Methanococcus Thermolithotrophicus Research Articles

Insertion of IF (insertion-in-flap) Domain into F36V to Provide Higher Activity of Peptidyl-propyl cis-trans Isomerase

Molecular chaperones are required to help nascent proteins fold properly or protect cells against various stresses by means of preventing their aggregation. Attempts to elucidate the molecular mechanisms by which chaperones can solubilize aggregation-prone proteins such as Aβ and αsynuclein (α-Syn) are relatively limited. So far, just a few publications have been written on the functional roles of chaperones such as Hsp104 and FKBP12 on controlling the aggregation of α-Syn into fibrillar structures. In our previous study, we described how functional Hsp104 protein not only inhibited α-Syn aggregation but also resolubilized aggregated protein in vitro. Recently, Engelborghs group has reported that FKBP12, containing the peptidyl-prolyl isomerase (PPIase) activity, accelerated fibril formation of α-Syn containing 5 Pro residues, which can be isomerized between cisand trans-conformations. Little is known about the molecular details that explain how molecular chaperones regulate fibril formation in many neurodegenerative diseases such as Alzheimer disease and Parkinson disease. In pursuit of developing a candidate regulator of fibril formation, we have attempted to engineer the FKBP system to acquire a higher activity of PPIase along with the chaperone activity. The IF (insertion-in-flap) domain with 61amino acids starting with AYG and ending with LKF was adopted from SlyD (sensitive-to-lysis) in Escherichia coli (E. coli) as well as from MtFKBP17 of Methanococcus thermolithotrophicus. The structural analysis of the IF domain, protruding from a loop of the FKBP domain near the PPIase catalytic site, indicated its hydrophobicity so that it can interact with unfolded proteins in the nonpolar region found in extended structures such as a β-sheet. Folding of the IF domain is influenced by the FKBP domain, in such a way that the guest IF domain of SlyD folds rapidly when the host FKBP domain of SlyD is folded and is unfolded when the host is unfolded. Construction of an FKBPIF hybrid replacing the flap by an IF domain was previously reported by the Schmid group. The resulting hybrid FKBPIF protein exhibited strong folding activity as well as chaperone activity. For example, upon the refolding of RCM-T1 (a disulfide-reduced and Scarboxymethylated form of a variant of RNase T1), the hybrid FKBPIF showed the kcat/Km enhanced by 200 fold. In monitoring chaperone activity, FKBPIF exhibited inhibition of CS (citrate synthase) aggregation whereas FKBP did not. The insertion effect of IF in FKBPIF was also confirmed in the inhibited aggregation of insulin reduced by dithiothreitol. Upon structural analysis of the binding site of FKBP for FK506, it was envisioned that it would be surrounded by hydrophobic residues such as F36. Further mutation of F36 to V36 yielded a better system in terms of fitting ligands through a protruding structural part. Using the structural contact between them, the Wandless group have developed a regulation system in which the stability of a certain domain

Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability

The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes.

Osmoadaptation and osmoregulation in archaea: update 2004.

The response of archaea to changes in external NaCl is reviewed and compared to what is known about osmoadaptation and osmoregulation in bacteria and eukaryotes. Cells placed in altered external NaCl exhibit short term and long term responses. The earliest events are likely to be water movement through aquaporin-like channels (efflux if external NaCl has been increased, influx into the cell if the external NaCl has been decreased) and ion movement (e.g., K+ moving in the direction opposite to water flow) through channels sensitive to osmotic pressure. A brief discussion of recent structures of homologues of these membrane proteins is presented. Accumulation of organic solutes, either by uptake from the medium or de novo synthesis, is triggered after these initial changes. Archaea have some unique organic solutes (osmolytes) that are not used by other organisms. These as well as other more common solutes have a role in stabilizing macromolecules from denaturation. Many osmolytes are distinguished by their stability in the cell and their lack of strong interactions with cellular components. A cell may respond by accumulating one or more temporary osmolytes, then over time readjust the intracellular solute distribution to what is optimal for cell growth under the new conditions. Coupled with the movement and accumulation of solutes is the induction of stress proteins (e.g., chaperonins) and, in some cases, transcriptional regulation of key enzymes. The response to NaCl stress of Methanococcus thermolithotrophicus is presented as an example of how one particular archaeon responds and adapts to altered osmotic pressure. The detailed response of many other archaea to osmotic stress will be needed in order to identify features (aside from some of the organic osmolytes) unique to the organisms in this kingdom.

