Recent Advances in Halophilic and Thermophilic Extremophiles

Research in Microbiology 161 (2010) 506e514 www.elsevier.com/locate/resmic

Exploring research frontiers in microbiology: recent advances in halophilic and thermophilic extremophiles

Beate Averhoff, Volker Mu¨ller*

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 far- reaching 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 from thermal springs in Yellowstone National Park (Brock and Freeze, 1969). This enzyme has made the polymerase chain Extremophilic prokaryotes are characterized by inhabiting reaction possible on a large, automated scale. With this enzyme, ecosystems that are, from a human perspective, extreme. Such sequencing genomes was made possible and it gave rise to the environments may have extremely high or low pH, high or low thousands of genomes that we have today, ranging from nano- temperatures, high salinity, high pressure and various combi- archaea to humans. nations thereof. Extremophilic microorganisms include The community of extremophilic researchers is rather large. members of all three domains of life, the Archaea, Bacteria and There are conferences that deal only with extremophiles; there Eukarya. Often, these microbes are not only challenged by one is an “International Society for Extremophiles”, and there is extreme, but multiple, and thus they are “polyextremophile” even a journal named “Extremophiles”, founded by Koki (Mesbah and Wiegel, 2008). Examples would be life at hot Horikoshi and now led by Garabed Antranikian. Research on alkaline springs or hypersaline and alkaline lakes or hot and extremophiles has been plentiful over the last few decades and acidic springs. Ever since extremophiles were discovered, their is as diverse as the organisms. It started with the isolation of physiology and their adaptation to the unhostile environment extremophiles and continued on by exploring their biochem- have attracted much interest. This was not only because of the istry, then into their genetics and regulation of their metabo- interest in their lifestyle, but also for exploring their biotech- lism. A couple of recent excellent books cover the entire nological potential. The enzyme Taq polymerase is a prime spectrum of extremophiles and describe their lifestyle (Gerday example of an enzyme from an extremophile, Thermus aqua- and Glansdorff, 2007; Garrett and Klenk, 2007; Oren, 2002). ticus, isolated in 1969 by Thomas D. Brock and Hudson Freeze Considering the wealth of information on extremophiles, on the one hand, and the limitation in space for this review, on the * Corresponding author. Tel.: þ49 69 79829509/507; fax: þ49 69 79829306. other, we will restrict ourselves to two examples and apologize E-mail addresses: [email protected] (B. Averhoff), vmueller@ to the readers and the community for not being able to cover bio.uni-frankfurt.de (V. Mu¨ller). the entire spectrum.

2. The molecular basis of salt adaptation in halophiles a genome-wide scale (Pflu¨ger et al., 2007). 84 genes of different functional categories, such as solute transport and þ Every living cell is challenged by changing water activities biosynthesis, Na export, stress response, ion, protein and in its ecosystem; thus, constant monitoring and adapting to phosphate transport, metabolic enzymes, regulatory proteins, changing water activities is a prerequisite for life. This is of DNA modification systems and cell surface modulators were particular importance for (moderate) halophiles. The biggest found to be more strongly expressed at high salinities. challenge is to adjust the turgor and living cells have devel- Moreover, 10 genes encoding different metabolic functions oped two principal strategies to re-establish turgor pressure including potassium uptake and ATP synthesis were reduced and to circumvent the detrimental consequences of water loss in expression under high salt. The overall expression profiles when exposed to increasing osmolality. On the one hand, there suggest that M. mazei is able to adapt to high salinities by is the “salt-in-cytoplasm”-strategy, which means that inorganic multiple upregulation of many different cellular functions, ions, mainly Kþ and Cl, accumulate in the cytoplasm until including protective pathways such as solute transport and the internal salt concentration is similar to the extracellular biosynthesis, import of phosphate, export of Naþ, and upre- one. This strategy is found in extremely halophilic Halobac- gulation of pathways for modification of DNA and cell surface teria (Archaea) and