Content uploaded by Donald A. Cowan
Author content
All content in this area was uploaded by Donald A. Cowan on Sep 29, 2014
Content may be subject to copyright.
CMLS, Cell. Mol. Life Sci. 57 (2000) 250–264
1420-682X/00/020250-15 $ 1.50+0.20/0
© Birkha¨user Verlag, Basel, 2000
Review
Biomolecular stability and life at high temperatures
R. M. Daniel
a,
* and D. A. Cowan
b
a
Thermophile Research Unit, Department of Biological Sciences, School of Science, University of Waikato,
Private Bag 3105, Hamilton (New Zealand), Fax +64 7 8384324, e-mail: r.daniel@waikato.ac.nz
b
Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT (United
Kingdom)
Received 13 July 1999; received after revision 30 September 1999; accepted 22 October 1999
Abstract. It is not clear what the upper temperature limit known to support life, but evidence concerning degrada-
tive reactions above 100 °C is slight. DNA duplexfor life is, or what specific factors will set this limit, but
it is generally assumed that the limit will be dictated by stability is apparently achieved at high temperature by
molecular instability. In this review, we examine the elevated salt concentrations, polyamines, cationic
thermal stability of two key groups of biological proteins, and supercoiling rather than manipulation of
molecules: the intracellular small molecules/metabolites C-G ratios. RNA stability seems dependent upon cova-
and the major classes of macromolecules. Certain small lent modification, although secondary structure is prob-
molecules/metabolites are unstable in vitro at the growth ably also critical. The diether-linked lipids, which make
up the monolayer membrane of most organisms growingtemperatures of the hyperthermophiles in which they are
above 85 °C are chemically very stable and seem poten-found. This instability appears to be dealt with in vivo
tially capable of maintaining membrane integrity at muchby a range of mechanisms including rapid turnover,
metabolic channelling and local stabilisation. Evidence higher temperatures. However, the in vivo implications
to date suggests that proteins have the potential to be of the in vitro instability of biomolecules are difficult to
stable at substantially higher temperatures than those assess, and in vivo data are rare.
Key words. Archaea; biomolecules; DNA; enzymes; lipids; membranes; proteins; RNA; stability; thermophiles;
thermostability.
Introduction
Studies of life at high temperatures (\ 75 °C) are com-
paratively recent, but have been a fertile field, providing
new impetus to discussions on the origin of life and
molecular (in)stability, and broadening the environmen-
tal ‘envelope’ within which we expect to find life. The
latter potentially provides a key set of parameters to
guide the current and future design of strategies for
detection of life on planets other than Earth.
In this review we will deal with the molecular factors
which are characteristic of organisms living at tempera-
tures above 75 °C and which might act as an upper
temperature limit. However, much of the focus will be
on the higher-temperature ranges, since the molecular
differences between low- and high-temperature life
manifest themselves most obviously above 90 °C. Life
above 75 °C is confined to the bacteria and the archaea,
and only members of the latter are capable of growth
above 95 °C. This makes it difficult in many cases to
separate those features that are related to a high-tem-
perature existence from those which are present for
* Corresponding author.
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 251Review Article
phylogenetic reasons. (We will not speculate on why no
eucaryotes exhibit thermophily, except to point out that
other major biochemical and physiological pathways
and properties are also absent from this group, including
methanogenesis and extreme halophilicity.) Taxonomic
trees constructed from ribosomal RNA (rRNA) se-
quences show the archaeal kingdom, with its high pro-
portion of thermophiles and hyperthermophiles, nearest
the root. Among the bacteria, Aquifex (t
max
=95 °C) is
more deeply rooted than Thermotoga (t
max
=90 °C),
which is more deeply rooted than Thermus (t
max
=
80 °C) [1, 2] (fig. 1), and other thermophilic bacteria
such as Dictyoglomus and Thermodesulfobacterium are
also deeply rooted. The simplest explanation of these
trees is a high-temperature origin for the earliest living
organisms, although the trees are a subject of consider-
able debate [3], and it is argued by some that the deep
branching of the hyperthermophilic bacterial 16S rRNA
sequences is an artefact arising from low rates of evolu-
tion. The evidence for a thermophilic origin of life is
therefore indicative rather than overwhelming. Protago-
nists of the ‘RNA world’ suggest that a high-tempera-
ture origin for life is unlikely because the nonliving
systems preceding such organisms are likely to have
been based on RNA, which is unstable at high tempera-
tures. However, only minor modifications are needed to
stabilise RNA in vitro and in vivo [4], and organisms
capable of growth at 110 °C do contain functional
RNA.
Arguments over the nature of the universal ancestor
notwithstanding, the thermophiles and hyperther-
mophiles have provided new insights into a wide range
Figure 1. Universal phylogenetic tree based on rRNA sequences (after [2]).
R. M. Daniel and D. A. Cowan Life at high temperatures252
Table 1. Metabolite and coenzyme stabilities.
% remaining afterMetabolite/coenzyme
3h/105 °C1h/95 °C
B5 n.d.NAD
85100FAD
75FMN 65
40Pyridoxal phosphate 0
100Glucose 100
100 70Glucose-6-phosphate
5090Glucose-1,6-diphosphate
100Gluconate 100
906-Phosphogluconate 100
100100Glycerate
1003-Phosphoglycerate 100
100100Acetate
B10Acetyl phosphate n.d.
100CoASH 45
75100Acetyl CoA
ATP 40 0
050ADP
60AMP 95
Solutions (10 mM) in distilled water containing 1 mM KI were
heated in sealed glass tubes, and degradation assessed by changes
to the electrospray mass spectrum [5].
ple, table 1). The known presence of these compounds
in hyperthermophiles suggests the existence of a num-
ber of mechanisms by which metabolite/coenzyme
thermal instability may be overcome at high growth
temperatures.
Microenvironment protection
The thermal stability of many metabolites is highly
dependent on conditions. For example, adenosine
5%-triphosphate (ATP) stability is greatly affected by pH
and metal ions [6, 7], so microenvironment protection is
possible. This may also be the case for NADH, which in
dilute buffer at around pH 7 has a half-life of only a
few minutes at 95 °C, but which at higher pH is much
more stable [8–10]. In any event, since ATP and
NAD(P) are both used as coenzymes in archaea grow-
ing at 105 °C, such organisms obviously have a way of
circumventing this instability, and the exploitation of
microsites which have less destabilising conditions is
one possibility.
Metabolic channelling
It has become clear over the last decade that cytoplasm
is not a homogeneous medium in which enzymes are
dissolved. But while the idea of cytoplasmic microstruc-
ture is now well accepted [11], its physiological implica-
tions are less clear. Nevertheless, a strong case has been
made that channelling of intermediates between physi-
cally associated enzymes which are sequential members
of a metabolic pathway can have major effects. These
include reduction in intermediate metabolite concentra-
tion and increase in pathway flux [12 –14]. Van de
Casteele et al. [15, 16] have suggested that the thermal
instability of carbamoyl phosphate can be circumvented
at high temperatures by the physical juxtaposition of
the enzyme producing the metabolite (carbamoyl phos-
phate synthetase) and the next enzyme in the metabolic
pathway (ornithine carbamoyl transferase), which con-
sumes the metabolite. Evidence for such a juxtaposition
has been obtained for Pyrococcus furiosus [17].
Given the extent of cytoplasm microstructure, and the
relatively undeveloped state of this field, enzyme-en-
zyme association may turn out to be a potent and
widespread mechanism by which metabolite instability
can be circumvented. Currently there is no evidence to
indicate that the phenomenon is more widespread in
thermophiles, but this is not necessarily an argument
against it.
Catalytic efficiency
Sterner et al. [18] have suggested that the thermal insta-
bility of phosphoribosyl anthranilate (an intermediate
in the tryptophan biosynthesis pathway; t
1/2
=39sat
of biochemical and physiological processes. Indeed,
studies on this relatively small and select group of
organisms may have a greater influence on our under-
standing of fundamental biomolecular processes than
virtually any other biotype.
While at this stage of our knowledge it may be most
rational to ask how life has managed to adapt to the
difficulties imposed by sub-80 °C temperatures, the
question most commonly asked of the hyperther-
mophiles (living optimally at between 85 and 113 °C)
is, What molecular adaptations are responsible for their
ability to survive and grow at such temperatures? Two
subsidiary and closely related questions are, What
molecular instability (or other process) dictates the up-
per limit of life? and Where key metabolic components
are known to be unstable at temperatures well below
this limit, how do hyperthermophiles retain metabolic
activity up to this limit? This review attempts to present
and discuss some of the current evidence bearing on
these questions.
Throughout, we have used the term ‘thermophile’ in the
general sense of growing optimally above 75 °C, and to
include hyperthermophiles growing optimally at tem-
peratures well above this.
Metabolism, metabolites and cofactors
Many low molecular weight metabolites and coenzymes
have quite short half-lives even at 95 °C (see, for exam-
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 253Review Article
80 °C) is overcome in Thermotoga maritima by the very
high efficiency of the phosphoribosyl anthranilate iso-
merase enzyme. The high efficiency is largely due to a
very low K
m
, although the k
cat
is relatively high.