Structures of Thermophilic and Mesophilic Adenylate Kinases from the Genus Methanococcus

The crystal structures of adenylate kinases from the thermophile Methanococcus thermolithotrophicus and the mesophile Methanococcus voltae have been solved to resolutions of 2.8 Å and 2.5 Å, respectively. The structures of the enzymes are similar to that of the adenylate kinase from archaeal Sulfolobus acidocaldarius in many respects such as the extended central β-sheets, the short LID domain, and the trimeric state. The analysis of unligated and AMP-bound subunits of M. voltae suggests that movements of two mobile domains are not independent of each other. The methanococcal structures are examined with respect to their lack of the “invariant” Lys residue within the phosphate-binding loop, and two Arg residues in the LID domain are proposed as substituting residues based on their conservation among archaeal adenylate kinases and mobility within the structures. Since S. acidocaldarius adenylate kinase has the invariant Lys residue as well as the two Arg residues, its phosphate-binding loop is examined and compared with those of other adenylate kinases. On the basis of the comparison and other available biochemical data, the unusual conformation of the Lys residue in S. acidocaldarius adenylate kinase is explained. Despite possessing 78% sequence identity, the methanococcal enzymes exhibit significantly different thermal stabilities. To study the determinants of thermostability, several structural features including salt-links, hydrogen bonds, packing density, surface to volume ratio and buried surface area are compared between the enzymes. From their difference in apolar buried surface area, hydrophobic interaction is proposed to be a basis for the disparate thermostabilities, and the corresponding free energy difference is also estimated. Results of previous mutational studies are interpreted in terms of the crystal structures, and support the importance of hydrophobic interactions in thermostability.

Three-dimensional Solution Structure of an Archaeal FKBP with a Dual Function of Peptidyl Prolyl cis– trans Isomerase and Chaperone-like Activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis– trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.

Morphological, small subunit rRNA, and physiological characterization of Trimyema minutum (Kahl, 1931), an anaerobic ciliate from submarine hydrothermal vents growing from 28 degrees C to 52 degrees C.

A thermophilic strain of Trimyema minutum was isolated from the hydrothermally heated sea floor at Vulcano Island (Italy) and cultivated monoxenically on Marinobacter sp. and Methanococcus thermolithotrophicus. It can be propagated strictly anaerobically and is sensitive to oxygen: if exposed to air at 48 degrees C all cells die within 60 min. It grows from 0.45-7.2% (w/v) salt and at pH 6.0-8.0. The isolate is the most extreme thermophilic ciliate which ever has been cultivated, exhibiting an optimal growth temperature of 48 degrees C (doubling time 6 h). Growth occurs between 28 degrees C and 52 degrees C. Trimyema minutum is redescribed using live observation and silver impregnation. Its morphology and the small subunit ribosomal RNA sequence is distinctly different from that of T. compressum, but morphology is highly similar to that of T. shoalsia Nerad et al. 1995, which is thus probably a junior synonym of T. minutum. To stabilize the bewildering species taxonomy in Trimyema, we suggest to recognize our population as a neotype of T. minutum.

Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

The Basal Transcription Factors TBP and TFB from the Mesophilic Archaeon Methanosarcina mazeii: Structure and Conformational Changes upon Interaction with Stress-gene Promoters

Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii , sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 °C).

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates β-glutamate and l-α-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N ϵ-acetyl-β-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K + occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K + is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K +, far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K + concentration (over 10–15 min), at which point synthesis and accumulation of primarily l-α-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30–100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion ( N ϵ-acetyl-β-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N ϵ-acetyl-β-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N ϵ-acetyl-β-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.

