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Research in Microbiology 161 (2010) 506e514
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Exploring research frontiers in microbiology: recent advances in halophilic
and thermophilic extremophiles
Beate Averhoff, Volker Müller*
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438,
Frankfurt am Main, Germany
Received 25 March 2010; accepted 11 May 2010
Available online 31 May 2010
Abstract
Extremophilic prokaryotes inhabit ecosystems that are, from a human perspective, extreme, and life in these environments requires farreaching cellular adaptations. Here, we will describe, for two examples (Thermus thermophilus, Halobacillus halophilus), how thermophilic or
halophilic bacteria adapt to their environment; we will describe the molecular basis of sensing and responding to hypersalinity and we will
analyze the impact and basis of natural competence for survival in hot environments.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Compatible solutes; DNA transport; Extremophiles; Halophiles; Natural competence; Thermophiles
1. Introduction
Extremophilic prokaryotes are characterized by inhabiting
ecosystems that are, from a human perspective, extreme. Such
environments may have extremely high or low pH, high or low
temperatures, high salinity, high pressure and various combinations thereof. Extremophilic microorganisms include
members of all three domains of life, the Archaea, Bacteria and
Eukarya. Often, these microbes are not only challenged by one
extreme, but multiple, and thus they are “polyextremophile”
(Mesbah and Wiegel, 2008). Examples would be life at hot
alkaline springs or hypersaline and alkaline lakes or hot and
acidic springs. Ever since extremophiles were discovered, their
physiology and their adaptation to the unhostile environment
have attracted much interest. This was not only because of the
interest in their lifestyle, but also for exploring their biotechnological potential. The enzyme Taq polymerase is a prime
example of an enzyme from an extremophile, Thermus aquaticus, isolated in 1969 by Thomas D. Brock and Hudson Freeze
* Corresponding author. Tel.: þ49 69 79829509/507; fax: þ49 69 79829306.
E-mail addresses: [email protected] (B. Averhoff), vmueller@
bio.uni-frankfurt.de (V. Müller).
0923-2508/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2010.05.006
from thermal springs in Yellowstone National Park (Brock and
Freeze, 1969). This enzyme has made the polymerase chain
reaction possible on a large, automated scale. With this enzyme,
sequencing genomes was made possible and it gave rise to the
thousands of genomes that we have today, ranging from nanoarchaea to humans.
The community of extremophilic researchers is rather large.
There are conferences that deal only with extremophiles; there
is an “International Society for Extremophiles”, and there is
even a journal named “Extremophiles”, founded by Koki
Horikoshi and now led by Garabed Antranikian. Research on
extremophiles has been plentiful over the last few decades and
is as diverse as the organisms. It started with the isolation of
extremophiles and continued on by exploring their biochemistry, then into their genetics and regulation of their metabolism. A couple of recent excellent books cover the entire
spectrum of extremophiles and describe their lifestyle (Gerday
and Glansdorff, 2007; Garrett and Klenk, 2007; Oren, 2002).
Considering the wealth of information on extremophiles, on
the one hand, and the limitation in space for this review, on the
other, we will restrict ourselves to two examples and apologize
to the readers and the community for not being able to cover
the entire spectrum.
B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
2. The molecular basis of salt adaptation in halophiles
Every living cell is challenged by changing water activities
in its ecosystem; thus, constant monitoring and adapting to
changing water activities is a prerequisite for life. This is of
particular importance for (moderate) halophiles. The biggest
challenge is to adjust the turgor and living cells have developed two principal strategies to re-establish turgor pressure
and to circumvent the detrimental consequences of water loss
when exposed to increasing osmolality. On the one hand, there
is the “salt-in-cytoplasm”-strategy, which means that inorganic
ions, mainly Kþ and Cl, accumulate in the cytoplasm until
the internal salt concentration is similar to the extracellular
one. This strategy is found in extremely halophilic Halobacteria (Archaea) and halophilic, anaerobic Haloanaerobiales
(Bacteria) (Galinski and Trüper, 1994; Ventosa et al., 1998).