halophilic, anaerobic Haloanaerobiales architecture. Taking this (and other studies) into account, it is (Bacteria)(Galinski and Tru¨per, 1994; Ventosa et al., 1998). obvious that a fine-tuned response to changing salinities On the other hand, the vast majority of prokaryotes cope with requires an elaborate regulatory network that, in addition, also increasing osmolarity by uptake or synthesis of compatible has to talk to other networks. Again, analyses of these solutes, which are defined as small, highly soluble, organic networks have only begun recently and we will describe one molecules which do not interfere with the central metabolism, that we discovered in the moderately halophilic bacterium even if they accumulate at high concentrations (Brown, 1976). Halobacillus halophilus. This strategy is widespread and evolutionarily well conserved in all three domains of life (Bohnert, 1995; Kempf and 2.1. The chloride regulon in the moderate halophile Bremer, 1998; Roeßler and Mu¨ller, 2001b; Saum and H. halophilus Mu¨ller, 2008a). However, the spectrum of compatible solutes used comprises only a limited number of compounds and these The moderately halophilic bacteria are a specialized group can be divided into 2 major groups: 1) sugars and polyols; and of organisms that require NaCl for growth (Ventosa et al., 2) a- and b-amino acids and their derivatives, including 1998). They grow at nearly the same rate over a rather wide methylamines. This limitation to a rather small number of range of external salt concentrations (0.5e2.5 M) which is compounds reflects the fundamental constraints on solutes evidence for effective mechanisms to cope with changing which are compatible with macromolecular and cellular external salinities. The aerobic, endospore-forming, Gram- functions (Le Rudulier et al., 1984). Most archaeal compatible positive bacterium H. halophilus has become a model system solutes resemble in structure their bacterial counterpart, with to study salt adaptation at various cellular levels (Mu¨ller and the difference that the majority of them carry a negative charge Saum, 2005). (Martin et al., 1999; Roeßler and Mu¨ller, 2001b). H. halophilus is the first organism for which a strict chlo- The uptake and biosynthesis of compatible solutes is ride dependence of growth was described (Claus et al., 1983; induced by high salinity or high osmolarity on both the DNA Roeßler and Mu¨ller, 1998). No growth was observed at a Cl and protein level. The pathways for the biosynthesis of various concentration of 0.2 M, but addition of chloride (to a medium solutes have been identified in different bacteria and archaea, with constant osmolarity) restored growth in a concentration- but how the environmental signal “salinity” is sensed and how dependent manner. Optimal growth occurs at 0.8e1.0 M Cl . this signal is transmitted to various output modules at the level Moreover, not only growth rates but also cell yields (final of gene, enzyme or transporter activation is completely optical densities) were strictly chloride-dependent (Roeßler obscure (Wood et al., 2001). This is even more important if and Mu¨ller, 1998). one considers that the overall cellular response of cells to What looked like an exotic phenotype at a first glance was hypersalinity is not only the accumulation of solutes but analyzed in more detail. To understand the function of chloride a reprogramming of cellular metabolism and structure in it was important to figure out physiological processes that are general. The first step towards unraveling the complexity of chloride-dependent. In addition to growth, germination of regulation could be by genome-wide expression profiling endospores as well as flagella production and motility were studies, but unfortunately, these have not been done with identified to be chloride-dependent (Dohrmann and Mu¨ller, (moderate) halophiles. However, there are a few studies 1999; Roeßler et al., 2000) and the very different functions of available on halotolerant organisms that actually demonstrate Cl (motility, flagellation, spore germination, growth) indicate their complexity (Weber and Jung, 2002; Steil et al., 2003; a role of the anion in gene or protein activation. To test this and Pflu¨ger et al., 2007). The methanogenic archaeon Meth- identify proteins regulated by Cl , two different lines of anosarcina mazei is a non-halophilic methanogen that can experiments were performed. First, a (very limited) proteome adapt to 800 mM NaCl (Roeßler and Mu¨ller, 2002). We have analyses revealed five proteins upregulated