As a group, enzymes from thermophiles tend to have
similar k
cat
values to their mesophilic homologues, and
there is likewise no evidence for systematically lower K
m
values for thermophilic enzymes. However, it must be
borne in mind that few systematic comparisons between
the kinetic properties of enzymes from thermophiles
and mesophiles have been carried out, and given the
wide variation among mesophilic enzymes, it would not
necessarily be easy to draw conclusions from such a
comparison. Nevertheless, on the basis of current evi-
dence an increase in catalytic efficiency in thermophilic
enzymes does not seem likely to be a widespread
strategy.
Substitution or deletion
In this case, the thermal instability of a metabolite is
circumvented by use of an alternate pathway or by
using a more stable alternative compound.
NAD(P) and non-haem iron protein. There is a strong
association between the use of non-haem iron proteins
instead of NAD(P) and thermophily [19]. NAD and
NAD(P) are unstable at 95 °C (t
1/2
2 min) [10],
whereas non-haem iron proteins have the potential to
be quite stable and functional at 100 °C [19, 20].
Makund and Adams [21] have shown that P. furiosus
oxidises glucose to pyruvate via a nonphosphorylated
Entner-Doudoroff pathway in which the redox reac-
tions are all catalysed by ferredoxin-linked oxidoreduc-
tases. In the less primitive archaea, Sulfolobus and
Thermoplasma [22], glucose is also catabolised via this
nonphosphorylated pathway. Glucose dehydrogenase
and glyceraldehyde dehydrogenase are NAD/P-linked
[23, 24], but pyruvate decarboxylation is ferredoxin-
linked [25].
Other examples of the tendency for non-haem iron
protein to replace NAD/P in more thermophilic organ-
isms include the finding [26, 27] that in Sulfolobus the
2-oxoglutarate oxido-reductase is ferredoxin-linked,
rather than NAD/P linked, the non-haem iron protein
linkage of the aldehyde oxidoreductase from ES-4 [28]
(already known in P. furiosus), the 2-keto isovalerate
ferredoxin oxidoreductase from several hyperther-
mophilic archaea [29] and the indole pyruvate oxidore-
ductase in P. furiosus [30]. The examples above are from
Archaea, but an increased dependence upon non-haem
iron protein rather than NAD has also now been shown
in one of the most deeply rooted and thermophilic of
the bacteria, Thermotoga. This occurs at the pyruvate-
ferredoxin oxidoreductase step, although the enzyme
mechanism is different to that found in Archaea [31].
Thus, although the use of non-haem iron proteins in
place of NAD(P) is associated with archaea, occurring
in methanogens and halophiles as well as in ther-
mophiles, it also occurs in thermophilic bacteria. This
suggests that although it seems likely to be a primitive
characteristic [19], there is a correlation with
thermophily.
Acetyl phosphate. Acetyl phosphate is particularly un-
stable relative to acetate and acetyl coenzyme A (CoA)
(table 1), and its use as a metabolic intermediate would
seem to pose particular difficulties in hyperther-
mophiles. Shafer et al. [32] have found that in hyper-
thermophiles the interconversion of acetyl CoA to
acetate is direct rather than proceeding via acetyl phos-
phate. So far it seems that the absence of acetyl phos-
phate from this pathway is an archaeal characteristic
rather than a thermophilic one, so it is not clear
whether this absence is imposed by thermophily, or if
the use of acetyl phosphate arose after the separation of
the other two kingdoms. Of course, if the universal
ancestor was a thermophile, and most closely related to
the archaeal kingdom, this question does not arise.
Other phosphorylated compounds. It is possible that the
presence of an Enter-Douderoff pathway based on non-
phosphorylated carbohydrates in thermophilic archaea
[33] arises because of the greater stability of these inter-
mediates compared with the phosphorylated forms.
However, the in vitro differences in stability are small
(table 1). Nevertheless, although the relative importance
of the phosphorylated and nonphosphorylated path-
ways is not clear within the thermophilic archaea, the
nonphosphorylated pathway appears to be confined to
this group.
The most important phosphorylated metabolites are of
course ATP and adenosine 5%-diphosphate (ADP). The
stability of this class of compounds is in the order
pyrophosphate (PP)/AMP\ ADP\ ATP, and ATP is
relatively unstable at 95 °C (table 1). There is some
evidence for the use of the more stable compounds (PP
and ADP) instead of ATP in thermophilic archaea. For
example, Kengan et al. [34] have found that in Pyrococ-
cus both hexokinase and PFK are, uniquely, ADP-
linked. In the case of hexokinase, glucose is normally
phosphorylated by ATP, which is less stable than ADP.
In the case of PFK, F-6-P is also normally phosphory-
lated by ATP; Siebers and Hensel [35] have shown that
in Thermoproteus tenax pyrophosphate is used, but this
is also the case in some primitive eukaryotic parasites.
The four mechanisms described here are by no means
mutually exclusive, and other mechanisms, such as
rapid synthesis, are also possible. In summary, some
key coenzymes and metabolites, including NAD(P),
acetyl phosphate and ATP are quite unstable above
100 °C. While the presence of some of these com-
pounds in archaea growing above 100 °C indicates the
existence of mechanisms for circumventing this instabil-
R. M. Daniel and D. A. Cowan Life at high temperatures254
ity in ‘modern’ organisms, there is also evidence that in
many thermophiles more stable compounds (e.g. non-
haem iron protein) replace some or all of the functions
of these [e.g. NAD(P)].
Enzymes and proteins
Denaturation
It has been known for some time that some enzymes
from thermophiles and hyperthermophiles (e.g. [36])
have significant half-lives at \ 100 °C (table 2). A
variety of detailed structural studies have compared
such proteins to much less stable variants from
mesophiles (e.g. [44 –46]). Considering these results as a
whole, the most significant finding is that although
there are structural differences between very stable and
‘normal’ enzymes, there is no pattern of systematic
structural differences. Within a closely related group of
enzymes there may be a pattern of changes differentiat-
ing the more and less stable variants, but for a different
group of enzymes the changes will be different: overall,
in structural terms the differences between very stable
and less stable enzymes are no greater than the differ-
ences within the groups [47, 48]. This finding is entirely
in keeping with early theoretical studies indicating that
the tertiary structures of proteins are only marginally
stable (e.g. [49]), and with results showing that only a
few amino acid substitutions are required to bring
about significant changes in thermal stability [50, 51].
The conformational stability of a protein (defined as the
difference in free energy between the folded and un-
folded states, DG) is the sum of a large number of weak,
noncovalent interactions, including hydrogen bonds,
salt bridges, van der Waals interactions and the hydro-
phobic effect, and the destabilising forces arising largely
from conformational entropy. The sum of these stabilis-
ing interactions is about 1 MJ mol
−1
. Destabilising
forces are of a similar magnitude, and DG, the differ-
ence between the two, is only of the order of 40 kJ
mol
−1
. Point mutational studies have shown that re-
placement of a single amino acid (and thus apparent
removal of a single stabilising interaction) can have a
significant effect on stability without any detectable
effect on the three-dimensional structure [52]. Such ‘in-
dividual’ interactions can contribute up to 25 kJ mol
−1
,
so it is evident that only a few additional interactions
will be needed to account for the additional stability of
enzymes from hyperthermophiles. However, the ther-
modynamic consequences of such a single amino acid
mutation are rarely limited to deletion (or addition) of
a single noncovalent interaction, since the global effect
on DG must include changes in the unfolded as well as
the folded state.
There is no evidence that in any protein all the amino
acid residues are participating in stabilising interactions.
Because of this, and because each additional interaction
can have such a marked stabilising effect, it is difficult
to determine what might be the upper limit for confor-
mational stability. The temperature dependence of hy-
drophobic interactions suggests that these may be weak
at 140 °C [53], so that this might seem to set an upper
limit. But the hydrophobic interaction and the magni-
tude of its contribution to protein stability is incom-
pletely understood [53, 54]. Since stability also depends
on other interactions, as well as on any factors that may
destabilise the unfolded state, then even if hydrophobic
interactions are much weaker above 140 °C, this may
not represent an upper temperature limit for protein
stability.
In general terms, there is strong evidence for an inverse
correlation of conformational stability with specific ac-
tivity, via molecular flexibility (e.g. [47, 55]). Whereas
enzyme activity is dependent upon flexibility, a less
flexible enzyme will be more stable. The broadest gen-
eral evidence for this is the finding that, at any given
temperature, as a group, enzymes from thermophiles
are more stable, less flexible and less active than those
from mesophiles [47, 48, 55– 59]. Together with the
general structural and functional identity of stable and
less stable enzymes, this leads to the view that the
instability of enzymes from mesophiles is a functional
requirement rather than because of any restraint on
achieving higher stability [47, 55]. It is required so that
enzymes have sufficient flexibility to perform their cata-
lytic functions: i.e. enzymes tend to be denatured at
temperatures not very far above their evolved or ‘de-
sign’ temperature because too much stability would
mean not enough flexibility for effective catalysis,
whereas too little stability would mean too short a
useful lifetime. An additional requirement for instability
can be inferred from the finding that, irrespective of
whether or not they are denatured, stable proteins are
more resistant to proteolysis [60]. A balance between
stabilizing and destabilizing interactions is required to
Table 2. Stability of some enzymes at 100 °C.
