The Archaeal Molecular Chaperone Machine: Peculiarities and Paradoxes

Corresponding author: Alberto J. L. Macario, Wadsworth Center, Room B-749, Division of Molecular Medicine, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509., macario{at}wadsworth.org (E-mail)

Communicating editor: W. B. WHITMAN

	ABSTRACT

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains.

	The players

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

The hsp70(dnaK) gene is widespread in nature and its protein product, the molecular chaperone Hsp70(DnaK), is highly conserved in sequence (GUPTA and SINGH 1994 ; RENSING and MAIER 1994 ; GUPTA 1998 ). In fact, this chaperone is considered one of the most-conserved proteins throughout evolution.

To exercise its chaperoning functions in polypeptide folding and refolding, Hsp70(DnaK) interacts with other proteins, the cochaperones Hsp40(DnaJ) and GrpE (HARTL and MARTIN 1995 ; MAYER and BUKAU 1998 ). The three molecules constitute the chaperone machine that assists other proteins to fold correctly or to regain the proper configuration after partial denaturation (GEORGOPOULOS and WELCH 1993 ; PARSELL and LINDQUIST 1993 ; GLOVER and LINDQUIST 1998 ; KEDZIERSKA et al. 1999 ).

Protein folding may also require the participation of another chaperoning system, the chaperonins that constitute the GroEL/S and TRiC structures in bacteria and eukaryotes, respectively (ROMMELAERE et al. 1993 ; KUBOTA et al. 1995 ; BUKAU and HORWICH 1998 ; RANSON et al. 1998 ; XU and SIGLER 1998 ). While TRiC resides in the eukaryotic-cell cytosol, GroEL/S occurs in the cytoplasm of bacteria and in the eukaryotic-cell organelles such as mitochondria and chloroplasts believed to have originated in bacterial endosymbionts (GUPTA and SINGH 1994 ; NELISSEN et al. 1995 ; HORST et al. 1997 ; PALMER 1997 ).

In summary, it is accepted that polypeptides have the information necessary for correct self-folding inscribed in the primary structure. However, in vivo, in the overcrowded environment inside the cell and its compartments, self-folding is inefficient and slow, with danger of misfolding and aggregation. Assistance must be provided to the new polypeptides so they will fold correctly and fast, without aggregation. Two important molecular teams are involved in assisting protein folding: the chaperone machine formed by Hsp70(DnaK), Hsp40(DnaJ), and GrpE, and the chaperonin system, which in bacteria consists of the GroEL and GroES proteins, and in the eukaryotic-cell organelles is represented by homologs of GroEL and GroES. TriC is the chaperonin system of the eukaryotic-cell cytosol.

	The action

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

Studies on the mechanism of protein folding and refolding after partial denaturation due to stress (temperature elevation, decrease in pH, etc.) by the chaperone machine alone or in cooperation with the chaperonin complex have been performed in vitro and in vivo using bacteria (e.g., Escherichia coli) and eukaryotes (e.g., Saccharomyces cerevisiae, and mammalian cell lines; references in MACARIO et al. 1999 ). Despite the large amount of information obtained, many details of the folding and refolding processes are incompletely understood. Still less is known about the chaperones and chaperonins in archaea, their functions and mechanism of action. It is important to remark, however, that becoming acquainted with what is known for archaea is extremely useful not only for deciding what to do—and how to do it—to increase our understanding of the archaeal chaperoning systems, but also for discovering new molecules and mechanisms that will enhance research with bacteria and eukaryotes. It is probable that by studying archaea one will unveil molecular mechanisms also present in bacteria and/or eukaryotes but modified by evolution, and not as easily detectable in the latter two groups of organisms.

The protein-folding pathways or networks that have been proposed are based, mostly, on studies done in the bacterium E. coli, and with the eukaryotic cytosol and organellar systems. As mentioned above, there are still many details that have not been elucidated. What follows is a brief and simplified account of the possible folding mechanisms that may be inferred from the data available at the present time.