in a chloride- used microarray studies to examine the effect of elevated dependent fashion (Roeßler and Mu¨ller, 2002). For some of salinities on the regulation of gene expression in M. mazei on these, chloride-dependent induction has been verified by other 508 B. Averhoff, V. Mu¨ller / Research in Microbiology 161 (2010) 506e514 means. For example, western blot analyses revealed that the ectoine, and their biosynthetic routes and regulation have been production of the structural component of the flagellum, identified (Saum et al., 2006; Saum and Mu¨ller, 2007a,b; flagellin, was impaired in the absence of Cl (Roeßler and Burkhardt et al., 2009). Based on our studies, the following Mu¨ller, 2002). However, upon addition of Cl the cellular model for long-term salt adaptation was proposed (Saum and flagellin pool increased in a concentration-dependent manner. Mu¨ller, 2008b). Challenged by low extracellular water activity Optimal flagellin production was achieved at 0.8e1.0 M Cl. caused by elevated NaCl concentrations, H. halophilus main- The same was observed for other proteins such as LuxS, tains a rather high internal Cl concentration in the molar a component of the quorum sensing system (Sewald et al., range, but additionally accumulates compatible solutes. At 2007). It plays a role in the biosynthesis of autoinducers of intermediate salinities (1.0e1.5 M NaCl), glutamate and the furanone family that are found in Gram-negative as well as glutamine are the main solutes and accumulate in response to in Gram-positive bacteria. external salinity. Upon further increase in salinity, cells have Second, the effect of Cl in gene transcription was analyzed. an interesting phenotype: the intracellular glutamine and To test whether transcription of fliC, the gene encoding the glutamate pools are not further increased, but the cells switch flagellum, was also chloride-dependent, it was cloned and to proline as the dominant compatible solute (at 2.0e3.0 M sequenced. Subsequent northern blot and RT-PCR analyses NaCl). with RNA from cells grown at different chloride concentrations The regulation of glutamate/glutamine and proline unequivocally demonstrated chloride-stimulated expression of biosynthesis was addressed on a molecular level. H. hal- fliC (Roeßler and Mu¨ller, 2002). In summary, these experiments ophilus has two isogenes each for a glutamate dehydrogenase, demonstrated that Cl influences the cellular flagellin pool by glutamate synthase and glutamine synthetase. Among these, acting at both the transcriptional and translational level, but the only glutamine synthetase 2 was regulated by salinity, but also effect on translation was much more pronounced. This was the and more importantly, by the chloride concentration (Saum first time that Cl dependence of gene expression and protein et al., 2006). As discussed above, the effect was much more production was shown in any prokaryote. pronounced at the enzyme actvity level than at the gene level. The fact that so many different physiological processes and Apparently, the first step of the cells is to sense salinity of the so many different proteins and genes are activated by Cl points environment by measuring the chloride concentration that then to a global Cl regulon active in H. halophilus (Saum and triggers synthesis of glutamine and glutamate via glutamate Mu¨ller, 2008b). What could be the function of the Cl -depen- synthetase 2. As soon as the glutamate concentration reaches dent regulatory network? At least one function must be essen- a value of around 0.2 M in the cells, glutamate induces tial to growth, since growth of H. halophilus is strictly Cl- expression of the proline biosynthesis operon and the switch dependent. One has to keep in mind that one essential function from glutamine/glutamate to proline is initiated. The molec- of moderate halophiles is to sense external salinity and to ular basis of the glutamate-induced switch to proline produc- respond to it on a transcriptional, translational and enzyme tion remains to be established. activity level to adjust the intracellular pool size of the Therefore, the most prominent task of the chloride regulon compatible solutes. H. halophilus apparently uses a mixture of in H. halophilus is to measure the chloride concentration and the “salt-in-cytoplasm” and “compatible solute” strategy in activate biosynthesis of compatible solutes, a prerequisite for osmoadaptation (Saum and Mu¨ller, 2008b). It was shown that life at elevated salinities. The chloride regulon not only the intracellular Cl