On the other hand, the vast majority of prokaryotes cope with
increasing osmolarity by uptake or synthesis of compatible
solutes, which are defined as small, highly soluble, organic
molecules which do not interfere with the central metabolism,
even if they accumulate at high concentrations (Brown, 1976).
This strategy is widespread and evolutionarily well conserved
in all three domains of life (Bohnert, 1995; Kempf and
Bremer, 1998; Roeßler and Müller, 2001b; Saum and
Müller, 2008a). However, the spectrum of compatible solutes
used comprises only a limited number of compounds and these
can be divided into 2 major groups: 1) sugars and polyols; and
2) a- and b-amino acids and their derivatives, including
methylamines. This limitation to a rather small number of
compounds reflects the fundamental constraints on solutes
which are compatible with macromolecular and cellular
functions (Le Rudulier et al., 1984). Most archaeal compatible
solutes resemble in structure their bacterial counterpart, with
the difference that the majority of them carry a negative charge
(Martin et al., 1999; Roeßler and Müller, 2001b).
The uptake and biosynthesis of compatible solutes is
induced by high salinity or high osmolarity on both the DNA
and protein level. The pathways for the biosynthesis of various
solutes have been identified in different bacteria and archaea,
but how the environmental signal “salinity” is sensed and how
this signal is transmitted to various output modules at the level
of gene, enzyme or transporter activation is completely
obscure (Wood et al., 2001). This is even more important if
one considers that the overall cellular response of cells to
hypersalinity is not only the accumulation of solutes but
a reprogramming of cellular metabolism and structure in
general. The first step towards unraveling the complexity of
regulation could be by genome-wide expression profiling
studies, but unfortunately, these have not been done with
(moderate) halophiles. However, there are a few studies
available on halotolerant organisms that actually demonstrate
their complexity (Weber and Jung, 2002; Steil et al., 2003;
Pflüger et al., 2007). The methanogenic archaeon Methanosarcina mazei is a non-halophilic methanogen that can
adapt to 800 mM NaCl (Roeßler and Müller, 2002). We have
used microarray studies to examine the effect of elevated
salinities on the regulation of gene expression in M. mazei on
507
a genome-wide scale (Pflüger et al., 2007). 84 genes of
different functional categories, such as solute transport and
biosynthesis, Naþ export, stress response, ion, protein and
phosphate transport, metabolic enzymes, regulatory proteins,
DNA modification systems and cell surface modulators were
found to be more strongly expressed at high salinities.
Moreover, 10 genes encoding different metabolic functions
including potassium uptake and ATP synthesis were reduced
in expression under high salt. The overall expression profiles
suggest that M. mazei is able to adapt to high salinities by
multiple upregulation of many different cellular functions,
including protective pathways such as solute transport and
biosynthesis, import of phosphate, export of Naþ, and upregulation of pathways for modification of DNA and cell surface
architecture. Taking this (and other studies) into account, it is
obvious that a fine-tuned response to changing salinities
requires an elaborate regulatory network that, in addition, also
has to talk to other networks. Again, analyses of these
networks have only begun recently and we will describe one
that we discovered in the moderately halophilic bacterium
Halobacillus halophilus.
2.1. The chloride regulon in the moderate halophile
H. halophilus
The moderately halophilic bacteria are a specialized group
of organisms that require NaCl for growth (Ventosa et al.,
1998). They grow at nearly the same rate over a rather wide
range of external salt concentrations (0.5e2.5 M) which is
evidence for effective mechanisms to cope with changing
external salinities. The aerobic, endospore-forming, Grampositive bacterium H. halophilus has become a model system
to study salt adaptation at various cellular levels (Müller and
Saum, 2005).
H. halophilus is the first organism for which a strict chloride dependence of growth was described (Claus et al., 1983;
Roeßler and Müller, 1998). No growth was observed at a Cl
concentration of 0.2 M, but addition of chloride (to a medium
with constant osmolarity) restored growth in a concentrationdependent manner. Optimal growth occurs at 0.8e1.0 M Cl.
