Structural distribution of stability in a thermophilic enzyme

Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.

Identification of thermophilic species by the amino acid compositions deduced from their genomes

The global amino acid compositions as deduced from the complete genomic sequences of six thermophilic archaea, two thermophilic bacteria, 17 mesophilic bacteria and two eukaryotic species were analysed by hierarchical clustering and principal components analysis. Both methods showed an influence of several factors on amino acid composition. Although GC content has a dominant effect, thermophilic species can be identified by their global amino acid compositions alone. This study presents a careful statistical analysis of factors that affect amino acid composition and also yielded specific features of the average amino acid composition of thermophilic species. Moreover, we introduce the first example of a ‘compositional tree’ of species that takes into account not only homologous proteins, but also proteins unique to particular species. We expect this simple yet novel approach to be a useful additional tool for the study of phylogeny at the genome level.

The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function of Mg2+ and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Archaeal diversity in two thermophilic chalcopyrite bioleaching reactors

This study used a culture-independent molecular approach to investigate the archaeal community composition of thermophilic bioleaching reactors. Two culture samples, MTC-A and MTC-B, grown with different concentrations of chalcopyrite (CuFeS2), a copper sulfidic ore, at a temperature of 78 degrees C and pH 1.6 were studied. Phylogenetic analysis of the 16S rRNA genes revealed that both cultures consisted of Archaea belonging to the Sulfolobales. The 16S rRNA gene clone library of MTC-A grown with 4% (w/v) chalcopyrite was dominated by a unique phylotype related to Sulfolobus shibatae (69% of total clones). The remaining clones were affiliated with Stygiolobus azoricus (11%), Metallosphaera sp. J1 (8%), Acidianus infernus (2%), and a novel phylotype related to Sulfurisphaera ohwakuensis (10%). In contrast, the clones from MTC-B grown with 12% (w/v) chalcopyrite did not appear to contain Sulfolobus shibatae-like organisms. Instead the bioleaching consortium was dominated by clones related to Sulfurisphaera ohwakuensis (73.9% of total clones). The remaining microorganisms detected were similar to those found in MTC-A.

Two-stage thermophilic-mesophilic anaerobic digestion of waste activated sludge-from a biological nutrient removal plant

A two-stage thermophilic-mesophilic anaerobic digestion pilot-plant was operated solely on waste activated sludge (WAS) from a biological nutrient removal (BNR) plant. The first-stage thermophilic reactor (HRT 2 days) was operated at 47, 54 and 60 degrees C. The second-stage mesophilic digester (HRT 15 days) was held at a constant temperature of 36-37 degrees C. For comparison with a single-stage mesophilic process, the mesophilic digester was also operated separately with an HRT of 17 days and temperature of 36-37 degrees C. The results showed a truly thermophilic stage (60 degrees C) was essential to achieve good WAS degradation. The lower thermophilic temperatures examined did not offer advantages over single-stage mesophilic treatment in terms of COD and VS removal. At a thermophilic temperature of 60 degrees C, the plant achieved 35% VS reduction, representing a 46% increase compared to the single-stage mesophilic digester. This is a significant level of degradation which could make such a process viable in situations where there is no primary sludge generated. The fate of the biologically stored phosphorus in this BNR sludge was also investigated. Over 80% of the incoming phosphorus remained bound up with the solids and was not released into solution during the WAS digestion. Therefore only a small fraction of phosphorus would be recycled to the main treatment plant with the dewatering stream.

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin

armful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d−1 for O. cf. ovata, 0.35 ± 0.01 d−1 for C. monotis and 0.33 ± 0.04 d−1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed.

Isolation and polyphasic characterization of a novel hyper catalase producing thermophilic bacterium for the degradation of hydrogen peroxide.

A newly isolated microbial strain of thermophilic genus Geobacillus has been described with emphasis on polyphasic characterization and its application for degradation of hydrogen peroxide. The validation of this thermophilic strain of genus Geobacillus designated as BSS-7 has been demonstrated by polyphasic taxonomy approaches through its morphological, biochemical, fatty acid methyl ester profile and 16S rDNA sequencing. This thermophilic species of Geobacillus exhibited growth at broad pH and temperature ranges coupled with production of extraordinarily high quantities of intracellular catalase, the latter of which as yet not been reported in any member of this genus. The isolated thermophilic bacterial culture BSS-7 exhibited resistance against a variety of organic solvents. The immobilized whole cells of the bacterium successfully demonstrated the degradation of hydrogen peroxide (H2O2) in a packed bed reactor. This strain has potential application in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to applications in the textile, paper, food and pharmaceutical industries.

Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1–2 copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.

Enzyme activities and biotechnological applications of cold-active microfungi

Fungi are eukaryotic organisms and considered to be less adaptable to extreme environments when compared to bacteria. While there are no thermophilic microfungi in a strict sense, some fungi have adapted to life in the cold. Cold-active microfungi have been isolated from the Antarctic and their enzyme activities explored with a view to finding new candidates for industrial use. On another front, environmental pollution by petroleum products in the Antarctic has led to a search for, and the subsequent discovery of, fungal isolates capable of degrading hydrocarbons. The work has paved the way to developing a bioremedial approach to containing this type of contamination in cold climates. Here we discuss our efforts to map the capability of Antarctic microfungi to degrade oil and also introduce a novel cold-active fungal lipase enzyme.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.