	Interaction

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

Protein biogenesis proceeds through a series of events that begins with the synthesis of a polypeptide in the ribosome—initiation-elongation-termination and release—accompanied and followed by maturation and progression to a final, functional, or native configuration, and by translocation to the place in the cell where the mature protein will reside and function (plasma membrane, periplasmic space, nucleus, nucleolus, mitochondria, chloroplasts, endoplasmic reticulum, peroxysome, cytosol).

Throughout virtually all the stages of protein biogenesis, the nascent as well as the completed protein interact with other molecules and structures within the cell, among which the chaperone machine and the chaperonin system are prominent. These interactions leading to correct protein folding may be described in a simplified manner as the following possible different series of events:

1. Chaperone machine only: A simple pathway would involve only the chaperone machine that would assist the nascent polypeptide to fold in the cytosol, without participation of chaperonins.

2. Chaperonins only: Another simple pathway would be that the newly made polypeptide would reach, unassisted, the open central cavity of the GroEL complex, enter into it, and become enclosed in it when the GroES ring occludes the opening of the cavity, which thus becomes a closed folding chamber. Within the chamber, as suggested by experimental data, the polypeptide finds itself isolated in a hydrophilic environment conducive to correct folding. Subsequently, the chamber opens and the folded protein emerges into the cytosol. If folding were incomplete, the polypeptide would reenter the now-open GroEL cavity for another folding cycle. This process would be repeated until correct folding was consummated.

3. Chaperone machine and chaperonins: A more complicated pathway would involve the chaperone machine and the chaperonin complex. The latter would receive, and admit into its central cavity, a polypeptide maintained in a "foldable" shape by the chaperone machine. This implies that the machine would assist the nascent polypeptide as it is being synthesized, or immediately after termination and release (see below, co- and post-translational folding), thus preventing misfolding and aggregation. In this scenario, the chaperone machine's function would be to keep the nascent polypeptide in an extended configuration until it reaches the folding cavity.

4. Prefoldin and TRiC: While the mechanism described above (point 3), is physiologically valid in bacteria, at least for a minority of its proteins, it may not apply to the eukaryotic-cell cytosol. It is unclear whether, or how, the chaperone machine cooperates with TRiC. This is a chaperonin system that seems to be involved in the biogenesis of only a few proteins, particularly those found in the cytoskeleton, like tubulin and actin, and its interaction with the chaperone machine has not been proven. Thus, other alternatives must exist in the eukaryotic-cell cytosol to ensure that nascent polypeptides are properly ushered into a folding chamber. Recently, another system has been described and named prefoldin (VAINBERG et al. 1998 ). It occurs in the eukaryotic cytosol, and its function would be that of handing nascent polypeptides to the TRiC complex for folding. Details are still unavailable, so it is not possible to propose a definite pathway that would involve prefoldin and TRiC in a physiologically meaningful manner.

5. Variation according to circumstance: In bacteria, which have on the average shorter and simpler proteins than eukaryotes, a relatively simple pathway involving post-translational folding in the cytoplasm may be sufficient (at least for a significant number of proteins). Bacterial proteins with only one functional domain, or with very few along their sequence touching each other side by side or separated by a small number of amino acids, would not require a highly complicated chaperoning pathway because they would not have a strong tendency to misfold. The assumption is that errors in self-folding are less likely with short, simple proteins. In contrast to bacteria, eukaryotes on average have long proteins with multiple domains that are contiguous, or whose components may be far apart in the primary structure but come into proximity after correct folding. The probability of folding error seems immensely greater for these proteins than for those from bacteria. Hence, the nascent polypeptide must, in eukaryotes, be assisted immediately during synthesis, before the growing string of amino acids entangles itself and others in the immediate surroundings. Cotranslational chaperoning would then be mandatory in eukaryotes, especially for proteins with multiple adjacent domains. Molecules with discontiguous domains would require assistance during and after translation from the chaperone machine and the chaperonin system.