concentration (Cli ) increases with the regulates solute uptake and synthesis, but also spore germi- external Cl concentration (Cle )(Roeßler and Mu¨ller, 1998). nation, flagellum synthesis and quorum sensing and possibly At suboptimal Cle concentrations, Cli is ten times lower than much more, such as, for example, respiration or cell wall Cle , at 0.5 M Cle the Cli concentration was 0.08 M, and in the composition (Fig. 1). Our model for the first time provides an range of 0.8e2.0 M the Cle /Cli gradient decreased to a nearly explanation of how the different players in the cell are constant value of 1.5e2. In other words, the internal Cl orchestrated to give the synchronized output “cellular adap- concentration at an external salinity of 2 M NaCl is 1 M. The tation to changing salinities”: by the action of the chloride þ counter-ion has not yet been determined but is likely to be K . regulon! Whether this is of general importance in prokaryotes H. halophilus, in addition, accumulates compatible solutes remains to be addressed in the future, but it should be noted in by uptake or biosynthesis. A very early and very important this context that a number of bacteria were shown to require observation was that the uptake of glycine betaine was strictly chloride for growth at elevated salinities (Mu¨ller and Oren, dependent on the Cl concentration (Roeßler and Mu¨ller, 2003; Roessler et al., 2003). 2001a). These experiments clearly revealed the first Cl- dependent osmolyte transporter in prokaryotes. Furthermore, 3. The role of horizontal DNA transfer in adaptation to these experiments corroborate the idea that the essential func- extreme environments tion of the chloride regulon is to sense external salt and to induce/activate systems involved in accumulation of compatible Microorganisms are able to exploit very different, often solutes. Unfortunately, the transporter(s) catalyzing Cl - extreme environments and therefore have evolved phenotypic dependent glycine betaine transport have not been identified yet. traits allowing adaptation and survival under extreme envi- H. halophilus synthesizes different solutes such as glycine ronmental conditions. This microbial adaptation and diversi- betaine (from choline), glutamate, glutamine, proline and fication can be achieved by gene mutations, differential gene B. Averhoff, V. Mu¨ller / 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 analyses such that there is substantial evidence for mobile transfer permitting the exchange of DNA among organisms of elements in alkaliphilic bacteria (Takami et al., 2000) and for different species. The latter is recognized as the major driving frequent genetic input via horizontal gene transfer in ther- force for bacterial adaptation and bacterial genome evolution. moacidophilic archaea important for the acidophilic survival This is concluded from the results of comparative genome strategy (Angelov and Liebl, 2006). Among the microorgan- analyses indicating that >20% of the total bacterial genes and isms thriving in extreme habitats, thermophiles and hyper- even >40% of the archaeal genomes have been horizontally thermophiles clearly stand out in terms of interdomain DNA transferred (Smith et al., 1997; Jain et al., 1999; Lawrence, transfer such as 24 and 16.2% of the genes in the hyper- 1999; Eisen, 2000; Ochman et al., 2000; Deppenmeier et al., thermophilic bacteria Thermotoga maritima and Aquifex 2002; Gogarten et al., 2002; Boucher et al., 2003; Daubin aeolicus, respectively, are suggested to be transferred from et al., 2003; Garcia-Vallve et al., 2003; Thomas and Nielsen, archaeal hyperthermophiles (Aravind et al., 1998; Nelson 2005). et al., 1999). Many of the transferred genes are thermophilic Horizontal gene transfer offers the advantage of gaining traits that are essential for survival under extreme conditions. substantial amounts of novel genetic information, e.g. meta- One prominent example is reverse gyrase, a hyperthermophile- bolic traits, resistance genes and pathogenicity determinants, specific protein suggested to be transferred as a thermoa- but the latter may lead to clinically relevant problems daptation trait from archaea to bacteria (Forterre et al., 2000). (Ochman et al., 2000; Gophna et al., 2004). Moreover, the Given these findings, hyperthermophilic bacteria are suggested transfer of DNA is not restricted to bacterial DNA, but also to play a central role in interdomain DNA transfer and are of permits horizontal gene transfer among organisms of different crucial importance for horizontal gene transfer of thermophilic domains (Aravind et al., 1998; Doolittle, 1999; Koonin et al., traits between hyperthermophiles. 