Enzyme (Source) T
1/2
at 100 °C
Cellobiohydrolase (Thermotoga) [37] \200 min
90 minb-Glucosidase (Thermotoga) [38]
20 minXylanase (Thermotoga) [39]
Xylosidase (Thermotoga) [38] 150 min
Esterase (Sulfolobus) [40] 60 min
Hydrogenase (Pyrococcus) [41] 120 min
360 minAmylase (Pyrococcus) [42]
DNA-dependent RNA polymerase \120 min
(Thermoproteus) [43]
T
1/2
values are half-lives of activity under specified, but not
identical, conditions.
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 255Review Article
meet the conflicting demands of stability on the one
hand, and catalytic function and cellular turnover on
the other. As a consequence, if we consider only confor-
mational stability, we may postulate that the reason we
have not found proteins that are stable much above
130 °C is because we have not found organisms grow-
ing much above 110 °C (rather than vice versa).
However, the influence of molecular flexibility on stabil-
ity and activity is poorly defined. Conformation stabil-
ity is a global property of an enzyme [49, 51], but it is
not clear whether the influence of flexibility on stability
is regional or global. It seems likely that flexibility at
particular points in the structure is much more critical
for stability than elsewhere. With respect to activity,
although certain local motions at or near the active site
must occur over time scales similar to those of substrate
turnover, it is not clear whether these are coupled to
faster motions, locally or globally. It has been found
that enzyme activity at low temperatures is unaffected
by the cessation of fast anharmonic global dynamics
(B 100 ps time scales) [61, 62]. It is difficult to under-
stand how dynamics mediate the apparent inverse cor-
relation between stability and activity if the dynamics
required for activity is local, while that required for
stability is global; but if this is the case, it will certainly
hold out good prospects for the engineering of enzymes
which are both more stable and more active. For a
more detailed discussion see [47, 61, 62].
Degradation
Thermophilic proteins are conformationally stable at
high temperatures [20, 36], but the effect of irreversible
degradative processes [63, 64] on these proteins at high
temperatures is unclear. In contrast to denaturation
(and leaving aside the special case of disulphide bonds),
the irreversible processes of protein inactivation arise
from changes in covalent bonding. The most common
at high temperatures are deamidation of the amide side
chain of Asn and Gln residues, succinimide formation
at Glu and Asp, and oxidation of His, Met, Cys, Trp
and Tyr. These reactions have high activation energies
and are thus greatly accelerated by high temperatures,
and so have the potential to play a particularly impor-
tant role in the inactivation of enzymes at high temper-
atures. At least some of the chemical mechanisms for
irreversible degradation in proteins require local molec-
ular flexibility. A survey of environments around Asp
and Asn resides in known three-dimensional protein
structures suggests that the rigidity of the folded protein
greatly decreases the intramolecular imide formation
necessary for degradation. In the numerous X-ray crys-
tal structures studied, the peptide-bond nitrogen could
not approach the side-chain carbonyl carbon closely
enough to form the succinimide ring [65]. At 37 °C the
rate of deamidation has been shown to be higher for
small peptides with high flexibility than for proteins
when comparing the same amino acid sequence [66],
and higher in denatured than in native proteins [67]. In
other words, the resistance to degradation of a protein
is linked to its conformational integrity. This contention
is supported by studies using thermally stable proteins
in which the conformation is known to be retained for
significant periods at the high temperatures at which
degradative reactions occur. Hensel et al. have shown
that the rate of deamidation for a thermostable glycer-
aldehyde phosphate dehydrogenase from Pyrococcus
woesei at 100 °C is increased after denaturation of the
enzyme [68] (and after dialysing away the denaturing
guanidinum hydrochloride). Furthermore, for the same
enzyme from Methanothermus fer6idus, deamidation oc-
curred more readily at 85 °C in a less stable chimaeric
form of the enzyme derived from a construct containing
both thermophilic and mesophilic genes. Similarly, the
addition of phosphate, known to stabilise the confor-
mation of the dehydrogenase, decreased the rate of
peptide bond hydrolysis at temperatures ranging from
85 to 100 °C [68]. Support for the view that the loss of
conformation precedes irreversible degradative reac-
tions comes from studies of deamidation (ammonia
release) and loss of activity of the very stable xylanase
from Thermotoga strain FjSS 3B1 in the range 95–
100 °C [47]. Both the onset and progress of deamida-
tion occurred later than those of activity loss, consistent
with a dependence of deamidation upon loss of
conformation.
In pig muscle myokinase, which is a very stable enzyme
despite its mesophilic origin, studies on peptide bond
hydrolysis give similar results. In the native and dena-
tured enzyme at 95 °C [47] the rate of peptide bond
hydrolysis is always lower than the rate of activity loss,
although agents which affect the rate of activity loss
such as SDS plus mercaptoethanol (faster) and sub-
strate (slower) have a similar effect on peptide bond
hydrolysis. These correlations are consistent with a de-
pendence of degradation on loss of conformation.
There is thus growing evidence that the degradative
reactions to which proteins are subject are slower or do
not occur in conformationally intact proteins, at least
up to 100 °C. In other words, the upper temperature
limit for protein stability may after all be determined by
the conformational integrity of the protein, although we
must bear in mind that few studies on conformational
or degradative stability above 100 °C have been made.
Nucleic acids
The question of how hyperthermophiles maintain the
structure and integrity of their DNA and RNA in vivo
R. M. Daniel and D. A. Cowan Life at high temperatures256
has been the focus of considerable research over the
past 20 years. Based on early studies of the effects of
high temperature on nucleic acids [69], it has been
reasonably assumed that hyperthermophiles, as com-
pared with mesophiles, would be required to cope with
a considerably greater burden of both chemical degra-
dation and duplex destabilisation.
At high temperatures DNA undergoes denaturation by
strand separation. However, it has long been known
that duplex stability in vitro could be manipulated over
a wide temperature range by addition of salts. The
discovery that some hyperthermophiles contained molar
concentrations of potassium di-inositol-1,1%-phosphate
[70] or tripotassium cyclic-2,3-diphosphoglycerate [71],
suggested a possible in vivo mechanism for stabilisation
of nucleic acid secondary structure. (It must be noted
that these compounds also stabilise protein conforma-
tion at high temperatures in vitro.) However, not all
hyperthermophiles contain high intracellular ion
concentrations.
Polycationic polyamines, which increase the melting
temperature of DNA and protect S. solfataricus
ribosomes from thermal inactivation in vitro [72],
have also been observed in hyperthermophiles [73].
Concentrations of putrescine (H
2
N-(CH
2
)
4
-NH
2
), sper-
midine (H
2
N-(CH
2
)
3
-NH-(CH
2
)
4
-NH
2
), norspermidine
(H
2
N-(CH
2
)
3
-NH-(CH
2
)
3
-NH
2
, thermospermine (H
2
N-
(CH
2
)
3
-NH-(CH
2
)
3
-NH-(CH
2
)
4
-NH
2
) and spermine
(H
2
N-(CH
2
)
3
-NH-(CH
2
)
4
-NH-(CH
2
)
3
-NH
2
)ofupto0.4
g% (d.w. cell biomass) were detected in various Sul-
folobus strains. A comprehensive analysis of the
polyamines in 75 bacterial and archaeal isolates from
mesophilic to hyperthermophilic sources has been car-
ried out [74]. The results showed that some polyamines
(norspermine and norspermidine) occurred only in the
hyperthermophilic archaea, but that there was no sig-
nificant correlation between total intracellular
polyamine concentration and the growth temperature of
the source organism. However, the hyperthermophilic
archaea were found typically to contain a greater diver-
sity of polyamines than other organisms.
Stabilisation of nucleic acid duplex structure may also
be achieved by increasing the G–C ratio, a strategy
which the hyperthermophiles appear to eschew, there
being no obvious correlation between percentage G +C
content and optimum growth temperature in even the
most hyperthermophilic of the archaea (table 3). Gro-
gan [82] notes that the molar percentage G+ Cinthe
16S RNAs of this same group of organisms is in all
cases significantly higher (typically 63–69%).
Alternative mechanisms for the stabilisation of DNA
secondary structure include supercoiling and associa-
tion with cationic proteins. The discovery of a novel
ATP-dependent topoisomerase I activity (‘reverse gy-
rase’, generating positive supercoils [83]) in all the hy-
Table 3. Molar percentage G+C values for hyperthermophilic
archaea.
ReferenceOrganism Mol % G+CT
opt
(°C)
Methanopyrus kandleri 6098 [75]
100Pyrobaculum islandicum [76]46
Pyrococcus abyssi 96 44–45 [77]
38100Pyrococcus furiosus [78]
59 [79]97Pyrodictium abyssi
[80]62105Pyrodictium occultum
[81]53106Pyrolobus fumarii
perthermophilic archaea tested [84] (and in some
hyperthermophilic bacteria [85]) led to the proposal that
this was a specific mechanism for DNA stabilisation,
and possibly a true ‘hyperthermophilic characteristic’
[86]. Subsequent studies (e.g. [87]) have shown that the
presence of positive supercoils per se was not specifi-
cally required for thermostabilisation of DNA. It there-
fore seems likely that the torsional constraints of
supercoiling, whether positive or negative, provide sub-
stantial but similar increases in T
m
. The true in vivo role
of topoisomerases in hyperthermophiles is currently un-
clear. Evidence that reverse gyrase and topoisomerase II
(relaxing) activities in Desulfurococcus amylolyticus were
regulated both by temperature and growth phase [88]
suggests that these enzymes play a complex but impor-
tant role in the superhelicity of the genome.