	Questions

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

What happens in archaea? What are the molecular chaperones and chaperonins of archaea? Are they of bacterial or eucaryal type? What are the mechanisms of protein folding in the various phylogenetic branches of this domain? And considering that heat is a protein denaturing agent, are there differences between mesophilic and extreme thermophilic species?

The above questions address problems of great importance in archaeal biology and evolution, with implications for the understanding of the stress response and protein folding in the other two phylogenetic domains also.

	THE ARCHAEAL CHAPERONE MACHINE

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

Distribution of hsp70(dnaK):
One of the major contributions to the field of molecular chaperones in archaea in recent years has been the discovery that, while some species have the hsp70(dnaK) gene, others do not (reviewed in MACARIO et al. 1999 ). The importance of this discovery is increasingly appreciated as more full-genome sequences become available and our knowledge of protein folding, chaperones, and chaperonins grows. If one considers the key role of Hsp70(DnaK) in protein biogenesis in organisms of the domains Bacteria and Eucarya, one wonders how a cell can thrive under physiologic conditions, and even survive stress, without this molecular chaperone. Intriguing questions immediately come to mind. What replaces (if anything) Hsp70(DnaK) in those archaeal organisms that lack it, so protein folding and refolding can proceed at a physiologic rate and in stressful situations?

This question has not been answered yet. It exemplifies one of the many aspects of archaeal biology whose elucidation is likely to impact on other fields beyond archaea. Furthermore, this particular problem cannot be investigated with any known bacterial or eukaryotic system, because all organisms in these two domains do have the gene, and sometimes more than one copy.

Another intriguing question is, what is the origin of the hsp70(dnaK) gene in those archaeal species that have it? Is it intrinsic to the archaea or foreign? These topics will be dealt with in a subsequent section.

The archaeal Hsp70(DnaK) is a bacterial protein:
All the archaeal Hsp70(DnaK) molecules sequenced and studied thus far resemble more closely those from bacteria than those from eukaryotes (GUPTA and SINGH 1994 ; RENSING and MAIER 1994 ; GUPTA 1998 ; GRIBALDO et al. 1999 ). This closeness is manifested by a high percentage of amino acid identity at comparable positions and by the sharing of structural motifs. Most remarkable, the archaeal protein was found to lack 23 amino acids in the N-terminal quadrant by comparison with the homologs from Gram-negative bacteria and eukaryotes (MACARIO et al. 1991 ). The deletion was proposed as a unique marker, distinctive of archaea and Gram-positive bacteria, a key evolutionary landmark indicating a common origin for these two groups of prokaryotes (GUPTA and SINGH 1992 , GUPTA and SINGH 1994 ). Although this hypothesis may be disputed, the deletion, or the lack of the insert typical of the Hsp70(DnaK) proteins from all organisms except archaea and Gram-positive bacteria, remains an outstanding feature of the archaeal chaperone. In addition to its potential evolutionary significance, one has to wonder whether the deletion has any functional implications.

The teammates:
The other components of the bacterial chaperone machine, in addition to Hsp70(DnaK), are Hsp40(DnaJ) and GrpE. Homologs of the latter two have been found in archaea (MACARIO et al. 1993 ; CONWAY DE MACARIO et al. 1994 ; BUSTARD and GUPTA 1997 ; SMITH et al. 1997 ). In fact, whenever the hsp70(dnaK) gene was found in an archaeon, hsp40(dnaJ) and grpE were also found if sufficient sequencing data were obtained, and the three genes were mapped to the same locus, near each other without intervening genes (see below).

There are, at the present time, three archaeal hsp70(dnaK) loci that have been sequenced to an extent that allows one to ascertain which genes are on both flanking regions of the three chaperone-machine genes. The loci are from the methanogens Methanosarcina mazei S-6 (CONWAY DE MACARIO et al. 1994 ), Methanosarcina thermophila TM-1 (J. HOFMAN-BANG, M. LANGE, E. CONWAY DE MÀCARIO, A. J. L. MÀCARIO and B. K. AHRING, unpublished results), and Methanobacterium thermoautotrophicum H (SMITH et al. 1997 ). In the three loci the gene organization is 5'-grpE-hsp70(dnaK)-hsp40(dnaJ)-3', the same as in many bacteria.