Moreover, not only growth rates but also cell yields (final
optical densities) were strictly chloride-dependent (Roeßler
and Müller, 1998).
What looked like an exotic phenotype at a first glance was
analyzed in more detail. To understand the function of chloride
it was important to figure out physiological processes that are
chloride-dependent. In addition to growth, germination of
endospores as well as flagella production and motility were
identified to be chloride-dependent (Dohrmann and Müller,
1999; Roeßler et al., 2000) and the very different functions of
Cl (motility, flagellation, spore germination, growth) indicate
a role of the anion in gene or protein activation. To test this and
identify proteins regulated by Cl, two different lines of
experiments were performed. First, a (very limited) proteome
analyses revealed five proteins upregulated in a chloridedependent fashion (Roeßler and Müller, 2002). For some of
these, chloride-dependent induction has been verified by other
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B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
means. For example, western blot analyses revealed that the
production of the structural component of the flagellum,
flagellin, was impaired in the absence of Cl (Roeßler and
Müller, 2002). However, upon addition of Cl the cellular
flagellin pool increased in a concentration-dependent manner.
Optimal flagellin production was achieved at 0.8e1.0 M Cl.
The same was observed for other proteins such as LuxS,
a component of the quorum sensing system (Sewald et al.,
2007). It plays a role in the biosynthesis of autoinducers of
the furanone family that are found in Gram-negative as well as
in Gram-positive bacteria.
Second, the effect of Cl in gene transcription was analyzed.
To test whether transcription of fliC, the gene encoding the
flagellum, was also chloride-dependent, it was cloned and
sequenced. Subsequent northern blot and RT-PCR analyses
with RNA from cells grown at different chloride concentrations
unequivocally demonstrated chloride-stimulated expression of
fliC (Roeßler and Müller, 2002). In summary, these experiments
demonstrated that Cl influences the cellular flagellin pool by
acting at both the transcriptional and translational level, but the
effect on translation was much more pronounced. This was the
first time that Cl dependence of gene expression and protein
production was shown in any prokaryote.
The fact that so many different physiological processes and
so many different proteins and genes are activated by Cl points
to a global Cl regulon active in H. halophilus (Saum and
Müller, 2008b). What could be the function of the Cl-dependent regulatory network? At least one function must be essential to growth, since growth of H. halophilus is strictly Cldependent. One has to keep in mind that one essential function
of moderate halophiles is to sense external salinity and to
respond to it on a transcriptional, translational and enzyme
activity level to adjust the intracellular pool size of the
compatible solutes. H. halophilus apparently uses a mixture of
the “salt-in-cytoplasm” and “compatible solute” strategy in
osmoadaptation (Saum and Müller, 2008b). It was shown that
the intracellular Cl concentration (Cl
i ) increases with the
)
(Roeßler
and Müller, 1998).
external Cl concentration (Cl
e
concentrations,
Cl
is
ten
times lower than
At suboptimal Cl
e
i
,
at
0.5
M
Cl
the
Cl
concentration
was
0.08
M, and in the
Cl
e
e
i
range of 0.8e2.0 M the Cl
/Cl
gradient
decreased
to a nearly
e
i
constant value of 1.5e2. In other words, the internal Cl
concentration at an external salinity of 2 M NaCl is 1 M. The
counter-ion has not yet been determined but is likely to be Kþ.
H. halophilus, in addition, accumulates compatible solutes
by uptake or biosynthesis. A very early and very important
observation was that the uptake of glycine betaine was strictly
dependent on the Cl concentration (Roeßler and Müller,
2001a). These experiments clearly revealed the first Cldependent osmolyte transporter in prokaryotes. Furthermore,
these experiments corroborate the idea that the essential function of the chloride regulon is to sense external salt and to
induce/activate systems involved in accumulation of compatible
solutes. Unfortunately, the transporter(s) catalyzing Cldependent glycine betaine transport have not been identified yet.
