DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher kcat/Km values than those previously determined, primarily as a result of better binding of substrate. The Km values for the two new reductases were 57 and 67 microM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum.

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif.

Extremophiles in astrobiology: per Ardua ad Astra.

As we consider the possibilities of finding life on other planets, it behooves us to evaluate what we know about the limits for life on planet Earth. In our continued exploration of Earth, we are finding microbes in a variety of unexpected habitats. In geothermal hot springs, we have discovered organisms thriving at temperatures near the boiling point of water and at pH values down to 0.5; in the deepest parts of the oceans, those that grow optimally at pressures above 1000 bars and die at pressures below 500 bars; and at the poles, those that grow below the freezing point of water and die at temperatures above 10 degrees C. All of these organisms are living proof that the biochemical "machinery" of life can be adapted to conditions that, from our anthropocentric perspective, appear to be extreme. By studying the molecular adaptations of extremophiles, we begin to identify the critical cellular components that expand the envelope for life. As an example, I will discuss what we have learned about the role of the proteins we call "heat shock proteins" in pushing the upper temperature limit of life and how our studies have provided a new perspective on the function of these proteins.

Chaperonin filaments: their formation and an evaluation of methods for studying them.

Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo (J. D. Trent et al., 1997, Proc. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATPgammaS, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.

Two-dimensional crystals of reconstituted beta-subunits of the chaperonin TF55 from Sulfolobus shibatae.

We have obtained 2-dimensional crystals of the beta-subunits of the chaperonin TF55 from Sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. The subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. From electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.

Chaperonin filaments: the archaeal cytoskeleton?

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent approximately 4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations >/= 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

Conformational cycle of the archaeosome, a TCP1-like chaperonin from Sulfolobus shibatae.

The major heat shock proteins in the archaeon Sulfolobus shibatae are similar to the cytosolic eukaryotic chaperonin and form an 18-subunit bitoroidal complex. Two sequence-related subunits constitute a functional complex, named the archaeosome. The archaeosome exists in two distinct conformational states that are part of chaperonin functional cycle. The closed archaeosome complex binds ATP and forms an open complex. Upon ATP hydrolysis, the open complex dissociates into subunits. Free subunits reassemble into a two-ring structure. The equilibrium between the complexes and free subunits is affected by ATP and temperature. Denatured proteins associate with both conformational states as well as with free subunits that form an intermediate complex. These unexpected observations suggest a new mechanism of archaeosome-mediated thermotolerance and protein folding.

The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettasomes and the eukaryotic TCP1 protein family and the distant relationship to the bacterial GroEL/HSP60 proteins.

Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.

Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.

A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1.

There is evidence to suggest that components of archaebacteria are evolutionarily related to cognates in the eukaryotic cytosol. We postulated that the major heat-shock protein of the thermophilic archaebacterium, Sulfolobus shibatae, is a molecular chaperone and that it is related to an as-yet unidentified chaperone component in the eukaryotic cytosol. Acquired thermotolerance in S. shibatae correlates with the predominant synthesis of this already abundant protein, referred to as thermophilic factor 55 (TF55). TF55 is a homo-oligomeric complex of two stacked 9-membered rings, closely resembling the 7-membered-ring complexes of the chaperonins, groEL, hsp60 and Rubisco-binding protein. The TF55 complex binds unfolded polypeptides in vitro and has ATPase activity-features consistent with its being a molecular chaperone. The primary structure of TF55, however, is not significantly related to the chaperonins. On the other hand, it is highly homologous (36-40% identity) to a ubiquitous eukaryotic protein, t-complex polypeptide-1 (TCP1). In Saccharomyces cerevisiae, TCP1 is an essential protein that may play a part in mitotic spindle formation. We suggest that TF55 in archaebacteria and TCP1 in the eukaryotic cytosol are members of a new class of molecular chaperones.

Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12.

The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins. In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, to a much lesser extent, two minor proteins (28 and 35 kilodaltons). Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant. They could not yet be related to known heat shock proteins. In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes. However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.

Possible artefactual basis for apparent bacterial growth at 250 degrees C.

Baross and Deming reported that thermophilic marine bacteria isolated from the vicinity of a submarine hot-spring grow at temperatures up to at least 250 degrees C. They did not, however, conduct the appropriate control experiments to eliminate the possibilities of chemical artefacts or contamination. Here, in experiments using the same growth medium, the same temperature and pressure apparatus and the same sampling and analytical procedures, we report results nearly identical to theirs. We conclude that their evidence indicating bacterial growth at 250 degrees C may be due to artefacts produced in the medium and to contaminants introduced during sample processing.

Marine snow: microplankton habitat and source of small-scale patchiness in pelagic populations.

In near-surface waters of the neritic zone, the fragile aggregate material called "marine snow" is enriched by a variety of planktonic organisms and detrital products of plankton. Here marine snow is a source of patchiness and taxonomic diversity for microplankton populations and is a likely food resource and recycling agent for fecal particles.