While the gene organization and the proteins encoded in these genes are close to bacterial homologs, the intergenic regions are not. The latter are in fact quite diverse, with one exception, i.e., the region between hsp70(dnaK) and hsp40(dnaJ). This and other features of the archaeal loci with potential evolutionary significance will be discussed below.

A hybrid appearance:
The hsp70(dnaK)-locus genes in archaea share bacterial and eukaryotic characteristics. As mentioned above, the proteins encoded in the genes closely resemble bacterial homologs. In contrast, the upstream flanking regions have no promoters or identifiable cis-acting regulatory signals like those known for stress genes in bacteria, and the promoters are typically archaeal—of eukaryotic type in sequence and location with respect to the transcription-initiation site (MACARIO and CONWAY DE MACARIO 1997 , MACARIO and CONWAY DE MACARIO 1999 ). In this regard, the archaeal stress genes that constitute the chaperone machine are similar to the other, nonstress genes studied in archaea (BROWN et al. 1989 ; ZILLIG et al. 1993 ).

	ARCHAEAL CHAPERONINS

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

Bacterial chaperone machine and eukaryotic chaperonin system in the same cell:
Another level of complexity, or another facet of peculiarity, is added to the archaeal complexion by the nature of its chaperonin system as compared to that of the chaperone machine. We have already discussed the hybrid character of the archaeal hsp70(dnaK)-locus genes and emphasized the similarity of their protein products (the chaperone machine) with bacterial homologs. It was concluded that the chaperone Hsp70(DnaK) and the cochaperones Hsp40(DnaJ) and GrpE are bacterial-like and, possibly, of bacterial origin.

In sharp contrast, the archaeal chaperonin system of the Hsp60 family of stress proteins, called thermosome, is more closely related to the eukaryotic homolog TRiC than to the bacterial GroEL/S counterpart (TRENT et al. 1991 ; ANDRA et al. 1996 ; KLUMPP and BAUMEISTER 1998 ).

The coexistence of a chaperone machine of bacterial type with a chaperonin system similar to that of eukaryotes appears as another incongruity of archaea. We must recognize, however, that we are scrutinizing archaea, only recently the focus of our attention, with the eyes and biases developed over many years of study of bacteria and eukaryotes; it is therefore not surprising that we tend to consider an incongruity anything that does not fit into the framework of knowledge derived from these previous studies.

In any event, these seemingly puzzling features raise interesting questions. Does the archaeal chaperone machine interact with the TRiC-like thermosome? Does the archaeal machine interact with another chaperonin system, unique to the archaea and yet to be identified, which would play a role similar to that reserved for the GroEL/S complex in bacteria?

These questions are made even more pertinent if one considers that, while the interaction of the chaperone machine with GroEL/S does play a role in protein folding and is physiologically significant in bacteria (GEORGOPOULOS and WELCH 1993 ; NETZER and HARTL 1998 ; KEDZIERSKA et al. 1999 ), an interaction between the eukaryotic Hsp70(DnaK) and its teammates such as Hsp40(DnaJ) and other cofactors with TRiC is doubtful, or at least not required to fold or refold the majority of proteins in the eukaryotic cytosol (VAINBERG et al. 1998 , and references therein; ROMMELAERE et al. 1999 ).