H. halophilus synthesizes different solutes such as glycine
betaine (from choline), glutamate, glutamine, proline and
ectoine, and their biosynthetic routes and regulation have been
identified (Saum et al., 2006; Saum and Müller, 2007a,b;
Burkhardt et al., 2009). Based on our studies, the following
model for long-term salt adaptation was proposed (Saum and
Müller, 2008b). Challenged by low extracellular water activity
caused by elevated NaCl concentrations, H. halophilus maintains a rather high internal Cl concentration in the molar
range, but additionally accumulates compatible solutes. At
intermediate salinities (1.0e1.5 M NaCl), glutamate and
glutamine are the main solutes and accumulate in response to
external salinity. Upon further increase in salinity, cells have
an interesting phenotype: the intracellular glutamine and
glutamate pools are not further increased, but the cells switch
to proline as the dominant compatible solute (at 2.0e3.0 M
NaCl).
The regulation of glutamate/glutamine and proline
biosynthesis was addressed on a molecular level. H. halophilus has two isogenes each for a glutamate dehydrogenase,
glutamate synthase and glutamine synthetase. Among these,
only glutamine synthetase 2 was regulated by salinity, but also
and more importantly, by the chloride concentration (Saum
et al., 2006). As discussed above, the effect was much more
pronounced at the enzyme actvity level than at the gene level.
Apparently, the first step of the cells is to sense salinity of the
environment by measuring the chloride concentration that then
triggers synthesis of glutamine and glutamate via glutamate
synthetase 2. As soon as the glutamate concentration reaches
a value of around 0.2 M in the cells, glutamate induces
expression of the proline biosynthesis operon and the switch
from glutamine/glutamate to proline is initiated. The molecular basis of the glutamate-induced switch to proline production remains to be established.
Therefore, the most prominent task of the chloride regulon
in H. halophilus is to measure the chloride concentration and
activate biosynthesis of compatible solutes, a prerequisite for
life at elevated salinities. The chloride regulon not only
regulates solute uptake and synthesis, but also spore germination, flagellum synthesis and quorum sensing and possibly
much more, such as, for example, respiration or cell wall
composition (Fig. 1). Our model for the first time provides an
explanation of how the different players in the cell are
orchestrated to give the synchronized output “cellular adaptation to changing salinities”: by the action of the chloride
regulon! Whether this is of general importance in prokaryotes
remains to be addressed in the future, but it should be noted in
this context that a number of bacteria were shown to require
chloride for growth at elevated salinities (Müller and Oren,
2003; Roessler et al., 2003).
3. The role of horizontal DNA transfer in adaptation to
extreme environments
Microorganisms are able to exploit very different, often
extreme environments and therefore have evolved phenotypic
traits allowing adaptation and survival under extreme environmental conditions. This microbial adaptation and diversification can be achieved by gene mutations, differential gene
B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
509
Fig. 1. The chloride regulon of H. halophilus. A summary of physiological processes, genes and enzymes/proteins that depend on Cl for activity, expression or
synthesis. The chloride transporter has not been identified. Glycine betaine uptake from the medium is chloride-dependent. EctABC, genes for the biosynthesis of
ectoine; glnA2, glutamine synthetase 2; proHJA genes for the biosynthesis of proline. YviD, YhfK and the potential N-acetyl-muraminidase were identified as
chloride-induced proteins in a proteomic screen, but their function is unknown. Chloride stimulates transcription, translation, enzyme and transporter activity, as
indicated by the sign Cl.
loss, intramolecular recombination and/or horizontal gene
transfer permitting the exchange of DNA among organisms of
different species. The latter is recognized as the major driving
force for bacterial adaptation and bacterial genome evolution.
This is concluded from the results of comparative genome
analyses indicating that >20% of the total bacterial genes and
even >40% of the archaeal genomes have been horizontally
transferred (Smith et al., 1997; Jain et al., 1999; Lawrence,
1999; Eisen, 2000; Ochman et al., 2000; Deppenmeier et al.,
2002; Gogarten et al., 2002; Boucher et al., 2003; Daubin
et al., 2003; Garcia-Vallve et al., 2003; Thomas and Nielsen,
2005).