2001; Jain et al., 2002; Gogarten and Townsend, 2005). Horizontal gene transfer is facilitated by three principal Particularly in extremophilic bacteria, horizontal gene mechanisms: conjugation, transduction and transformation. In transfer is suggested to be a very important force for adapta- recent years, it has become evident that particularly natural tion. This suggestion is supported by comparative genome competence for DNA transformation, which describes the 510 B. Averhoff, V. Mu¨ller / Research in Microbiology 161 (2010) 506e514 uptake and incorporation of naked DNA, is a major contributor highest transformation frequencies; 1 out of 10e100 cells to horizontal exchange of genetic information between takes up free DNA (Koyama et al., 1986). To gain insights into bacteria and is recognized as an important mechanism for the physiology of DNA uptake in thermophilic bacteria, we genome plasticity over evolutionary history (Lorenz and chose T. thermophilus HB7 as the model bacterium and Wackernagel, 1994; Chen and Dubnau, 2003, 2004; Chen studied the kinetics of DNA uptake. These studies revealed et al., 2005). Moreover, the growing evidence that natural that Thermus takes up DNA extremely fast with a maximal transformation is not restricted to prokaryotic DNA, but also velocity of 40 kb s 1 per cell (Schwarzenlander and Averhoff, mediates transfer of transgenic plant DNA to bacteria suggests 2006). This is much faster than DNA uptake in mesophilic that natural transformation is the most versatile mechanism of transformable bacteria, such as Bacillus subtilis (4 kb s1), or DNA transfer (de Vries et al., 2001). Natural transformation is Haemophilus influenzae (16 kb s1 per cell) (Deich and Smith, a powerful mechanism for generating genetic diversity, 1980; Dubnau, 1991). evolution of metabolic traits, spreading advantageous alleles In addition to its extreme efficiency, the DNA transporter in and mediating some forms of antigenic variation and the T. thermophilus HB27 was found to exhibit an extraordinarily impact of natural transformation in horizontal gene transfer is broad substrate spectrum. In contrast to the DNA uptake supported by the finding that the ability to take up free DNA is systems in Neisseria gonorrhoeae and H. influenzae, where widely distributed among representatives of very different specific DNA uptake sequences (DUS) are recognized by the phylogenetic and trophic groups. Presently, transformability cell surface binding/uptake system (Goodman and Scocca, has been found in about 90 species from all major taxonomic 1988; Elkins et al., 1991; Smith et al., 1995; Treangen et al., groups (Brigulla and Wackernagel, 2010). 2008), the DNA uptake system of T. thermophilus does not Thermus thermophilus has become a model system to study display sequence specificity. Moreover, the DNA translocator natural transformation in thermophiles (Averhoff, 2009). of T. thermophilus takes up DNA from members of all three T. thermophilus HB27 shares its natural habitat with other domains, Bacteria, Archaea, and Eukarya (Schwarzenlander thermophilic bacteria as well as archaea. The high abundance and Averhoff, 2006; Schwarzenlander et al., 2008). Taken of thermophilic archaea together with the broad substrate together, the high efficiency and the extraordinarily broad specificity of the HB27 DNA translocator might have triggered substrate specificity of the T. thermophilus HB27 DNA uptake interdomain gene transfer between T. thermophilus and ther- system indicate the great impact of this DNA transporter upon mophilic archaea. The presence of several characteristic thermoadaptation of T. thermophilus HB27 and interdomain archaeal genes in the T. thermophilus genome, such as DNA transfer in hot environments. a tungsten-containing aldehyde ferredoxin oxidoreductase, The availability of the T. thermophilus HB27 genome a potassium uptake protein, a peptide chain release factor 1, sequence initiated our molecular and biochemical studies of a DNA modification methylase and two membrane proteins the natural transformation system of T. thermophilus HB27, (Omelchenko et al., 2005) corresponds with this suggestion. unraveling numerous competence proteins and giving first Moreover, some proteins, such as a phosphoglycerate mutase, insights into the function of the components of a DNA a SAR1-like GTPase and a predicted DNA modification transporter in a thermophile. Mutant studies led to the