DNA topology is also affected by interaction with
cationic proteins, numerous examples of which have
been identified in hyperthermophiles (table 4). These
small basic proteins bind DNA in vitro with substantial
increases in T
m
, and have been variously shown to bend
DNA [89] or form nucleosome structures [96]. For
example, the HMf family of archaeal histones (origi-
nally identified in M. fer6idus) are homologues of eu-
caryal nucleosome core histones and have been shown
to bind to and compact archaeal DNA both in vitro
and in vivo [97–99]. Histone-like proteins from Sul-
folobus [100] have no eucaryal homologues but, like the
HMf proteins, also compact DNA and increase the T
m
of DNA in vitro. The driving force for the evolution of
these proteins may therefore have more to do with
DNA packaging and nucleosome formation [101] than
DNA stabilisation. For more detail on DNA topology
and nucleosome structure, the reader is directed to a
recent review [97].
The structural stabilisation of ribonucleic acids in hy-
perthermophiles is particularly important in transfer
RNAs (tRNAs), where there is a requirement for the
maintenance of a complex three-dimensional structure
in the absence of other macromolecular associa-
tions. The strategy employed by the hyperthermophilic
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 257Review Article
Table 4. Hyperthermophilic histone-like proteins.
Protein Source organism Homologues References
[89]Eucaryal nucleosome core histones H2A, H2B, H3 andMethanothermus fer6idusHMf family
H4
Methanobacterium thermoau- [90]
totrophicum
Thermococcus zilligii [91]
[92]Methanopyrus kandleri
Pyrococcus spp. (HPy) [93]
[94]Sulfolobus acidocaldarius Eukaryal SH3 domainsSac family
DNABPII E. coli HU histone-like proteins [95]Thermotoga maritima
archaea would seem to be largely one of posttranscrip-
tional modification. Studies of modified sugars and
bases in thermophilic archaea and bacteria have re-
vealed a number of novel modifications, some of which
are archaea-specific [102]. The importance of these
modification with respect to thermostability is not clear,
although it was noted [102] that the greatest number of
different ribose methylated nucleosides were observed in
the most hyperthermophilic organism (Pyrodictium oc-
cultum, T
opt
=105 °C) whereas the least thermophilic
organisms (Thermoplasma acidophilum (55 °C) and
Methanobacterium thermoautotrophicum (65 °C) con-
tained the fewest. Clearer evidence for the role of post-
transcriptional modification of ribonucleotides is found
in the study of Edmonds et al. [103], which reported the
liquid chromatography/mass spectrometry analysis of
the ribonucleosides from Pyrococcus furiosus grown at
70, 85 and 100 °C. Three modified nucleosides (fig. 2)
were shown to increase in relative abundance as a
function of cell culture temperature. Each of these nu-
cleosides was localised to regions where it contributed
to conformational rigidity [4].
The requirement for specific stabilisation of rRNA at
hyperthermophilic temperatures remains uncertain, pos-
sibly because the complex topology and RNA-protein
interactions of these molecules reduces the potential
impact of deleterious conformational changes. How-
ever, posttranscriptional modifications in the 16S and
23S rRNAs of Sulfolobus solfataricus have been investi-
gated [103]. Ribose O-2% methylations occurred most
frequently, and for some nucleotides, successive in-
creases in the degree of methylation ( \ 10%) were
detected in cells grown at 63, 75 and 83 °C. The au-
thors tentatively concluded that these modifications
may play a role in ‘the secondary and tertiary stabilisa-
tion of rRNA’.
Chemical degradation of bases at high temperature is
potentially a serious threat to the genetic stability of
hyperthermophiles. The most common covalent modifi-
cations are the loss of bases from one strand to generate
apurinic or apyrimidinic sites, and base deaminations
(particularly deamination of cytosine and 5-methyl cy-
tosine). It has been suggested that both processes
should be greatly enhanced at high temperatures, chem-
ical damage to the genome of a hyperthermophile grow-
ing at 100 °C being possibly as much as 3000-fold more
rapid than in an Escherichia coli genome at 37 °C [104].
The authors are not aware of any experimental verifica-
Figure 2. Modified nucleosides implicated in stabilisation of hy-
perthermophile tRNA.
R. M. Daniel and D. A. Cowan Life at high temperatures258
tion of these calculations. Not all the known enzymes
required for the repair of these modifications have been
investigated in hyperthermophiles, but archaeal homo-
logues of well-characterised bacterial and eucaryal
DNA repair systems continue to be discovered. For
example, homologues of homologous recombination
components recA/RAD
51
[105] and a putative SOS
repair system component (dinF homologue [106]) have
been identified in Pyrococcus, whereas nucleotide exci-
sion repair has been demonstrated in Methanobacterium
thermoautotrophicum extracts [107]. Uracil DNA glyco-
sylase activity (the repair enzyme for cytosine deamina-
tion) has also been detected in a number of
hyperthermophilic bacteria and archaea [108]. The im-
portance of DNA repair mechanisms in hyperther-
mophiles is reviewed in more detail by Grogan [82].
Lipids and membranes
The basic components of the thermophilic microbial cell
membrane, the membrane lipids, play a key role in
thermophily. The maintenance of membrane fluidity,
transport functions, intracellular solute concentrations,
chemiosmotic gradients and membrane protein stability
are but a few examples of the functions of these
molecules. Furthermore, the membrane composition
must adapt to fluctuations in environmental tempera-
ture in order to retain these functions while still possess-
ing the chemical stability necessary to avoid
degradation at high temperature (e.g. in Pyrodictium
abyssi, T
max
=110 °C) and by combinations of high
temperature and very low pH (e.g. Sulfolobus solfatari-
cus, T
max
=85 °C, pH
min
=1.5).
The molecular strategies employed by the two phyloge-
netically distinct groups of hyperthermophiles, the cren-
archaeota (archaea) and the thermotogales and
aquificales (bacteria) for maintaining stable and adapt-
able membranes are substantially different. In the ther-
mophilic archaea, more stable ether bonds replace the
ester linkages of bacterial and eukaryotic cells, and the
‘bilayer’ is replaced by a monolayer generated by C
40
transmembrane phytanyl chains. As ether linkages are
not restricted to the archaea, but are also found in some
thermophilic bacteria, the presence of these stable
chemical structures may be a true ‘thermophilic’ adap-
tation. For a detailed analysis of the composition of
archaeal membrane lipids, the reader is directed to
several recent reviews [109–111].
The hyperthermophilic bacteria show evidence of lipid
composition mimicking that in the archaea, possible
support for the contention that hyperthermophily
evolved independently in the different lineages [112].
The hyperthermophilic bacteria variously possess di-
ether links with fatty alcohols, tetraesters and long-
chain aliphatic diols. All are heavily decorated with a
wide variety of phospho-, glyco-, sulfoglyco-, sulfophos-
pho- and phosphoglyco-derivatives.
Lipids of the thermophilic archaea
The ‘core’ lipids of the archaea are largely based on
saturated isoprenoid chains linked to a glycerol back-
bone by ether bonds. Common structures include the
monomeric diphytanylglycerol ethers (fig. 3a) and the
dimeric dibiphytanyldiglycerol tetraethers and dibiphy-
tanyl glycerol nonitol tetraethers (fig. 3b). The former,
termed archaeols, are found in all archaea, whereas the
latter, termed caldarchaeols and nonitolcaldarchaeols,
are found only in the thermophilic archaea. The caldar-
chaeols and nonitolcaldarchaeols exhibit further modifi-
cation by containing up to four cyclopentane rings in
each of the C
40
biphytanyl chains (figs 3c– f). The addi-
tion of cyclic structures in the transmembrane portion
of the lipid appears to be a thermoadaptive response,
resulting in enhanced membrane packing and reduced
membrane fluidity [113]. However, not all hyperther-
mophilic archaea contain cyclopentane-modified dibi-
phytanyldiglycerol tetraethers. It is suggested, though
not proven, that in such organisms the polar head
groups contribute to membrane rigidity [114, 115].
With the publication of lipid analyses of an increasingly
diverse range of hyperthermophilic archaea, the diver-
sity of lipids is rapidly increasing with, for example, the
identification of fatty acids in the marine archaeon P.
furiosus, unsaturated diether lipids in Methanopyrus
kandleri [116], 36-membered macrocyclic diether lipids
in Methanococcus jannaschii (fig. 3g) [117] and te-
traether lipids containing a covalent cross-link at the
centre of the isoprenoid chains in Methanothermus fer-
6idus (fig. 3h) [118]. There is currently no suggestion
that any of these more exotic structures have a specific
role in thermophily.