Horizontal gene transfer offers the advantage of gaining
substantial amounts of novel genetic information, e.g. metabolic traits, resistance genes and pathogenicity determinants,
but the latter may lead to clinically relevant problems
(Ochman et al., 2000; Gophna et al., 2004). Moreover, the
transfer of DNA is not restricted to bacterial DNA, but also
permits horizontal gene transfer among organisms of different
domains (Aravind et al., 1998; Doolittle, 1999; Koonin et al.,
2001; Jain et al., 2002; Gogarten and Townsend, 2005).
Particularly in extremophilic bacteria, horizontal gene
transfer is suggested to be a very important force for adaptation. This suggestion is supported by comparative genome
analyses such that there is substantial evidence for mobile
elements in alkaliphilic bacteria (Takami et al., 2000) and for
frequent genetic input via horizontal gene transfer in thermoacidophilic archaea important for the acidophilic survival
strategy (Angelov and Liebl, 2006). Among the microorganisms thriving in extreme habitats, thermophiles and hyperthermophiles clearly stand out in terms of interdomain DNA
transfer such as 24 and 16.2% of the genes in the hyperthermophilic bacteria Thermotoga maritima and Aquifex
aeolicus, respectively, are suggested to be transferred from
archaeal hyperthermophiles (Aravind et al., 1998; Nelson
et al., 1999). Many of the transferred genes are thermophilic
traits that are essential for survival under extreme conditions.
One prominent example is reverse gyrase, a hyperthermophilespecific protein suggested to be transferred as a thermoadaptation trait from archaea to bacteria (Forterre et al., 2000).
Given these findings, hyperthermophilic bacteria are suggested
to play a central role in interdomain DNA transfer and are of
crucial importance for horizontal gene transfer of thermophilic
traits between hyperthermophiles.
Horizontal gene transfer is facilitated by three principal
mechanisms: conjugation, transduction and transformation. In
recent years, it has become evident that particularly natural
competence for DNA transformation, which describes the
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B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
uptake and incorporation of naked DNA, is a major contributor
to horizontal exchange of genetic information between
bacteria and is recognized as an important mechanism for
genome plasticity over evolutionary history (Lorenz and
Wackernagel, 1994; Chen and Dubnau, 2003, 2004; Chen
et al., 2005). Moreover, the growing evidence that natural
transformation is not restricted to prokaryotic DNA, but also
mediates transfer of transgenic plant DNA to bacteria suggests
that natural transformation is the most versatile mechanism of
DNA transfer (de Vries et al., 2001). Natural transformation is
a powerful mechanism for generating genetic diversity,
evolution of metabolic traits, spreading advantageous alleles
and mediating some forms of antigenic variation and the
impact of natural transformation in horizontal gene transfer is
supported by the finding that the ability to take up free DNA is
widely distributed among representatives of very different
phylogenetic and trophic groups. Presently, transformability
has been found in about 90 species from all major taxonomic
groups (Brigulla and Wackernagel, 2010).
Thermus thermophilus has become a model system to study
natural transformation in thermophiles (Averhoff, 2009).
T. thermophilus HB27 shares its natural habitat with other
thermophilic bacteria as well as archaea. The high abundance
of thermophilic archaea together with the broad substrate
specificity of the HB27 DNA translocator might have triggered
interdomain gene transfer between T. thermophilus and thermophilic archaea. The presence of several characteristic
archaeal genes in the T. thermophilus genome, such as
a tungsten-containing aldehyde ferredoxin oxidoreductase,
a potassium uptake protein, a peptide chain release factor 1,
a DNA modification methylase and two membrane proteins
(Omelchenko et al., 2005) corresponds with this suggestion.
Moreover, some proteins, such as a phosphoglycerate mutase,
a SAR1-like GTPase and a predicted DNA modification
methylase, are only shared by T. thermophilus, Deinococcus
radiodurans and representatives of archaea and eukaryotes
(Omelchenko et al., 2005). Another good example for interkingdom gene transfer in T. thermophilus is the ATP synthase.