	EVOLUTION

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

The have and have nots:
If one adopts as a data-organizing framework the rRNA-based tree of life that classifies all extant organisms into three main phylogenetic lineages or domains, Bacteria, Archaea, and Eucarya (WOESE et al. 1990 ), it becomes apparent that organisms of the three domains do have the hsp70(dnaK) gene, except some archaeal species. We cannot yet discern the principles ruling the distribution of the gene among archaea because of insufficient data. Some hints are emerging, though. For example, while all the extreme thermophiles studied lack the gene (LANGE et al. 1997 ; KAWARABAYASI et al. 1998A , KAWARABAYASI et al. 1998B ; GRIBALDO et al. 1999 ), the extreme halophiles have it (GUPTA and SINGH 1992 , GUPTA and SINGH 1994 ; GRIBALDO et al. 1999 ), and the same is true for all the Methanosarcina species, meso- and thermophilic (CLARENS et al. 1995 ; and E. CONWAY DE MÀCARIO and A. J. L. MÀCARIO, unpublished results), but contrary to all the Methanococcus species (BULT et al. 1996 ; LANGE et al. 1997 ; GRIBALDO et al. 1999 ) examined up to the present time (Table 1). Interestingly, all extreme thermophilic bacteria investigated do have the gene (DECKERT et al. 1998 ; GRIBALDO et al. 1999 ), which is the opposite of the situation in extreme thermophilic archaea.

Is the absence of hsp70(dnaK) an inherent characteristic of archaea, or is it due to gene loss?
A recent analysis of Hsp70(DnaK) proteins from a series of organisms representing the three phylogenetic domains suggests that the gene found in some archaeal species is not aboriginal but foreign—namely, that it was received from another lineage via lateral transfer (GRIBALDO et al. 1999 ). If lateral gene transfer was the mechanism that brought hsp70(dnaK) into the chromosome of certain methanogens (Table 1), many details, which are not easily apparent at the present time, remain to be elucidated to explain features seemingly inherent to that event. For example, explanations are needed for the presence of the grpE and hsp40(dnaJ) genes near hsp70(dnaK), the preservation of their organization in the locus, and the conservation, or lack thereof, of the intergenic regions. Also, if one assumes that the genes came en bloc from a bacterium, one would have to explain why there are no bacterial promoters in the expected locations, where one finds archaeal (eukaryotic-type) promoters instead (MACARIO and CONWAY DE MACARIO 1997 , MACARIO and CONWAY DE MACARIO 1999 ).

One for all and all for one:
The data indicate that whenever the hsp70(dnaK) gene occurs in a genome, the other two members of the chaperone machine are also present. While the proteins encoded are of the bacterial type and are conserved according to comparative analyses, the intergenic regions are not, except perhaps that between hsp70(dnaK) and hsp40(dnaJ). These two genes appear often in the order 5'hsp70(dnaK)-hsp40(dnaJ)-3', and are separated by only a couple of hundred base pairs at most. These features of the archaeal loci are also observed in bacteria with few exceptions as exemplified by Lactococcus lactis (EATON et al. 1993 ), which tends to support the idea that the two genes evolved together and were transferred as a unit from a bacterium to an archaeon.

The nonaligned:
GrpE is considerably less conserved than the other two components of the chaperone machine. This is demonstrated by the low percentage of amino acid identity when compared to bacterial homologs (CONWAY DE MACARIO et al. 1994 ; NAYLOR et al. 1996 , NAYLOR et al. 1998 ), and by the variable location of the grpE gene in the chromosome. In the archaea investigated, grpE is part of the hsp70(dnaK) locus and is located upstream of this gene. The same is the case for many bacterial loci, but there are exceptions that confirm the low degree of conservation of grpE. For example, this gene is not part of the hsp70(dnaK) locus in E. coli (BLATTNER et al. 1997 ), Vibrio harveyi (KLEIN et al. 1998 ), or Porphyromonas gingivalis (YOSHIDA et al. 1999 ). Furthermore, although grpE is in the hsp70(dnaK) locus in Mycobacterium tuberculosis (COLE et al. 1998 ) and in Streptomyces coelicolor (BRANS et al. 1996 ), it does not lie upstream of hsp70(dnaK) but in between this gene and hsp40(dnaJ).