iden- methylase, are only shared by T. thermophilus, Deinococcus tification of 16 competence genes organized into seven distinct radiodurans and representatives of archaea and eukaryotes chromosomal loci (Friedrich et al., 2001, 2002, 2003). The (Omelchenko et al., 2005). Another good example for inter- identified genes were assigned to three different groups, kingdom gene transfer in T. thermophilus is the ATP synthase. including DNA translocator specific proteins, type IV pili This ATP synthase is encoded by nine genes organized in one (Tfp)-related proteins and non-conserved proteins. Three of gene cluster, which was apparently taken up from Eur- the deduced proteins (ComEA, ComEC, DprA) were found to yarchaeota (Mu¨ller and Gru¨ber, 2003; Mu¨ller et al., 2005). be similar to conserved DNA translocator proteins of natural Taken together, the abundance of genes from members of transformation systems in mesophilic bacteria, whereas nine other domains and the presence of potentially transferred were similar to proteins of Tfp or type II protein secretion thermophilic traits in the genome of T. thermophilus suggest systems, including the four pilin-like proteins PilA1, PilA2, that horizontal gene transfer has played a major role in ther- PilA3, PilA4, a leader peptidase (PilD), a traffic-NTPase moadaptation of T. thermophilus. protein (PilF), an inner membrane protein (PilC), a PilM- homologue and a secretin-like protein (PilQ). The latter was 3.1. Natural transformation in thermophiles: a unique detected in the outer membrane and found to be essential for feature of Thermus DNA binding which, together with the similarities of PilQ to secretins, led to the suggestion that PilQ forms a homopoly- Despite the impact of natural transformation in genome meric channel guiding the DNA translocator through the outer evolution and bacterial adaptation to very different, often membrane of T. thermophilus (Rumszauer et al., 2006; extreme environments, DNA transporters of extremophilic Schwarzenlander et al., 2008). In addition to these conserved bacteria have attracted less attention than might be expected competence proteins, four novel competence proteins, ComZ, from their important role in adaptation of archaea and bacteria PilN, PilO and PilW were identified; the latter three were to extreme environments. Among the transformable bacteria localized in the inner membrane (Rumszauer et al., 2006). The known to date, the thermophile T. thermophilus exhibits unique cell envelope (Quintela et al., 1995) and the B. Averhoff, V. Mu¨ller / Research in Microbiology 161 (2010) 506e514 511 peptidoglycan (Caston et al., 1993)ofT. thermophilus together mutants with morphologically intact pili (Friedrich et al., with the thermophilic lifestyle might have triggered the 2001). This finding suggests that the Thermus PilF is func- evolution of these unique proteins. tionally similar to the gonococcal retraction ATPase PilT, The similarities of several competence proteins of T. ther- whose mutation did result in loss of competence and retraction mophilus to proteins of Tfp systems led to two fundamental of pili without affecting the pilus structures on the surface of questions: 1) Does T. thermophilus HB27 exhibit pilus struc- the gonococcal cells (Wolfgang et al., 1998; Maier et al., tures on the surface? And if yes, 2) are the T. thermophilus 2002). Due to these findings it is tempting to speculate that HB27 pili functionally linked to DNA uptake? DNA uptake in Thermus cells requires a dynamic DNA Electron microscopic studies revealed that T. thermophilus translocator and pulling of the DNA through the cell wall and HB27 carries individual pilus structures 6 nm in diameter and periplasm via PilF-powered retraction of the DNA 1e3 mm in length. The absence of these pilus structures in translocator. transformation-defect mutants carrying disruptions in the Two proteins, ComEA and ComEC, are the most likely competence genes of the pilMQ-operon, the prepilin-pepti- candidates for transport of DNA across the inner membrane. dase, pilC and pilA4 suggest a functional linkage of the These proteins are unrelated to Tfp systems, but are conserved Thermus DNA uptake machinery and pili (Friedrich et al., in all transformation machineries in different Gram-positive 2002, 2003). The question of whether the Thermus pili and Gram-negative bacteria known to date (Chen and Dubnau, themselves are implicated in DNA uptake or not cannot be 2003; Averhoff, 2004). ComEA is a soluble but membrane- answered yet. But since Tfp are thin structures of