Isolation, characterization, and survival strategies of Thermotoga sp. strain PD524, a hyperthermophile from a hot spring in Northern Thailand.

A hyperthermophilic Thermotoga sp. strain PD524 was isolated from a hot spring in Northern Thailand. Cells were long-curved rods (0.5-0.6 × 2.5-10 µm) surrounded by a typical outer membrane toga. Strain PD524 is aero-tolerant at 4 °C but is aero-sensitive at 80 °C. A heat resistant subpopulation was observed in late-stationary phase. Cells from late-stationary phase were revealed remarkably less sensitive to 0.001 % SDS treatment than cells from exponential phase. The temperature range for growth was 70-85 °C (opt. temp. 80 °C), pH range was 6-8.5 (opt. pH 7.5-8.0), and NaCl range of 0 to <10 g/L (opt. 0.5 g/L). Glucose, sucrose, maltose, fructose, xylose, mannose, arabinose, trehalose, starch, and cellobiose were utilized as growth substrates. Growth was inhibited by S(o). Growth yield was stimulated by SO 4 (=) but not by S2O 3 (=) and NO3 (-). Analysis of 16S rRNA gene sequence (KF164213) of strain PD524 revealed closest similarity (96 %) to Thermotoga maritima MSB8(T), T. neapolitana NES(T), T. petrophila RKU-1(T), and T. naphthophila RKU-10(T).

Archaeal-like chaperonins in bacteria.

Chaperonins (CPN) are ubiquitous oligomeric protein machines that mediate the ATP-dependent folding of polypeptide chains. These chaperones have not only been assigned stress response and normal housekeeping functions but also have a role in certain human disease states. A longstanding convention divides CPNs into two groups that share many conserved sequence motifs but differ in both structure and distribution. Group I complexes are the well known GroEL/ES heat-shock proteins in bacteria, that also occur in some species of mesophilic archaea and in the endosymbiotic organelles of eukaryotes. Group II CPNs are found only in the cytosol of archaea and eukaryotes. Here we report a third, divergent group of CPNs found in several species of bacteria. We propose to name these Group III CPNs because of their distant relatedness to both Group I and II CPNs as well as their unique genomic context, within the hsp70 operon. The prototype Group III CPN, Carboxydothermus hydrogenoformans chaperonin (Ch-CPN), is able to refold denatured proteins in an ATP-dependent manner and is structurally similar to the Group II CPNs, forming a 16-mer with each subunit contributing to a flexible lid domain. The Group III CPN represent a divergent group of bacterial CPNs distinct from the GroEL/ES CPN found in all bacteria. The Group III lineage may represent an ancient horizontal gene transfer from an archaeon into an early Firmicute lineage. An analysis of their functional and structural characteristics may provide important insights into the early history of this ubiquitous family of proteins.

Extracellular Fe(III) reductase structure reveals a modular organization enabling S-layer insertion and electron transfer to insoluble substrates.

The thermophilic anaerobic Gram-positive bacterium Carboxydothermus ferrireducens utilizes insoluble Fe(III) oxides as electron acceptors in respiratory processes using an extracellular 11-heme cytochrome c OmhA as a terminal reductase. OmhA is able to transfer electrons to soluble and insoluble Fe(III) compounds, substrates of multiheme oxidoreductases, and soluble electron shuttles. The crystal structure of OmhA at 2.5 Å resolution shows that it consists of two functionally distinct parts: the cytochrome с electron transfer and the S-layer binding domains. Nonaheme C-terminal subdomain of the cytochrome с domain is structurally similar to the extracellular multiheme cytochrome OcwA from the metal-reducing Gram-positive bacterium "Thermincola potens." S-layer binding domain of OmhA is responsible for interaction with the S-layer that surrounds the Carboxydothermus ferrireducens cell envelope. The structural foundations enabling the embedding of extracellular multiheme cytochromes to the S-layer of a Gram-positive-type cell wall and putative electron transfer pathways to insoluble minerals are discussed.

Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaeal chaperonin.

Amyloid depositions containing exceptionally stable ß-sheet rich protein aggregates, called fibrils are associated with prevalent and incurable neurodegenerative diseases. Chaperones are proteins that facilitate protein folding in both eukaryotes and prokaryotes. We found that a cold-adapted mutant ATP-dependant chaperonins (Hsp60) from a hyperthermophilic archaeon binds to and fragments insulin fibrils very rapidly with local targeted entry points. Individual fragments swell and the fibrillar ß-sheet is quickly transformed into a mix of α-helical and unordered protein structures. After further incubation, the fragments coalesced, forming large amorphous aggregates with poly-disperse topologies. This finding represents a new approach to the disassembly of refractory protein aggregates under physiological conditions.