This ATP synthase is encoded by nine genes organized in one
gene cluster, which was apparently taken up from Euryarchaeota (Müller and Grüber, 2003; Müller et al., 2005).
Taken together, the abundance of genes from members of
other domains and the presence of potentially transferred
thermophilic traits in the genome of T. thermophilus suggest
that horizontal gene transfer has played a major role in thermoadaptation of T. thermophilus.
3.1. Natural transformation in thermophiles: a unique
feature of Thermus
Despite the impact of natural transformation in genome
evolution and bacterial adaptation to very different, often
extreme environments, DNA transporters of extremophilic
bacteria have attracted less attention than might be expected
from their important role in adaptation of archaea and bacteria
to extreme environments. Among the transformable bacteria
known to date, the thermophile T. thermophilus exhibits
highest transformation frequencies; 1 out of 10e100 cells
takes up free DNA (Koyama et al., 1986). To gain insights into
the physiology of DNA uptake in thermophilic bacteria, we
chose T. thermophilus HB7 as the model bacterium and
studied the kinetics of DNA uptake. These studies revealed
that Thermus takes up DNA extremely fast with a maximal
velocity of 40 kb s1 per cell (Schwarzenlander and Averhoff,
2006). This is much faster than DNA uptake in mesophilic
transformable bacteria, such as Bacillus subtilis (4 kb s1), or
Haemophilus influenzae (16 kb s1 per cell) (Deich and Smith,
1980; Dubnau, 1991).
In addition to its extreme efficiency, the DNA transporter in
T. thermophilus HB27 was found to exhibit an extraordinarily
broad substrate spectrum. In contrast to the DNA uptake
systems in Neisseria gonorrhoeae and H. influenzae, where
specific DNA uptake sequences (DUS) are recognized by the
cell surface binding/uptake system (Goodman and Scocca,
1988; Elkins et al., 1991; Smith et al., 1995; Treangen et al.,
2008), the DNA uptake system of T. thermophilus does not
display sequence specificity. Moreover, the DNA translocator
of T. thermophilus takes up DNA from members of all three
domains, Bacteria, Archaea, and Eukarya (Schwarzenlander
and Averhoff, 2006; Schwarzenlander et al., 2008). Taken
together, the high efficiency and the extraordinarily broad
substrate specificity of the T. thermophilus HB27 DNA uptake
system indicate the great impact of this DNA transporter upon
thermoadaptation of T. thermophilus HB27 and interdomain
DNA transfer in hot environments.
The availability of the T. thermophilus HB27 genome
sequence initiated our molecular and biochemical studies of
the natural transformation system of T. thermophilus HB27,
unraveling numerous competence proteins and giving first
insights into the function of the components of a DNA
transporter in a thermophile. Mutant studies led to the identification of 16 competence genes organized into seven distinct
chromosomal loci (Friedrich et al., 2001, 2002, 2003). The
identified genes were assigned to three different groups,
including DNA translocator specific proteins, type IV pili
(Tfp)-related proteins and non-conserved proteins. Three of
the deduced proteins (ComEA, ComEC, DprA) were found to
be similar to conserved DNA translocator proteins of natural
transformation systems in mesophilic bacteria, whereas nine
were similar to proteins of Tfp or type II protein secretion
systems, including the four pilin-like proteins PilA1, PilA2,
PilA3, PilA4, a leader peptidase (PilD), a traffic-NTPase
protein (PilF), an inner membrane protein (PilC), a PilMhomologue and a secretin-like protein (PilQ). The latter was
detected in the outer membrane and found to be essential for
DNA binding which, together with the similarities of PilQ to
secretins, led to the suggestion that PilQ forms a homopolymeric channel guiding the DNA translocator through the outer
membrane of T. thermophilus (Rumszauer et al., 2006;
Schwarzenlander et al., 2008). In addition to these conserved
competence proteins, four novel competence proteins, ComZ,
PilN, PilO and PilW were identified; the latter three were
localized in the inner membrane (Rumszauer et al., 2006). The
unique cell envelope (Quintela et al., 1995) and the
B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
peptidoglycan (Caston et al., 1993) of T. thermophilus together
with the thermophilic lifestyle might have triggered the
evolution of these unique proteins.