several mm anchored protein predicted to bind incoming DNA in the in length without any long axial hole, it is more likely that the periplasm and subsequently delivering the DNA to the inner long pilus structures themselves are not implicated in DNA membrane DNA transporter ComEC, a large protein with 677 uptake. Thus, it is more conceivable that either only the lower residues (Mr 72,000) and six membrane-spanning helices part of the pilus spanning the cell periphery or a distinct DNA (Friedrich et al., 2001). One strand of the DNA is transported translocator comprising components playing a dual role in through ComEC, while the other strand is degraded by a thus DNA uptake and Tfp mediate DNA uptake. far unidentified nuclease. DNA transport through ComEC is þ Inactivation of the Thermus traffic ATPase PilF, which is driven by ATP hydrolysis or mH (Fig. 2). Unfortunately, essential for natural transformation, led to non-competent 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 þ driven by ATP or DmH. 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. 512 B. Averhoff, V. Mu¨ller / Research in Microbiology 161 (2010) 506e514 available due to the fact that purification of the native of many extremophiles due to a lack of genetic systems. complexes/proteins as well as heterologous overproduction Future research must attempt to close these methodological failed (Draskovic and Dubnau, 2005). However, a T. thermo- gaps. Furthermore, we have learned a lot already about genes, philus comEC mutant was not affected in DNA binding and their expression, proteins, their mode of action and their transport into a DNase-resistant form (Schwarzenlander et al., biotechnological application and all of these data give clues to 2008) suggesting that a defect still allows for transport through the extremophilic lifestyle. However, it should be kept in mind the outer membrane, resulting in accumulation of DNA in the that all these data have been obtained with pure cultures and thick cell wall and/or the periplasm. The unique competence planktonic cells. Microbiologists have only recently addressed proteins PilM, PilN, PilO, and PilW, which have been local- the more relevant lifestyle of microbes in nature, biofilms and ized in the inner membrane, are suggested to be involved in genome-wide expression studies already revealed a different the assembly of the DNA translocator complex in the inner expression profile in biofilms versus planktonic cells. More- membrane (Fig. 2). over, it is obvious that biofilms may also protect against The identification of conserved competence proteins in extreme physicochemical conditions such as heat, pH or salt. T. thermophilus and the functional link to pili biogenesis For example, the pH or salt concentration in a biofilm may which is also found for the DNA transporter in Gram-negative differ substantially from that of the surrounding environment. mesophilic bacteria underlines the structural similarities of the It is tempting to speculate that extremophiles behave differ- DNA uptake machineries of Gram-negative mesophilic and ently in biofilms and by various types of interaction with their extremely thermophilic bacteria. On the other hand, the biotic and abiotic environment. These are the interesting tasks essential role of several novel competence genes in DNA for the future that will help to unravel the beauty of extrem- transport argues for unique features the of T. thermophilus ophilic prokaryotes. HB27 DNA translocator differing from transformation systems of mesophilic bacteria. The question of whether these Acknowledgements features were triggered by the extreme environment or are due to the phylogenetic position of Thermus and/or the distinct Work from the authors’ laboratories is supported by the features of the cell envelope and murein layer is open, and Deutsche Forschungsgemeinschaft. We are indebted to our further research will focus on this important question. coworkers for their excellent contributions. Furthermore, the extreme habitat of T. thermophilus might have triggered the evolution of stable DNA translocator References complexes. This will hopefully lead to the isolation of intact heteropolymeric transporter subcomplexes opening new Angelov, A., Liebl, W., 2006. Insights into extreme thermoacidophily based on avenues to gain functional and structural insights into these genome analysis of Picrophilus torridus and other thermoacidophilic unique transport machineries. archaea. J. Biotechnol. 126, 3e10. 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