	CONCLUSION

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

It is not yet possible to ascertain in what way, or why, some archaeal species acquired the hsp70(dnaK) and the other two components of the chaperone machine triad, hsp40(dnaJ) and grpE, while other species did not. Also, data are still insufficient to determine the overall distribution of hsp70(dnaK) in the various archaeal branches, and to establish whether or not this gene is always accompanied by the other two. Ideally, representatives of all the archaeal branches, and several species for each genus, should be tested to elucidate the evolutionary history of the chaperone machine in archaea. In addition, it would help considerably to understand this history if the genes' itineraries could be traced within groups of organisms, bacteria and archaea, that inhabit the same ecological niche or that are presumed to have shared the same ecosystem in the past. These ecosystem-related studies would be useful to draw conclusions about whether or not lateral gene transfer did occur more often between organisms that shared an ecological niche than between species with different habitats.

It is difficult, if not impossible, at the present time to distinguish between the two models for the distribution of hsp70(dnaK) in archaea: gene loss in the primitive archaeal lineage or gene acquisition in some but not all subsequent branches. Random, scattered gene loss in some branches would seem less likely to have happened than random gene gain. However, gene loss cannot be excluded as a possible cause of gene absence in some of the extant archaeal species. There is no known natural law that would preclude it, however improbable the event might appear to us.

A molecular phylogenetic analysis based on sequence comparisons (see, e.g., GRIBALDO et al. 1999 ) is a viable alternative to generate possible scenarios, but one must make critical assumptions concerning the rRNA-based phylogenetic tree and its reliability to resolve early branchings and concerning the location of the tree's root. It is generally acknowledged now that early branchings are not well resolved by rRNA comparisons (MARTIN 1996 ; WOESE 1998A ) and that tree-rooting efforts generate different results depending on the protein gene and the method used (BROWN and DOOLITTLE 1997 ; FORTERRE 1997 ). It would, therefore, seem that the molecular phylogenetic approach for elucidating the evolution of the chaperone machine, or that of the hsp70(dnaK) gene alone, will have to be postponed until more genes from a variety of organisms are sequenced, and tree-making and tree-rooting procedures are more accurate than those available today.

On the basis of our current knowledge of the distribution of hsp70(dnaK) (Table 1) and a recent version of the rRNA-based phylogenetic tree (WOESE 1998B ), it is tempting to speculate about possible scenarios. Assuming that the Archaea was a gene-less lineage from the beginning, it would seem possible that the gene transfer event that brought the gene(s) into archaea occurred relatively late. Perhaps it happened in the Euryarchaeota branch after the separation of Archaeoglobus, which does not have the genes (KLENK et al. 1997 ), and before the radiation of Thermoplasmae, Methanosarcinae, and the extreme halophiles, which have hsp70(dnaK) (Table 1). However, this seemingly straightforward picture fails to explain the absence of the gene in Methanospirillum hungateii (LANGE et al. 1997 ). Methanospirilla originated from the Euryarchaeota branch after Archaeoglobus and should have the gene according to the above proposal. It should be mentioned, though, that failure to detect the gene in M. hungateii may be attributed to the method used, namely Southern blotting with a heterologous probe, which could have produced a false negative result. So, a final answer to the question about the gene's absence will come only when the M. hungateii's genome is sequenced or if the gene is cloned from this organism.

The proposal that the gene transfer event occurred after the Archaeoglobus divergence is also supported by the observation that the three species of Methanococcus (a branch that separated before Archaeoglobus did) investigated up to this moment do not have the gene either, and the same is true for Methanopyrus and Methanothermus (Table 1). Once again, this proposal, though satisfying at first glance, is obscured by the fact that Methanobacterium, which also originated before Archaeoglobus, does have the three genes (Table 1). These problems are only a sample of the very exciting challenges that are posed by archaea and suggest ways one might follow for advancing our understanding of the archaeal chaperone machine.

	ACKNOWLEDGMENTS

Work in the authors' laboratories was supported in part by a grant from the United States Department of Energy.

Manuscript received March 26, 1999; Accepted for publication May 3, 1999.

	LITERATURE CITED

TOP ABSTRACT The players The action Interaction Questions THE ARCHAEAL CHAPERONE MACHINE ARCHAEAL CHAPERONINS EVOLUTION CONCLUSION LITERATURE CITED

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