The ether lipids of the thermophilic archaea are typi-
cally glycosylated at C
3
and C
6
of the glycerol and
nonitol backbones, respectively. It has been suggested
[119] that interglycosyl headgroup hydrogen-bonding
interactions further stabilise the membrane structure,
possibly by reducing lateral lipid mobility. The C
1
atom
of the ‘opposite’ glycerol backbone is phosphorylated,
typically with P-inositol, or P-ethanolamine. The orien-
tation of the phosphate groups to the inner face of the
archaeal membrane results in a high negative charge
density on the inner membrane surface. Without shield-
ing or charge compensation, this high anionic charge
density might be expected to destabilise the membrane.
Although nothing is known of the mechanisms prevent-
ing such putative destabilisation, the high intracellular
K
+
concentration of some hyperthermophiles may well
be implicated in preventing this destabilising effect.
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 259Review Article
Figure 3. Archaeal lipid architecture. (a) Diphytanyl glycerol diethers, (b) dibiphytanyl diglycerol tetraethers, (c–f) internal cyclisation
in dibiphytanyl diglycerol tetraethers, (g) macrocyclic diphytanyl glycerol diether, (h) internal covalent cross-linking in dibiphytanyl
diglycerol tetraether.
R. M. Daniel and D. A. Cowan Life at high temperatures260
Membrane stability
While few if any studies have been carried out on the
chemical stability of purified ether lipids, the stability of
artificial membranes (liposomes) has attracted consider-
able attention. Whether membrane fluidity is driven by
‘homeoviscous adaptation’ (maintenance of constant
membrane fluidity) [120] or ‘homeophasic adaptation’
(maintenance of a liquid-crystal state) [121], the mem-
branes of hyperthermophiles have clearly evolved mech-
anisms for maintaining a liquid crystal state at very
high temperatures. Thermoadaptative mechanisms in
bacteria include alterations in acyl chain length, satura-
tion, branching and/or cyclisation [122]. Only in ar-
chaea are membrane-spanning lipids employed.
Membranes composed of C
40
membrane spanning (bo-
laform amphiphilic) tetraether lipids maintain a con-
stant thickness of 2.5–3.0 nm [113], somewhat thinner
than typical C
18
phosphodiester bilayer membranes.
Nevertheless, these archaeal membranes are much more
physically stable than those formed from phosphodi-
esters. For example, large (600 nm) vesicles generated
from T. acidophilum ether lipids were found to be more
resistant to physical disruption by high temperature and
surface active agents such as phenol, alcohols and deter-
gents than dipalmitoyl phosphatidylcholine vesicles
[123]. In a more extensive study of liposome stability,
Sprott et al. [124] compared liposomes prepared from
the ether lipid extracts of a number of archaea with egg
phosphatidylcholine and dipalmitoyl phosphatidyl-
choline liposomes. In virtually all instances, the ar-
chaeal ester lipid liposomes showed higher levels of
stability to temperature, pH, serum proteins and long-
term oxidative effects than the ester lipid vesicles. Per-
haps not surprisingly, the former were also highly
resistant to the addition of phospholipases. The use of
fluorescent probe techniques to measure the thermal
stability of S. acidocaldarius lipid liposomes over a
temperature range of 25 –85 °C showed little variation
in proton permeability across the temperature range
[125]. The low permeability of these membranes was
attributed to steric hindrance by the methyl side groups
of the hydrophobic chains. X-ray analysis of Langmuir-
Blodgett films of S. solfataricus lipids showed decreas-
ing order with increasing temperature, but no loss of
periodic organisation at temperatures below 100 °C
[126].
The remarkable physical and chemical stability of ar-
chaeal ether lipid liposomes, attributed to the presence
of the ether linkage, to the intimate packing of the
phytanyl chains and to the reduced degree of molecular
translational freedom in a boliform structure, has pro-
vided a significant incentive to the practical utilisation
of such liposomes. A number of applications, including
drug delivery systems [124] and bioelectronics compo-
nents [127], have been proposed.
Conclusions
Since the discovery in the late 1960s of microorganisms
living at temperatures above 70 °C, numerous labora-
tories around the world have focussed specifically on
the biochemistry and physiology of life at high tempera-
tures. So where do we now stand in terms of our
understanding of the molecular and physiological basis
of high-temperature life processes and the factors which
dictate the upper limits? We must conclude that we still
lack a clear understanding of the mechanisms by which
thermophiles and hyperthermophiles address the issue
of biomolecule stability at high temperatures. Despite
research efforts spanning nearly 30 years and the huge
expansion of our understanding of high-temperature
microbial ecology, physiology, biochemistry and genet-
ics, there are many fundamental issues still to be re-
solved in this field.
One major difficulty is the shortage of data on
biomolecule stability above 100 °C. A second is that
most of the available data is, unsurprisingly, for in vitro
rather than in vivo conditions. Given the complexity of
intracellular conditions in terms of potentially stabilis-
ing environments and interactions, in vitro data cannot
be more than a very rough guide to the true stabilities
of molecules in vivo. We already know that several vital
biomolecules which are quite unstable in vitro at
100 °C are present and apparently participating in the
metabolism of organisms growing at 100–113 °C. This
observation underlines the necessity for caution in ap-
plying in vitro data to judgements on which factors
might limit life at high temperatures. Nevertheless, from
the data available it is difficult to see how protein or
lipid/membrane stability is likely to be a limiting factor
below, say, 140 °C. Indeed, the evidence to date sug-
gests that associations with such macromolecular struc-
tures may be a strategy for stabilising more
thermolabile molecules. The issue is much less clear-cut
in respect of nucleic acids and their polymers, but
mechanisms for stabilising these, including covalent
modification and association with proteins, do seem to
be available. For low molecular weight metabolites
there is potentially an even wider range of mechanisms
for stabilisation or circumventing instability. However,
the difficulty for the cell may lie in the range of func-
tions and properties of such molecules. A relatively
modest range of generic stabilising strategies (i.e. addi-
tional weak interactions, covalent modification, associa-
tion with other macromolecules) seems sufficient for
each of the major classes of macromolecules of the cell.
It is not obvious that the variety of stabilisation strate-
gies needed to deal with the wide range of chemistries
and functions of small molecules is sufficient to cope
with the effects of temperatures above 115 °C. How-
ever, this reservation may simply indicate how difficult
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 261Review Article
it is to avoid our inherent ‘mesocentric’ bias. Almost all
the biochemistry we know has been learnt from organ-
isms very well adapted to life at 20–40 °C.
There is still a strong tendency to see high temperature
as an obstacle to be overcome by thermophiles, rather
than as presenting advantages, such as faster (nonen-
zymic) reaction rates, lowered viscosity, faster diffusion
and raised solubilities. While it is obvious that some
intracellular components, essential in mesophiles, seem
to be unstable at 100 °C, it is not inconceivable that the
instability of these or other molecules is an advantage in
organisms evolved to exploit this. We should also re-
member that, from an organismal point of view, hyper-
thermophiles living at 100–110 °C are as well adapted
to their immediate environments as are mesophiles liv-
ing at 37 °C.
Finally, we stress that the question, ‘‘What molecules or
molecular factors will be responsible for establishing the
upper temperature limit of life?,’’ is still a very open
one. Not only are the in vitro data available to us very
uneven in coverage, but more seriously, the in vivo data
are entirely inadequate. To remedy the latter will re-
quire considerable experimental ingenuity and a better
knowledge of intracellular conditions than we currently
possess.
Acknowledgements. We thank the Royal Society of New Zealand
for the award of a James Cook Fellowship to R.M.D., and the
BBSRC and EU for research funding support to D.A.C.
1 Woese C. R. (1987) Bacterial evolution. Microbiol. Rev. 51:
221– 271
2 Pace N. R. (1997) A molecular view of microbial diversity
and the biosphere. Science 276: 734–740
3 Pennisi E. (1999) Is it time to uproot the tree of life? Science
284: 1305–1307
4 Kowalak J. A., Dalluge J. J., McCloskey J. A. and Stetter K.
O. (1994) The role of post-transcriptional modifications in
stabilization of transfer RNA from hyperthermophiles. Bio-
chemistry 33: 7869–7876
5 Daniel R. M. (1996) Primitive coenzymes and metabolites in
archaeal/thermophilic metabolic pathways. In: Ther-
mophiles: The Keys to Molecular Evolution and the Origin
of Life? pp. 299 – 310, Weigel J. and Adams M. W. W. (eds),
Taylor and Francis, London
6 Tetas M. and Lowenstein J. M. (1963) The effect of bivalent
metal ions on the hydrolysis of adenosine di- and triphos-
phate. Biochemistry 2: 350–357
7 Ramirez F., Marecek J. F. and Szamosi J. (1980) Magne-
sium and calcium ion effects on hydrolysis rates of
adenosine-5%-triphosphate. J. Org. Chem. 45: 4748–4752
8 Lowry O. H., Passonneau J. V. and Rock M. K. (1961) The
stability of pyridine nucleotides. J. Biol. Chem. 236: 2756–
2759
9 Walsh K. A. J., Daniel R. M. and Morgan H. W. (1983) A
soluble NADH dehydrogenase from Thermus aquaticus
strain T351. Biochem. J. 201: 427–433
10 Hudson R. D., Ruttersmith L. D. and Daniel R. M. (1993)
Glutamate dehydrogenase from the extremely thermophilic
achaebacterial isolate AN1. Biochim. Biophys. Acta 1202:
244– 250
11 Alhabori M. (1995) Microcompartmentation, metabolic
channelling and carbohydrate metabolism. Int. J. Biochem.
