. This strategy (A) of increasing
thermostability of thermolabile enzymes by increasing intracellular ionic strength as required,
seems to avoid some disadvantages of the alternative strategy (B), making intrinsically thermostable
enzymes. Thermostable enzymes reach their optimal activity only at a respective high temperature
and their temperature dependence of activity is
mostly higher than that of less thermostable enzymes [5,14,15]. We would therefore predict that
thermophilic organisms following strategy B would
(b)
%
100 ~j
•~
~
III~
Tm
oC
t
6O
°-
100
20O
3OO
-
mM
Fig. 2a and b. Effectiveness of K3cDPG in stabilizing the
G A P D H (a) and M D H (b) from Mt. fervidus against heat
denaturation (30 min incubation at 90 ° C). (I) K3cDPG; (A)
potassium phosphate; (zx) sodium phosphate; ( 0 ) potassium
sulfate; (O) sodium sulfate; (O) potassium chloride; (©)
sodium chloride.
0~
04
o6
0s
10
~5
Fig. 3. Dependence of the T,n of the DNA from Mt. fert,idus
on K~cDPG compared to KCI. (A) KacDPG; (O) KCI.
79
only be able to c o m p e t e at high t e m p e r a t u r e s
w i t h i n a n a r r o w range, whereas organisms following the more flexible strategy A should be able to
m a i n t a i n a b a l a n c e d physiology over a wider temperature range.
ACKNOWLEDGEMENTS
W e t h a n k S. F a b r y , V. D o e r i n g a n d B. Sam for
p r e p a r i n g G A P D H a n d M D H from Mt. fervidus,
S. L a u m a n n a n d M. Poignee for technical assistance a n d J. T r e n t for critical reading of the
m a n u s c r i p t . We also t h a n k J. W i n t e r a n d N. Weiss
for allowing us to use the flame p h o t o m e t e r a n d
the Gilford spectrophotometer. T h a n k s are also
due to K.O. Stetter a n d W. Zillig for s t i m u l a t i n g
discussions a n d P. Schoenheit for valuable hints
for d e t e r m i n i n g the intracellular volume. R.H.
thanks W. Zillig for the o p p o r t u n i t y of working in
his laboratory.
The work was s u p p o r t e d b y grants from the
Deutsche Forschungsgemeinschaft.
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