Dennis L. Maeder

Genomic sequence of hyperthermophile, Pyrococcus furiosus: implications for physiology and enzymology.

Publisher Summary Microorganisms that are able to grow at temperatures above 90° are called as “hyperthermophiles.” They form a diverse group consisting of autotrophic and heterotrophic prokaryotes, including several bacteria, although the majority of hyperthermophiles are Archaea. Most of the conventional tools of genetic and physiological analysis are either not effective or very difficult to apply to these microorganisms because of their unusual growth conditions. As a result, relatively slow progress has characterized the field since its inception. A new paradigm has characterized the field recently, with the availability of complete genome sequences of five hyperthermophiles. A unique resource for comparative studies of hyperthermophiles—namely, the complete genomic sequences of three species in the genus Pyrococcus, is now accessible. Although these strains are quite similar in their fermentative, sulfur-reducing growth physiology and optimal growth temperatures, which are in the range 98-100°, significant issues of genome divergence are emerging from the ongoing study of their genomic sequences.

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough

Abstract A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395 m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98°C (maximum growth temperature 102°C), but capable of prolonged survival at 105°C. Optimum growth was at pH 7 (range 5–8) and NaCl concentration 2.4% (range 1%–5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn2+-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA–DNA hybridization (<63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp. nov.

Evidence of recent lateral gene transfer among hyperthermophilic Archaea

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full‐length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose‐binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus.

The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105 degrees C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105 degrees C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56 degrees C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50 degrees C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.

Multi-subunit assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular protection at high temperature.

Abstract. The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, α-crystallin-like protein in response to exposure to extreme temperatures, above 103°C. The P. furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes. Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions. We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly. The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics. The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP. The amino terminal domain was not required for inter-species thermotolerance.

A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 degrees C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S(o)), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10-100 microM sodium tungstate in S(o)-free medium. However, P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcus furiosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.

Novel evolutionary histories and adaptive features of proteins from hyperthermophiles

The hyperthermophiles include both bacteria and archaea, although the majority of isolates growing above 100 degreesC are archaea. Newly described adaptive features of hyperthermophiles include proteins stable to 200 degreesC, nucleosomes, chaperonins and high-capacity DNA modifying enzymes. The ongoing release of genomic sequence data from hyperthermophiles will continue to accelerate the discovery of novel proteins.

Novel Chaperonins in a Prokaryote

Group II chaperonins belong to the Hsp60 family occurring in archaea and eukaryotes. The archaeal chaperonins build the thermosome, which is similar to the eukaryotic CCT (chaperonin-containing TCP-1). Eukaryotes have eight subunits, and up until now, it was thought that archaea had between one and three subunits, depending on the species. We now report two novel subunits, termed Hsp60-4 and Hsp60-5, in the archaeon Methanosarcina acetivorans, which also has Hsp60-1, Hsp60-2, and Hsp60-3 with orthologs in Methanosarcinae. Hsp60-4 and Hsp60-5 occur only in M. acetivorans, which makes this organism unique in that it has the highest number of chaperonin subunits ever described for an archaeon. Evolutionary analysis suggests that either Hsp60-4 or Hsp60-5 paralogs have arisen by gene duplication with vastly increased accepted substitution rates or that they represent ancestral types found only in this species.

[3] Glutamate dehydrogenases from hyperthermophiles

Publisher Summary This chapter focuses on a key enzyme of nitrogen metabolism in hyperthermophiles, glutamate dehydrogenase (GDH). GDHs are widely distributed enzymes involved in ammonia assimilation and catabolism of glutamate in microorganisms. GDH catalyses the reversible oxidative deamination of glutamate to 2-oxoglutarate and ammonia using NAD or NADP as cofactors. GDH represents an enzymatic link between major catabolic and biosynthetic pathways via the tricarboxylic acid (TCA) cycle intermediate 2-oxoglutarate. Analysis of GDH structures has provided the basis for comparative studies of extreme protein thermostability in this relatively complex enzyme. The chapter provides an overview of the purification and properties of thermostable GDHs and a review of the contributions that these studies have made to the understanding of the basis for exceptional enzyme stability.