Cell Biol. 27: 123–132
12 Kholodenko B. M., Westerhoff H. V. and Cascante M.
(1996) Effect of channeling on the concentration of bulk-
phase intermediates as cytosolic proteins become more con-
centrated. Biochem. J. 313: 921–926
13 Mendes P., Kell D. B. and Westerhoff H. V. (1996) Why and
when channelling can decrease pool size at constant net flux
in a simple dynamic channel. Biochim. Biophys. Acta 1289:
175– 186
14 Kholodenko B. M., Sakamoto N., Puigjaner J., Westerhoff
H. V. and Cascante M. (1996) Strong control on the transit
time in metabolic channelling. FEBS Lett. 389: 123–125
15 Van de Casteele M., Demarez M., Legrain C., Glansdorff N.
and Pierard A. (1990) Pathways of arginine biosynthesis in
extremely thermophilic archaeo-and eubacteria. J. Gen. Mi-
crobiol. 136: 1177–1183
16 Van de Casteele M., Legrain C., Desmarez L., Chen P. G.,
Pierard A. and Glansdorf N. (1997) Molecular physiology of
carbamoylation under extreme conditions – What can we
learn from extreme thermophilic microorganisms. Comp.
Biochem. Physiol. 118: 463–473
17 Legrain C., Demarez M., Glansdorff N. and Pierard A.
(1995) Ammonia-dependent synthesis and metabolic chan-
nelling of carbamoyl phosphate in the hyperthermophilic
archaeon Pyrococcus furiosus. Microbiology 141: 1093–1099
18 Sterner R., Kleeman G. R., Szadkowski H., Lustig A.,
Hennig M. and Kirschner K. (1996) Phosphoribosyl an-
thranilate isomerase from Thermotoga maritima is an ex-
tremely stable and active homodimer. Protein Sci. 5:
2000– 2008
19 Daniel R. M. and Danson M. J. (1995) Did primitive
microrganisms use nonhaem iron proteins in place of NAD/
P? J. Mol. Evol. 40: 559–563
20 Adams M. W. W. (1993) Enzymes and proteins from organ-
isms that grow near and above 100 °C. Ann. Rev. Micro-
biol. 47: 627–658
21 Mukund S. and Adams M. W. W (1991) The novel tungsten-
iron-sulfur protein of the hyperthermophilic archaebac-
terium, Pyrococcus furiosus, is an aldehyde ferredoxin
oxidoreductase; evidence for its participation in a unique
glycolytic pathway. J. Biol. Chem. 266: 14208–14216
22 Kjems J., Larsen N., Dalgaard J. Z., Garrett R. A. and
Stetter K. O. (1992) Phylogenetic relationships among the
hyperthermophilic Archaea determined from partial 23S
rRNA gene sequences. Syst. Appl. Microbiol. 15: 203–208
23 DeRosa M., Gambacorta A., Nicholaus B., Giardina P.,
Paerio E. and Buonocore V. (1984) Glucose metabolism in
the extreme thermoacidophilic archaebacterium Sulfolobus
solfataricus. J. Biochem. 224: 407–414
24 Budgen N. and Danson M. J. (1986) Metabolism of glucose
via a modified Entner-Doudoroff pathway in the ther-
moacidophilic archaebacterium Thermoplasma acidophilum.
FEBS Lett. 196: 207–210
25 Kerscher L. and Oesterhelt D. (1982) Pyruvate ferridoxin
oxidoreductase – new findings on an ancient enzyme. Trends
Biochem. Sci. 7: 371–374
26 Iwasaki T., Wakagi T., Isogai Y., Tanaka K., Iizuka T. and
Oshima T. (1994) Functional and evolutionary implications
of a [3Fe-4s] cluster of the dicluster-type ferredoxin from the
thermoacidophilic archeon, Sulfolobus sp. strain 7. J. Biol.
Chem. 269: 29444–29450
27 Iwasakii T., Wakagi T. and Oshima T. (1995) Ferredoxin-de-
pendant redox system of a thermoacidophilic archeon, Sul-
folobus sp. strain 7. J. Biol. Chem. 270: 17878–17883
28 Johnson J. L., Rajagopalan K. V., Makund S. and Adams
M. W. W. (1993) Identification of molybdopterin as the
organic component of the tungsten cofactor in four enzymes
from hyperthermophilic archaea. J. Biol. Chem. 268: 4848–
4853
29 Heider J., Mai X. and Adams M. W. W. (1996) Characteri-
sation of 2-ketoisovalerate ferredoxin oxidoreductase, a new
R. M. Daniel and D. A. Cowan Life at high temperatures262
and reversible coenzyme A-dependant enzyme involved in
peptide fermentation by hyperthermophilic archaea. J. Bact.
178: 780–787
30 Mai X. and Adams M. W. W. (1994) Indolepyruvate ferre-
doxin oxidoreductase from the hyperthermophilic archaeon,
Pyrococcus furiosus, a new enzyme involved in peptide fer-
mentation. J. Biol. Chem. 269: 16726–16732
31 Smith E. T., Blamey J. M. and Adams M. W. W. (1994)
Pyruvate ferredoxin oxidoreductases of the hyperther-
mophilic archaeon Pyrococcus furiosus and the hyperther-
mophilic bacterium Thermotoga maritima have different
catalytic mechanisms. Biochemistry 33: 1008–1016
32 Schafer T., Selig M. and Schonheit P. (1993) Acetyl-CoA
synthetase (ADP-forming) in archaea, a novel enzyme in-
volved in acetate formation and ATP synthesis. Arch. Mi-
crobiol. 159: 72–83
33 Danson M. J. (1988) Archaebacteria, The comparative enzy-
mology of their central metabolic pathways. Adv. Microbial
Physiol. 29: 165–231
34 Kengan S. W. M., de Bok F. A. M., van Loo N. D.,
Dijkema C., Stams A. J. M. and de Vos W. M. (1994)
Evidence for the operation of a novel Emden-Meyerhof
pathway that involves ADP-dependent kinases during sugar
fermentation by Pyrococcus furiosus. J. Biol. Chem. 269:
17537– 17541
35 Siebers B. and Hensel R. (1993) Glucose catabolism of the
hyperthermophilic Archaeum Thermoproteus tenax. FEMS
Microbiol. Lett. 111: 1–8
36 Coolbear T., Daniel R. M. and Morgan H. W. (1992) The
enzymes from extreme thermophiles. Adv. Biochem. Eng.
Biotechnol. 45: 57–98
37 Ruttersmith L. D. and Daniel R. M. (1991) Thermostable
cellobiohydrolase from the thermophilic eubacterium Ther-
motoga sp. Strain FjSS3-B1. Biochem. J. 277: 887–890
38 Ruttersmith L. D. and Daniel R. M. (1993) Thermostable
b-glucosidase and b-xylosidase from Thermotoga sp. Strain
FjSS3-B1. Biochim. Biophys. Acta 1156: 167–172
39 Simpson H. D., Hanfler U. R. and Daniel R. M. (1991) An
extremely stable xylanase from the thermophilic eubacterium
Thermotoga. Biochem. J. 277: 413–417
40 Sobek H. and Gorisch H. (1988) Purification and character-
isation of a heat-stable esterase from the thermoacidophilic
archaebacterium Sulfolobus acidocaldarius. Biochem J. 250:
453– 458
41 Bryant F. O. and Adams M. W. W. (1989) Characterisation
of hydrogenase from the hyperthermophilic archaebacterium
Pyrococcus furiosus. J. Biol. Chem. 264: 5070–5079
42 Koch R., Zablowski P., Spreinat A. and Antranikian G.
(1990) Extremely thermostable amylolytic enzyme from the
archaebacterium Pyrococcus furiosus. FEMS Microbiol. Rev.
71: 21–26
43 Prangshivilli D., Zillig W., Gievl A., Biesert L. and Holz I.
(1982) DNA dependant RNA polymerases of ther-
moacidophilic archaebacteria. Eur. J. Biochem. 122: 471 –
477
44 Russell R. J. M., Gerike U., Danson M. J., Hough D. W.
and Taylor G. L. (1998) Structural adaptation of the cold
active citrate synthase from an Antarctic bacterium. Struc-
ture 6: 351–361
45 Blake P. R., Park J. B., Bryant F. O., Aono S., Magnuson J.
K., Eccleston E. et al. (1991) Determinants of protein hyper-
thermostability. 1. Purification, amino acid sequence and
secondary structure from NMR of the rubredoxin from the
hyperthermophilic archaebacterium, Pyrococcus furiosus.