FK506-binding protein-type peptidyl-prolyl cis– trans isomerase from a halophilic archaeum, Halobacterium cutirubrum

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis– trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3 kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent.

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.

Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (Σ(T%-E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16 812 and 17 694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3 721 for N-PPIase and 5 285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.

FK506 binding protein from a thermophilic archaeon, methanococcus thermolithotrophicus, has chaperone-like activity in vitro

The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.

Archaeal peptidyl prolyl cis-trans isomerases (PPIases).

PPIases are ubiquitous in living organisms. While 3 families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases. Among 2 cyclophilins found in Archaea, only Halobacterium cyclophilin (HcCyP19) has been characterized. It is a cyclosporin A (CsA) sensitive CyP with a MW of 19.4kDa. The PPIase activity and CsA sensitivity of this CyP is higher at higher salt concentration in the medium. No parvulin or its homolog has been found in Archaea. Two types of FKBPs, 26-30kDa long type and 17-18 kDa short type FKBP, have been found in Archaea. While the N-terminal regions of these 2 type FKBPs are similar to each other, the long type archaeal FKBP has an additional ca. 100 amino-acid sequence at its C-terminal region. In comparison with human HsFKBP12, the N-terminal region of the archaeal FKBP has 2 insertion sequences in the regions corresponding to Bulge and Flap of HsFKBP12. A short type archaeal FKBP from Methanococcus thermolithotrophicus has been shown to have not only a PPIase activity but also a chperone like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates. Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the Flap seemed to be important.

Osmoadaptation and osmoregulation in archaea.

The response of archaea to changes in external NaCl is reviewed and compared to what is known about osmoadaptation and osmoregulation in bacteria and eukaryotes. Cells placed in altered external NaCl exhibit short term and long term responses. The earliest events are likely to be water movement through aquaporin-like channels (efflux if external NaCl has been increased, influx into the cell if the external NaCl has been decreased) and ion movement (e.g., K+ moving in the direction opposite to water flow) through channels sensitive to osmotic pressure. Accumulation of organic solutes, either by uptake from the medium or de novo synthesis, is triggered after these initial changes. Archaea have some unique organic solutes (osmolytes) that are not used by other organisms. These as well as other more common solutes have a role in stabilizing macromolecules from denaturation. Many osmolytes are distinguished by their stability in the cell and their lack of strong interactions with cellular components. A cell may respond by accumulating one or more temporary osmolytes, then over time readjust the intracellular solute distribution to what is optimal for cell growth under the new conditions. Coupled with the movement and accumulation of solutes is the induction of stress proteins (e.g., chaperonins) and, in some cases, transcriptional regulation of key enzymes. The response to NaCl stress of Methanococcus thermolithotrophicus is presented as an example of how one particular archaeon responds and adapts to altered osmotic pressure. Clearly, the detailed response of other archaea to osmotic stress will be needed in order to identify features (aside from some of the organic osmolytes) unique to the organisms in this kingdom.

FK506 Binding Protein from a Thermophilic Archaeon, Methanococcus thermolithotrophicus, Has Chaperone-like Activity in Vitro

The in vitro protein folding activity of an FKBP (FK506 binding protein, abbreviated to MTFK) from a thermophilic archaeon, Methanococcus thermolithotrophicus, was investigated. MTFK exhibited FK506 sensitive PPIase (peptidyl prolyl cis-trans isomerase) activity which accelerated the speed of ribonuclease T1 refolding, which is rate-limited by isomerization of two prolyl peptide bonds. In addition, MTFK suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. We called this activity of MTFK the chaperone activity. The chaperone activity of MTFK was also inhibited by FK506. Alignment of the amino acid sequences of MTFK with human FKBP12 showed that MTFK has two insertion sequences, consisting of 13 and 44 amino acids, at the N- and C-termini, respectively [Furutani, M., Iida, T., Yamano, S., Kamino, K., and Maruyama, T. (1998) J. Bacteriol. 180, 388-394]. To study the relationship between chaperone and PPIase activities of MTFK, mutant MTFKs with deletions of these insertion sequences or with amino acid substitutions were created. Their PPIase and chaperone activities were measured using a synthetic oligopeptide and denatured rhodanese as the substrates, respectively. The far-UV circular dichroism spectra of the wild type and the mutants were also analyzed. The results suggested that (1) the PPIase activity did not correlate with chaperone activity, (2) both insertion sequences were required for MTFK to take a proper conformation, and (3) the insertion sequence (44 amino acids) in the C-terminus was important for the chaperone activity.