Detection of circular polarization in light scattered from photosynthetic microbes.

The identification of a universal biosignature that could be sensed remotely is critical to the prospects for success in the search for life elsewhere in the universe. A candidate universal biosignature is homochirality, which is likely to be a generic property of all biochemical life. Because of the optical activity of chiral molecules, it has been hypothesized that this unique characteristic may provide a suitable remote sensing probe using circular polarization spectroscopy. Here, we report the detection of circular polarization in light scattered by photosynthetic microbes. We show that the circular polarization appears to arise from circular dichroism of the strong electronic transitions of photosynthetic absorption bands. We conclude that circular polarization spectroscopy could provide a powerful remote sensing technique for generic life searches.

The genomic basis of trophic strategy in marine bacteria.

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean's free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.

Oligomerization of an archaeal group II chaperonin is mediated by N-terminal salt bridges.

Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, (21)GRDAQRMNIL(30) was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21-50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.

A modulator domain controlling thermal stability in the Group II chaperonins of Archaea.

Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122°C. The C-terminal 15-25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif ((528)EKEKEKEGEK5(37)) proved to be a key domain for stabilization at or near 100°C. Mutations "tuned" the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca(2+), especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca(2+). The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.

Aspartic acid racemization and repair in the survival and recovery of hyperthermophiles after prolonged starvation at high temperature.

Long-term survivability is well-known for microorganisms in nutrient-depleted environments, but the damage accrued by proteins and the associated repair processes during the starvation and recovery phase of microbial life still remain enigmatic. We focused on aspartic acid (Asp) racemization and repair in the survival of Pyrococcus furiosus and Thermococcus litoralis under starvation conditions at high temperature. Despite the dramatic decrease of viability over time, 0.002% of P. furiosus cells (2.1×103 cells/mL) and 0.23% of T. litoralis cells (2.3×105 cells/mL) remained viable after 25 and 50 days, respectively. The D/L Asp ratio in the starved cells was approximately half of those from the autoclaved cells, suggesting that the starving cells were capable of partially repairing racemized Asp. Transcriptomic analyses of the recovered cells of T. litoralis indicated that the gene encoding Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) might be involved in the repair of damaged proteins by converting D-Asp back to L-Asp during the resuscitation of starved cells. Collectively, our results provided evidence that Asp underwent racemization in the surviving hyperthermophilic cells under starved conditions and PIMT played a critical role in the repair of abnormal aspartyl residues during the initial recovery of starved, yet still viable, cells.

Improved folding of recombinant protein via co-expression of exogenous chaperones.

Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon.

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.

'That which does not kill us only makes us stronger': the role of carbon monoxide in thermophilic microbial consortia.

Carbon monoxide (CO), while a potent toxin, is also a key intermediate in major autotrophic pathways such as methanogenesis and acetogenesis. The ability of purple sulfur bacteria to use CO as an energy source was first described by Uffen in 1976. The prototype extremely thermophilic carboxydotroph Carboxydothermus hydrogenoformans was described in 1991. Eight bacteria and one archaeon that utilize CO have since been isolated and described from diverse geothermal environments. They derive energy from the oxidation of CO with water to form CO(2) and H(2). Most of these isolates thrive with headspace CO partial pressures around 1 atm, which is grossly elevated relative to CO concentrations in geothermal effluents. To account for this, we suggest that under consortial growth conditions the carboxydotrophs occupy microniches in which biogenic CO accumulates locally to high concentrations. CO oxidizers dissipate these potentially toxic CO hot spots with the production of H(2), CO(2) and acetate whose subsequent oxidation fuels other thermophiles. The identification of genes related to anaerobic CO oxidation in many metagenomic databases attests to widespread distribution of carboxydotrophs. Current evidence suggests that CO-oxidizing bacteria and archaea hold a vital niche in thermophilic ecosystems.

An exceptionally stable Group II chaperonin from the hyperthermophile Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus (Pf) grows optimally at 100 degrees C and encodes single genes for the Group II chaperonin (Cpn), Pf Cpn and alpha-crystallin homolog, the small Heat shock protein (sHsp). Recombinant Pf Cpn is exceptionally thermostable and remained active in high ionic strength, and up to 3M guanidine hydrochloride (Gdn-HCl). Pf Cpn bound specifically to denatured lysozyme and ATP addition resulted in protection of lysozyme from aggregation and inactivation at 100 degrees C. While complexed to heat inactivated lysozyme, Pf Cpn showed enhanced thermostability and ATPase activity, and increased the optimal temperature for ATPase activity from 90 to 100 degrees C. Protein substrate binding also stabilized the 16-mer oligomer of Pf Cpn in 3M Gdn-HCl and activated ATPase hydrolysis in 3-5M Gdn-HCl. In addition, Pf Cpn recognized and refolded the non-native lysozyme released from Pf sHsp, consistent with the inferred functions of these chaperones as the primary protein folding pathway during cellular heat shock.