The similarities of several competence proteins of T. thermophilus to proteins of Tfp systems led to two fundamental
questions: 1) Does T. thermophilus HB27 exhibit pilus structures on the surface? And if yes, 2) are the T. thermophilus
HB27 pili functionally linked to DNA uptake?
Electron microscopic studies revealed that T. thermophilus
HB27 carries individual pilus structures 6 nm in diameter and
1e3 mm in length. The absence of these pilus structures in
transformation-defect mutants carrying disruptions in the
competence genes of the pilMQ-operon, the prepilin-peptidase, pilC and pilA4 suggest a functional linkage of the
Thermus DNA uptake machinery and pili (Friedrich et al.,
2002, 2003). The question of whether the Thermus pili
themselves are implicated in DNA uptake or not cannot be
answered yet. But since Tfp are thin structures of several mm
in length without any long axial hole, it is more likely that the
long pilus structures themselves are not implicated in DNA
uptake. Thus, it is more conceivable that either only the lower
part of the pilus spanning the cell periphery or a distinct DNA
translocator comprising components playing a dual role in
DNA uptake and Tfp mediate DNA uptake.
Inactivation of the Thermus traffic ATPase PilF, which is
essential for natural transformation, led to non-competent
511
mutants with morphologically intact pili (Friedrich et al.,
2001). This finding suggests that the Thermus PilF is functionally similar to the gonococcal retraction ATPase PilT,
whose mutation did result in loss of competence and retraction
of pili without affecting the pilus structures on the surface of
the gonococcal cells (Wolfgang et al., 1998; Maier et al.,
2002). Due to these findings it is tempting to speculate that
DNA uptake in Thermus cells requires a dynamic DNA
translocator and pulling of the DNA through the cell wall and
periplasm via PilF-powered retraction of the DNA
translocator.
Two proteins, ComEA and ComEC, are the most likely
candidates for transport of DNA across the inner membrane.
These proteins are unrelated to Tfp systems, but are conserved
in all transformation machineries in different Gram-positive
and Gram-negative bacteria known to date (Chen and Dubnau,
2003; Averhoff, 2004). ComEA is a soluble but membraneanchored protein predicted to bind incoming DNA in the
periplasm and subsequently delivering the DNA to the inner
membrane DNA transporter ComEC, a large protein with 677
residues (Mr 72,000) and six membrane-spanning helices
(Friedrich et al., 2001). One strand of the DNA is transported
through ComEC, while the other strand is degraded by a thus
far unidentified nuclease. DNA transport through ComEC is
driven by ATP hydrolysis or mHþ (Fig. 2). Unfortunately,
biochemical evidence in support of this hypothesis is not
Fig. 2. Model for DNA uptake in T. thermophilus HB27. DNA is bound to the secretin (PilQ) complex or to a thus far unknown DNA binding protein close to the
PilQ complex in the outermost cell layer, which is comprised of an S-layer and lipids. Subsequently, the DNA is either transported along a DNA transformation
shaft made up of the pilin PilA4 and pilin-like proteins PilA1, PilA2 and PilA3, which is guided through the outer membrane by the secretin ring. Retraction of the
DNA transporter, powered by the motor ATPase PilF pulls the DNA through the cell wall layers and into the periplasmic space. After binding to the inner
membrane-anchored receptor protein ComEA, the DNA is delivered to the inner membrane channel likely made by the polytopic inner membrane protein ComEC.