Analysis of Thermal Stabilizing Interactions in Mesophilic and Thermophilic Adenylate Kinases from the GenusMethanococcus

Adenylate kinases (ADKs) from four closely related methanogenic members of the Archaea (the mesophile Methanococcus voltae (MVO), the thermopile Methanococcus thermolithotrophicus (MTH), and the extreme thermopiles Methanococcus igneus (MIG) and Methanococcus jannaschii (MJA)) were characterized for their resistance to thermal denaturation. Despite possessing between 68 and 81% sequence identity, the methanococcal ADKs significantly differed in their stability against thermal denaturation, with melting points ranging from 69 to 103 degrees C. The high sequence identity between these organisms allowed regions of the MVO and MJA ADKs to be exchanged, producing chimeric ADKs with significantly altered thermal stability. Up to a 20 degrees C increase or decrease in stability was achieved for chimeric ADKs, whereas 88% of the original protein sequence was maintained. Based on our previous structural modeling studies, we conclude that cooperative interactions within the hydrophobic protein core play an integral role in determining the differences in structural stability observed between the methanococcal ADKs. From comparisons of the effects of temperature on protein unfolding and optimal enzymatic activity, we also conclude that thermostability and enzymatic temperature optima are influenced differently by molecular modifications and thus that the protein flexibility required for activity and stability, respectively, is not unconditionally linked within the methanococcal ADKs.

Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO 2 and H 2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-α-glutamate, β-glutamate, and N ϵ-acetyl-β-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of α-glutamate (over a 30-min time-scale) with smaller amounts of β-glutamate and little if any of the zwitterion N ϵ-acetyl-β-lysine. This is a marked contrast to adapted growth in high NaCl where N ϵ-acetyl-β-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced β-glutamate and N ϵ-acetyl-β-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.

Group II Chaperonin in a Thermophilic Methanogen,Methanococcus thermolithotrophicus: CHAPERONE ACTIVITY AND FILAMENT-FORMING ABILITY

A gene encoding 544 amino acids for a subunit of group II chaperonin (thermosome) was cloned from a thermophilic methanogen, Methanococcus thermolithotrophicus. The deduced amino acid sequence showed 66.5, 56.1, and 20.1% similarities to those of Methanopyrus kandleri and Thermoplasma acidophilum and group I chaperonin of Escherichia coli, respectively. We call this chaperonin MTTS (M. thermolithotrophicus thermosome). The MTTS gene was expressed in E. coli. The purified recombinant MTTS seemed to be monomeric on gel filtration in the absence of Mg2+ and ATP. The monomer assembled to an oligomer (complex) in the presence of 50 mM MgCl2, 0.25 mM ATP, and 0.3 M (NH4)2SO4. It was eluted immediately before the elution volume of E. coli GroEL tetradecamer on gel filtration with a TSKgel G3000SWXL column. This reconstructed MTTS complex showed the cylindrical structure with two stacked rings in electron microscopy. The MTTS complex formed filamentous structures in the presence of Mg2+ and ATP at the protein concentration above 3.0 mg/ml. This filament formation was reversible. The MTTS filament was dissociated to the complex by dilution to the protein concentration of 0.2 mg/ml, even in the presence of Mg2+ and ATP. The MTTS complex exhibited weak ATPase activity with the hydrolysis rate of 74 mol of ATP hydrolysis/mol of MTTS complex/min at 70 degreesC. The MTTS complex promoted the refolding of chemically denatured thermophilic archaeal citrate synthase and glucose dehydrogenase at 50 degreesC in an ATP-dependent fashion. The analysis of nucleotide specificity of chaperone activity of MTTS suggested that it was coupled with hydrolysis of ATP, CTP, or UTP.