Biochemistry 30: 10885–10891
46 Auerbach G., Ostendorp R., Prade L., Korndorfer I., Dams
T., Huber R. et al. (1998) Lactate dehydrogenase from the
hyperthermophilic bacterium Thermotoga maritima. Struc-
ture 6: 769–781
47 Daniel R. M., Dines M. and Petach H. H. (1996) The
denaturation and degradation of stable enzymes at high
temperatures. Biochem. J. 317: 1–11
48 Jaenicke R. and Bohm G. (1998) The stability of proteins in
extreme environments. Curr. Opin. Struct. Biol. 8: 738 – 748
49 Brandts J. F. (1967) Heat effects on proteins and enzymes.
In: Thermobiology, pp. 25–72, Rose A. H. (ed.), Academic
Press, New York
50 Langridge J. (1968) Genetic and enzymatic experiments re-
lating to the tertiary structure of beta-galactosidase. J. Bacte-
riol 96: 1711–1717
51 Matthews B. W. (1995) Studies on protein stability with T4
lysozyme. Adv. Protein Chem. 46: 249–278
52 Grutter M. G., Hawkes R. B. and Matthews B. W. (1979)
Molecular basis of thermostability in the lysozyme from
bacteriophage T4. Nature 277: 667–669
53 Creighton T. E. (1993) Proteins, 2nd edn, W. H. Freeman,
New York
54 Finney J. L. (1996) Hydration processes in biological and
macromolecular systems. Faraday Discuss. 103: 1–18
55 Daniel R. M., Bragger J. and Morgan H. W. (1990) En-
zymes from extreme environments. In: Biocatalysis, pp. 243 –
254, Abramowicz D. A. (ed.), Van Nostrand Reinhold, New
York
56 Wrba A., Schweiger A., Schultes V., Jaenicke R. and Zavod-
szky P. (1990) Extremely thermostable D-glyceraldehyde-3-
phosphate dehydrogenase from the eubacterium Thermotoga
maritima. Biochemistry 29: 7584–7592
57 Varley P. G. and Pain R. H. (1991) Relationship between
stability, dynamics and enzyme activity in 3-phosphoglycer-
ate kinases from yeast and Thermus thermophilus. J. Mol.
Biol. 220: 531–538
58 Shoichet B. K., Baase W. A., Kuroki R. and Matthews B.
W. (1995) A relationship between protein stability and
protein function. Proc. Natl. Acad. Sci. USA 92: 452–456
59 Zavodsky P., Kardos J., Svingor A. and Petsko G. A. (1998)
Adjustment of conformational flexibility is a key event in the
thermal adaptation of proteins. Proc. Natl. Acad. Sci. USA
95: 7406–7411
60 Daniel R. M., Cowan D. A., Morgan H. W. and Curran M.
P. (1982) A correlation between protein thermostability and
resistance to proteolysis. Biochem. J. 207: 641–644
61 Daniel R. M., Smith J. C., Ferrand M., Hery S., Dunn R.
and Finney J. L. (1998) Enzyme activity below the dynami-
cal transition at 200K. Biophys. J. 75: 2504–2507
62 Daniel R. M., Finney J. L., Reat V., Dunn R., Ferrand M.
and Smith J. C. (1999) Enzyme dynamics and activity:
timescale dependence of dynamical transitions in glutamate
dehydrogenase solution. Biophys. J. 77: 2184–2190
63 Ahern T. J. and Klibanov A. M. (1985) The mechanism of
irreversible enzyme inactivation at 100 °C. Science 228:
1280– 1283
64 Zale S. E. and Kilbanov A. M. (1986) Why does ribonucle-
ase irreversibly inactivate at high temperatures? Biochemistry
25: 5432–5444
65 Clarke S. (1987) Propensity for spontaneous succinimide
formation from aspartyl and asparaginyl residues in cellular
proteins. Peptide Protein Res. 30: 808–821
66 Brennan T. V., Anderson J. W., Jua Z., Waygood E. B. and
Clarke S. (1994) Repair of spontaneously deamidated HPR
phosphocarrier protein catalysed by the L-isoaspartate-(D-
aspartate) O-methyltransferase. J. Biol. Chem. 269: 24586–
24595
67 Wearne S. J. and Creighton T. E. (1989) Effect of protein
conformation on rate of deamidation: Ribonuclease A.
Proteins Struct. Funct. Genet. 5: 8–12
68 Hensel R., Jakob I., Scheer H. and Lottspeich R. (1992)
Proteins from hyperthermophilic archaea: stability towards
covalent modification of the polypeptide chain. Biochem.
Soc. Symp. 58: 127–133
69 Marmur J. and Doty P. (1962) Determination of the base
composition of deoxyribonucleic acid from its thermal de-
naturation temperature. J. Mol. Biol. 5: 109–118
70 Scholz S., Sonnenbichler J., Schafer W. and Hensel R. (1992)
Di-myo-inositol-1,1%-phosphate: a new inositol phosphate
isolated from Pyrococcus woesei. FEBS Lett. 306: 239–242
CMLS, Cell. Mol. Life Sci. Vol. 57, 2000 263Review Article
71 Hensel R. and Konig H. (1988) Thermoadaptation of
methanogenic bacteria by intracellular ion concentration.
FEMS Microbiol. Lett. 49: 75–79
72 Friedman S. M. (1986) Properties of the translational ma-
chinery from a sulfur-dependent archaebacterium. System.
Appl. Microbiol. 7: 325–329
73 Friedman S. M. and Oshima T. (1989) Polyamines of sulfur-
dependent archaebacteria and their role in protein synthesis.
J. Biochem. 105: 1030–1033
74 Kneifel H., Stetter K. O., Andreesen J. R., Weigel J., Ko¨nig
H. and Schoberth S. M. (1986) Distribution of polyamines in
representative species of archaebacteria. System. Appl. Mi-
crobiol. 7: 241–245
75 Kurr M., Huber R., Ko¨nig H., Jannasch H. W., Fricke H.,
Trincone A. et al. (1991) Methanopyrus kandleri gen. nov.
and sp. nov. represents a novel group of hyperthermophilic
methanogens growing at 110 °C. Arch. Microbiol. 156:
239– 247
76 Huber R., Kristjansson J. K. and Stetter K. O. (1987)
Pyrobaculum gen. nov., a new genus of neutrophilic, rod-
shaped archaebacteria from continental solfataras growing
optimally at 100 °C. Arch. Microbiol. 149: 95 – 101
77 Erauso G., Reysenbach A. L., Godfroy A., Meunier J.-R.,
Crump B., Partensky F. et al. (1993) Pyrococcus abyssi sp.
nov., a new hyperthermophilic archaeon isolated from a
deep-sea hydrothermal vent. Arch. Microbiol. 160: 338 – 349
78 Fiala G. and Stetter K. O. (1986) Pyrococcus furiosus sp.
nov. represents a novel genus of marine heterotrophic ar-
chaebacteria growing optimally at 100 °C. Arch. Microbiol.
156: 56–61
79 Pley U., Schipka J., Gambacorta A., Jannasch H. W., Fricke
H., Rachel R. et al. (1991) Pyrodictium abyssi sp. nov.
represents a novel heterotrophic marine archaeal hyperther-
mophile growing at 110 °C. System. Appl. Microbiol. 14:
245– 253
80 Stetter K. O., Konig H. and Stackebrandt E. (1983) Pyrodic-
tium gen. nov., a new genus of submarine disc-shaped sulfur
reducing archaebacteria growing optimally at 105 °C. Sys-
tem. Appl. Microbiol. 4: 535–551
81 Blo¨chl E., Rachel R., Burggraf S., Hafenbradl D., Jannasch
H. W. and Stetter K. O. (1997) Pyrolobus fumarii, gen. and
sp. nov., represents a novel group of archaea, extending the
upper temperature limit for life to 113 degrees C. Ex-
tremophiles 1: 14–21
82 Grogan D.W. (1998) Hyperthermophiles and the problem of
DNA stability. Mol. Microbiol. 28: 1043–1049
83 Kikuchi A. and Asai K. (1984) Reverse gyrase – a topoiso-
merase which introduces positive superhelical turns into
DNA. Nature 309: 677–681
84 De la Tour C. B., Portemer C., Nadal M., Stetter K. O.,
Forterre P. and Duguet M. (1990) Reverse gyrase, a hall-
mark of the hyperthermophilic archaebacteria. J. Bacteriol.
172: 6803–6808
85 Guipaud O., Marguet E., Knoll K. M., Bouthier de la Tour
C. and Forterre P. (1997) Both DNA gyrase and reverse
gyrase are present in the hyperthermophilic bacterium Ther-
motoga maritima. Proc. Natl. Acad. Sci. USA 94: 10606–
10611
86 Lopez-Garcia P. (1996) DNA topoisomerases, temperature
adaptation and early diversification of life. In: Thermophiles:
The Keys to Molecular Evolution and the Origin of Life? pp.
201– 216, Wiegel J. and Adams M. W. W. (eds), Taylor and
Francis, Philadelphia
87 Marguet E. and Forterre P. (1994) DNA stability at temper-
atures typical for hyperthermophiles. Nucleic Acids Res. 22:
1681– 1686
88 Slesarev A. I., Zaitev D. A., Zopylov V. M., Stetter K. O.
and Kozyvakin S. A. (1991) DNA topoisomerase-III from
extremely thermophilic archaebacteria: ATP-independent
type-I topoisomerase from Desulfurococcus amylolyticus
drives extensive unwinding of closed circular DNA at high
temperature. J. Biol. Chem. 266: 12321–12328
89 Sandman K., Krzycki J. A., Dobrinski B., Lurz R. and
Reeve J. N. (1990) HMf, a DNA-binding protein isolated
from the hyperthermophilic archaeon Methanothermus fer-
6idus, is most closely related to histones. Proc. Natl. Acad.