During transport through the inner membrane, one strand of the DNA is degraded by a thus far unidentified nuclease. DNA transport through the ComEC channel is
driven by ATP or Dmþ
H . Five novel inner membrane-associated proteins, PilM, PilN, PilO, PilW and ComZ, are suggested to be components of the DNA translocator
platform implicated in the assembly of the transporter, and/or assist transport of DNA across the inner membrane channel. OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane, ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.
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B. Averhoff, V. Müller / Research in Microbiology 161 (2010) 506e514
available due to the fact that purification of the native
complexes/proteins as well as heterologous overproduction
failed (Draskovic and Dubnau, 2005). However, a T. thermophilus comEC mutant was not affected in DNA binding and
transport into a DNase-resistant form (Schwarzenlander et al.,
2008) suggesting that a defect still allows for transport through
the outer membrane, resulting in accumulation of DNA in the
thick cell wall and/or the periplasm. The unique competence
proteins PilM, PilN, PilO, and PilW, which have been localized in the inner membrane, are suggested to be involved in
the assembly of the DNA translocator complex in the inner
membrane (Fig. 2).
The identification of conserved competence proteins in
T. thermophilus and the functional link to pili biogenesis
which is also found for the DNA transporter in Gram-negative
mesophilic bacteria underlines the structural similarities of the
DNA uptake machineries of Gram-negative mesophilic and
extremely thermophilic bacteria. On the other hand, the
essential role of several novel competence genes in DNA
transport argues for unique features the of T. thermophilus
HB27 DNA translocator differing from transformation
systems of mesophilic bacteria. The question of whether these
features were triggered by the extreme environment or are due
to the phylogenetic position of Thermus and/or the distinct
features of the cell envelope and murein layer is open, and
further research will focus on this important question.
Furthermore, the extreme habitat of T. thermophilus might
have triggered the evolution of stable DNA translocator
complexes. This will hopefully lead to the isolation of intact
heteropolymeric transporter subcomplexes opening new
avenues to gain functional and structural insights into these
unique transport machineries.
4. Concluding remarks
The extraordinary traits of proteins from extremophiles
have promoted their biotechnological application. The
increasing understanding of gene expression in extremophiles
and the availability of genetic systems have supported the
improvement of proteins from extremophiles, such as thermozymes from thermophiles and overproduction of thermostable and thermoactive enzymes in thermophiles, such as Mndependent catalase and DNA polymerase in Thermus (Hidalgo
et al., 2004; Moreno et al., 2005). These applications
demonstrate the high biotechnological potential and relevance
of thermophiles, i.e. Thermus representatives as alternative
cell factories for the optimization and overproduction of
thermozymes with great biotechnological impact.
These two examples of extremophile research were chosen
because they are central research themes in the laboratories of
the authors. Of course, there are many more interesting topics
in extremophile research and the time is ripe to address these
by novel techniques such as “Omics“ technologies. We still
need more genome sequences that reflect the diversity of
extremophiles and metagenome analyses will certainly
provide more data on the ecological relevance of certain
extremophiles. What is still lagging behind is genetic analyses
of many extremophiles due to a lack of genetic systems.
Future research must attempt to close these methodological
gaps. Furthermore, we have learned a lot already about genes,
their expression, proteins, their mode of action and their
biotechnological application and all of these data give clues to
the extremophilic lifestyle. However, it should be kept in mind
that all these data have been obtained with pure cultures and
planktonic cells. Microbiologists have only recently addressed
the more relevant lifestyle of microbes in nature, biofilms and
genome-wide expression studies already revealed a different
expression profile in biofilms versus planktonic cells. Moreover, it is obvious that biofilms may also protect against
extreme physicochemical conditions such as heat, pH or salt.
For example, the pH or salt concentration in a biofilm may
differ substantially from that of the surrounding environment.
It is tempting to speculate that extremophiles behave differently in biofilms and by various types of interaction with their
biotic and abiotic environment. These are the interesting tasks
for the future that will help to unravel the beauty of extremophilic prokaryotes.
Acknowledgements
Work from the authors’ laboratories is supported by the
Deutsche Forschungsgemeinschaft. We are indebted to our
coworkers for their excellent contributions.
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