Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates.

Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T(opt) = 55 °C), Caldicellulosiruptor obsidiansis (T(opt) = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T(opt) = 100 °C) and Thermotoga maritima (T(opt) = 80 °C). Multi-well plates were incubated at various temperatures for approximately 72-120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD(600). Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

Expression and characterization of an extremely thermostable ß-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638.

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This ß-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both ß-galactosidase and ß-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-ß-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable ß-glycosidase may be useful for food and pharmaceutical applications.

Preliminary neutron crystallographic analysis of selectively CH3-protonated deuterated rubredoxin from Pyrococcus furiosus.

Neutron crystallography is used to locate H atoms in biological materials and can distinguish between negatively scattering hydrogen-substituted and positively scattering deuterium-substituted positions in isomorphous neutron structures. Recently, Hauptman & Langs (2003; Acta Cryst. A59, 250-254) have shown that neutron diffraction data can be used to solve macromolecular structures by direct methods and that solution is aided by the presence of negatively scattering H atoms in the structure. Selective-labeling protocols allow the design and production of H/D-labeled macromolecular structures in which the ratio of H to D atoms can be precisely controlled. Methyl selective-labeling protocols were applied to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into deuterated rubredoxin from Pyrococcus furiosus (PfRd). Here, the production, crystallization and preliminary neutron analysis of a selectively CH3-protonated deuterated PfRd sample, which provided a high-quality neutron data set that extended to 1.75 A resolution using the new LADI-III instrument at the Institut Laue-Langevin, are reported. Preliminary analysis of neutron density maps allows unambiguous assignment of the positions of H atoms at the methyl groups of the valine and leucine residues in the otherwise deuterated rubredoxin structure.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus.

The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 A resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.