Sci. USA 87: 5788–5791
90 Tabassum R., Sandman K. M. and Reeve J. N. (1992) HMt,
A histone-related protein from Methanobacterium thermoau-
totrophicum Delta-H. J. Bacteriol. 174: 7890–7895
91 Ronimus R. S. and Musgrave D. R. (1996) Purification and
characterization of a histone-like protein from the Archaeal
isolate AN1, a member of the Thermococcales. Molecular
Microbiol. 20: 77–86
92 Kozyavkin S. A., Krah R., Gellert M., Stetter K. O., Lake J.
A. and Slesarev A. I. (1994) A reverse gyrase with an
unusual structure – a type-I DNA topoisomerase from the
hyperthermophile Methanopyrus kandleri is a 2-subunit
protein. J. Biol. Chem. 269: 11081–11089
93 Sandman K., Grayling R. A., Dobrinski B., Lurz R. and
Reeve J. N. (1994) Growth-phase-dependent synthesis of
histones in the archaeon Methanothermus fer6idus. Proc.
Natl. Acad. Sci. USA 917: 12624–12628
94 Grote M., Dijk J. and Reinhardt R. (1986) Ribosomal and
DNA-binding proteins of the thermoacidophilic archaebac-
terium Sulfolobus acidocaldarius. Biochim. Biophys. Acta
873: 405–413
95 Kim C. W., Markiewicz P., Lee J. J., Schierle C. F. and
Miller J. H. (1993) Studies of the hyperthermophile Thermo-
toga maritima by random sequencing of cDNA and genomic
libraries – identification and sequencing of the TrpEg (D)
operon. J. Mol. Biol. 231: 960–981
96 Pereira S. L., Grayling R. A., Luyrz R. and Reeve J.N.
(1997) Archaeal nucleosomes. Proc. Natl. Acad. Sci. USA
94: 12633–12637
97 Grayling R. A., Sandman K. and Reeve J. N. (1996) DNA
stability and DNA binding proteins. In: Advances in Protein
Chemistry, vol. 48, pp. 437–467, Adams M. W. W. (ed.)
Academic Press, San Diego
98 Soares D., Dahlke I., Li W.-T., Sandman K., Hethke C.,
Thomm M. et al. (1998) Archaeal histone stability, DNA
binding and transcription inhibition above 90 °C. Ex-
tremophiles 2: 75–81
99 Sandman K. and Reeve J. N. (1999) Archaeal nucleosome
positioning by CTG repeats. J. Bacteriol. 181: 1035–1038
100 Green G. R., Searcy D. G. and DeLange R. J. (1983)
Histone-like protein in the archaebacterium Sulfolobus
acidocaldarius. Biochim. Biophys. Acta 741: 251–257
101 Pereira S. L. and Reeve J. N. (1998) Histones and nu-
cleosomes in Archaea and Eukarya: a comparative analysis.
Extremophiles 2: 141–148
102 Edmonds C. G., Crain P. F., Gupta R., Hashizume T.,
Hocart C. H., Kowalak J. A. et al. (1991) Posttranscrip-
tional modification of tRNA in thermophilic archaea (ar-
chaebacteria). J. Bacteriol. 173: 3138–3148
103 Noon K. R., Bruenger E. and McCloskey J. A. (1998)
Posttranscriptional modifications in 16S and 23S rRNAs of
the archaeal hyperthermophile Sulfolobus solfataricus.J.
Bacteriol. 180: 2883–2888
104 Lindahl T. (1993) Instability and decay of the primary
structure of DNA. Nature 362: 709–715
105 Rashid N., Morikawa M., Nagahisa K., Kanaya S. and
Imanaka T. (1997) Characterisation of a RecA/RAD51 ho-
mologue from the hyperthermophilic archaeon Pyrococcus
sp. Nucleic Acids Res. 25: 719–726
106 Bouyoub A., Barbier G., Querellou J. and Forterre P. (1995)
A putative SOS repair gene (dinF-like) in a hyperther-
mophilic archaeon. Gene 167: 147–149
107 O
8
ru¨nc¸ M., Becker D. F., Ragsdale S. W. and Sancar A.
(1998) Nucleotide excision repair in the third kingdom. J.
Bacteriol. 180: 5796–5798
108 Koulis A., Cowan D. A., Pearl L. H. and Savva R. (1996)
Uracil DNA glycosylase activities in hyperthermophilic Ar-
chaea and Bacteria. FEMS Microbiol. Lett. 143: 267–271
R. M. Daniel and D. A. Cowan Life at high temperatures264
109 De Rosa M., Gambacorta A. and Gliozzi A. (1986) Struc-
ture, biosynthesis and physical properties of archaebacterial
lipids. Microbiol. Rev. 50: 70–80
110 Langworthy T. A. and Pond J. L. (1986) In: Thermophiles:
General Molecular and Applied Microbiology, pp. 107–134
Brock T. D. (ed.) Wiley-Interscience, New York
111 Kates M. (1993) Membrane lipids of archaea. In: The Bio-
chemistry of Archaea (Archaebacteria), pp. 261–296, Kates
M., Kushner D. J. and Matheson A. T. (eds), Elsevier,
Amsterdam
112 Forterre P. (1998) Were our ancestors actually hyperther-
mophiles? Viewpoint of a devil’s advocate. In: Thermophiles:
The Keys to Molecular Evolution and the Origins of Life,
pp. 137–146, Weigel J. and Adams M. W. W. (eds), Taylor
and Francis, London
113 Gliozi A, Paoli G., De Rosa M. and Gambacorta A. (1983)
Effect of isoprenoid cyclization on the transition tempera-
ture of lipids in thermophilic archaebacteria. Biochim. Bio-
phys. Acta 735: 234–242
114 Lattuati A., Guezennec J., Metzger P. and Largeau C. (1998)
Lipids of Thermococcus hydrothermalis, an archaean isolated
from a deep-sea hydrothermal vent. Lipids 33: 319–326
115 van de Vossenburg J. L. C. M., Driessen A. J. M., Zillig W.
and Konings W. N. (1998) Bioenergetics and cytoplasmic
membrane stability of the extremely acidophilic, ther-
mophilic archaeon Picrophilus oshimae. Extremophiles 2:
67–74
116 Hafenbradl D., Keller M. and Stetter K. O. (1993) Lipid
analysis of Methanopyrus kandleri. FEMS Microbiol. Lett.
136: 199–202
117 Arakawa K., Eguchi T. and Kakinuma K. (1998) Tightly
packed membranes composed of 36-membered macrocyclic
diether phospholipids found in archaea growing under deep-
sea hydrothermal vents. Chem. Lett. P: 901–902
118 Morii H., Eguchi T., Nishihara M., Kakinuma K., Konig H.
and Koga Y. (1998) A novel ether core lipid with H-shaped
C
80
-isoprenoid hydrocarbon chain from the hyperther-
mophilic methanogen Methanothermus fer6idus. Biochim.
Biophys. Acta 1390: 339–345
119 van de Vossenberg J. L. C. M., Dreissen A. J. M. and
Konings W. M. (1998) The essence of being extremophilic:
the role of the unique archaeal membrane lipids. Ex-
tremophiles 2: 163–170
120 Sinensky M. (1974) Homeoviscous adaptation – a homeo-
static process that regulates the viscosity of membrane lipids
in Escherichia coli. Proc. Natl. Acad. Sci. USA 71: 522– 525
121 McElhaney R. (1989) The influence of membrane lipid com-
position and physical properties of membrane structure and
function in Acholeplasma laidlawii. Crit. Rev. Microbiol. 17:
1–32
122 Tolner B., Poolman B. and Konings W. N. (1998) Adapta-
tion of micro-organisms and their transport systems to high
temperatures. Comp. Biochem. Physiol. 118A: 423–428
123 Ring K., Henkel B., Valenteijn A. and Guterman R. (1986)
Studies on the permeability and stability of liposomes
derived from a membrane spanning bipolar archaebacterial
tetraetherlipid. In: Liposomes as drug carriers, pp. 101 –
2123, Schmitd K. H. (ed.), Thieme, Stuttgart
124 Choquet C. G., Patel G. B., Beveridge T. J. and Sprott G.
D. (1974) Stability of pressure-extruded liposomes made
from archaeobacterial ether lipids. Appl. Microbiol. Biotech-
nol. 42: 375–384
125 Komatsu H. and Chong P. L.-G. (1998) Low permeability
of liposomal membranes composed of bipolar tetraether
lipids from thermoacidophilic archaebacterium Sulfolobus
acidocaldarius. Biochemistry 37: 107–115
126 Dante S., Maccioni E., Rustichelli F., Accossato P. and
Nicolini C. (1995) Langmuir-Blodgett films of bipolar lipids
from thermophilic archaea. Mater. Sci. Engin. C. 3: 13–21
127 De Rosa M., Morana A., Riccio A., Gambacorta A., Trin-
cone A. and Incani O. (1994) Lipids of the Archaea: a new
tool for bioelectronics. Biosens. Bioelectron. 9: 669–675
.