Bridging human chaperonopathies and microbial chaperonins.

Chaperonins are molecular chaperones that play critical physiological roles, but they can be pathogenic. Malfunctional chaperonins cause chaperonopathies of great interest within various medical specialties. Although the clinical-genetic aspects of many chaperonopathies are known, the molecular mechanisms causing chaperonin failure and tissue lesions are poorly understood. Progress is necessary to improve treatment, and experimental models that mimic the human situation provide a promising solution. We present two models: one prokaryotic (the archaeon Pyrococcus furiosus) with eukaryotic-like chaperonins and one eukaryotic (Chaetomium thermophilum), both convenient for isolation-study of chaperonins, and report illustrative results pertaining to a pathogenic mutation of CCT5.

Aspartic acid racemization constrains long-term viability and longevity of endospores.

Certain microorganisms survive long periods of time as endospores to cope with adverse conditions. Since endospores are metabolically inactive, the extent of aspartic acid (Asp) racemization will increase over time and might kill the spores by preventing their germination. Therefore, understanding the relationship between endospore survivability and Asp racemization is important for constraining the long-term survivability and global dispersion of spore-forming bacteria in nature. Geobacillus stearothermophilus was selected as a model organism to investigate racemization kinetics and survivability of its endospores at 65°C, 75°C and 98°C. This study found that the Asp racemization rates of spores and autoclaved spores were similar at all temperatures. The Asp racemization rate of spores was not significantly different from that of vegetative cells at 65°C. The Asp racemization rate of G. stearothermophilus spores was not significantly different from that of Bacillus subtilis spores at 98°C. The viability of spores and vegetative cells decreased dramatically over time, and the mortality of spores correlated exponentially with the degree of racemization (R2 = 0.9). This latter correlation predicts spore half-lives on the order of hundreds of years for temperatures typical of shallow marine sediments, a result consistent with studies about the survivability of thermophilic spores found in these environments.

A Multipronged Method for Unveiling Subtle Structural-Functional Defects of Mutant Chaperone Molecules Causing Human Chaperonopathies.

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.

Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901.

We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a "minimal" model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.

Life on the fringe: microbial adaptation to growth on carbon monoxide.

Microbial adaptation to extreme conditions takes many forms, including specialized metabolism which may be crucial to survival in adverse conditions. Here, we analyze the diversity and environmental importance of systems allowing microbial carbon monoxide (CO) metabolism. CO is a toxic gas that can poison most organisms because of its tight binding to metalloproteins. Microbial CO uptake was first noted by Kluyver and Schnellen in 1947, and since then many microbes using CO via oxidation have emerged. Many strains use molecular oxygen as the electron acceptor for aerobic oxidation of CO using Mo-containing CO oxidoreductase enzymes named CO dehydrogenase. Anaerobic carboxydotrophs oxidize CO using CooS enzymes that contain Ni/Fe catalytic centers and are unrelated to CO dehydrogenase. Though rare on Earth in free form, CO is an important intermediate compound in anaerobic carbon cycling, as it can be coupled to acetogenesis, methanogenesis, hydrogenogenesis, and metal reduction. Many microbial species-both bacteria and archaea-have been shown to use CO to conserve energy or fix cell carbon or both. Microbial CO formation is also very common. Carboxydotrophs thus glean energy and fix carbon from a "metabolic leftover" that is not consumed by, and is toxic to, most microorganisms. Surprisingly, many species are able to thrive under culture headspaces sometimes exceeding 1 atmosphere of CO. It appears that carboxydotrophs are adapted to provide a metabolic "currency exchange" system in microbial communities in which CO arising either abiotically or biogenically is converted to CO 2 and H 2 that feed major metabolic pathways for energy conservation or carbon fixation. Solventogenic CO metabolism has been exploited to construct very large gas fermentation plants converting CO-rich industrial flue emissions into biofuels and chemical feedstocks, creating renewable energy while mitigating global warming. The use of thermostable CO dehydrogenase enzymes to construct sensitive CO gas sensors is also in progress.

Corrigendum: Regulation of multiple carbon monoxide consumption pathways in anaerobic bacteria.

[This corrects the article on p. 147 in vol. 2, PMID: 21808633.].