Global harmonization of vaccine testing requirements: Making elimination of the ATT and TABST a concrete global achievement.

This one-day symposium organized by Humane Society International (HSI) brought together 18 international experts from Argentina, Brazil, China, Europe, India, Russia, South Africa and the United States to discuss the elimination of the abnormal toxicity test (ATT) from the testing requirements for human vaccines as well as the target animal batch safety test (TABST) and the laboratory animal batch safety test (LABST) for veterinary vaccines. Participants reported on country-specific regulatory requirements and, where present, the perspectives on waiver and elimination of those tests. In addition, the attendees, with HSI in the role of facilitator, moved to define the barriers to the complete elimination or waiving of these tests. This report expounds the outcomes of the symposium, and introduces a proposed roadmap - populated with country specific activities - for the elimination of these tests.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli.

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.

Whole Genome Comparison of Thermus sp. NMX2.A1 Reveals Principle Carbon Metabolism Differences with Closest Relation Thermus scotoductus SA-01.

Genome sequencing of the yellow-pigmented, thermophilic bacterium Thermus sp. NMX2.A1 resulted in a 2.29 Mb draft genome that encodes for 2312 proteins. The genetic relationship between various strains from the genus Thermus was assessed based on phylogenomic analyses using a concatenated set of conserved proteins. The resulting phylogenetic tree illustrated that Thermus sp. NMX2 A.1 clusters together with Thermus scotoductus SA-01, despite being isolated from vastly different geographical locations. The close evolutionary relationship and metabolic parallels between the two strains has previously been recognized; however, neither strain's genome data were available at that point in time. Genomic comparison of the Thermus sp. NMX2.A1 and T. scotoductus SA-01, as well as other closely related Thermus strains, revealed a high degree of synteny at both the genomic and proteomic level, with processes such as denitrification and natural cell competence appearing to be conserved. However, despite this high level of similarity, analysis revealed a complete, putative Calvin-Benson-Bassham (CBB) cycle in NMX2.A1 that is absent in SA-01. Analysis of horizontally transferred gene islands provide evidence that NMX2 selected these genes due to pressure from its HCO3 (-) rich environment, which is in stark contrast to that of the deep subsurface isolated SA-01.

Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas.

Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.1 km. During sampling in Beatrix gold mine (1.4 km beneath the surface), central South Africa, CaCO3 stalactites growing on the mine tunnel ceiling were collected and observed, in two cases, to contain a living obligate brackish water/marine nematode species, Monhystrella parvella. After sterilization of the outer surface, mineral layers were physically removed from the outside to the interior, and DNA extracted. Based upon 16S and 18S rRNA gene sequencing, Archaea, Bacteria, and Eukarya in different combinations were detected for each layer. Using CT scan and electron microscopy the inner structure of CaCO3 and salt stalactites were analyzed. CaCO3 stalactites show a complex pattern of lamellae carrying bacterially precipitated mineral structures. Nematoda were clearly identified between these layers confirming that bacteria and nematodes live inside the stalactites and not only in the central straw. Salt stalactites exhibit a more uniform internal structure. Surprisingly, several Bacteria showing highest sequence identities to marine species were identified. This, together with the observation that the nematode M. parvella recovered from Beatrix gold mine stalactite can only survive in a salty environment makes the origin of the deep subsurface colonization enigmatic. The possibility of a Permian origin of fracture fluids is discussed. Our results indicate stalactites are suitable for biodiversity recovery and act as natural traps for microorganisms in the fissure water long after the water that formed the stalactite stopped flowing.

Analysis of genomic rearrangements, horizontal gene transfer and role of plasmids in the evolution of industrial important Thermus species.

BACKGROUND: Bacteria of genus Thermus inhabit both man-made and natural thermal environments. Several Thermus species have shown biotechnological potential such as reduction of heavy metals which is essential for eradication of heavy metal pollution; removing of organic contaminants in water; opening clogged pipes, controlling global warming among many others. Enzymes from thermophilic bacteria have exhibited higher activity and stability than synthetic or enzymes from mesophilic organisms. RESULTS: Using Meiothermus silvanus DSM 9946 as a reference genome, high level of coordinated rearrangements has been observed in extremely thermophilic Thermus that may imply existence of yet unknown evolutionary forces controlling adaptive re-organization of whole genomes of thermo-extremophiles. However, no remarkable differences were observed across species on distribution of functionally related genes on the chromosome suggesting constraints imposed by metabolic networks. The metabolic network exhibit evolutionary pressures similar to levels of rearrangements as measured by the cross-clustering index. Using stratigraphic analysis of donor-recipient, intensive gene exchanges were observed from Meiothermus species and some unknown sources to Thermus species confirming a well established DNA uptake mechanism as previously proposed. CONCLUSION: Global genome rearrangements were found to play an important role in the evolution of Thermus bacteria at both genomic and metabolic network levels. Relatively higher level of rearrangements was observed in extremely thermophilic Thermus strains in comparison to the thermo-tolerant Thermus scotoductus. Rearrangements did not significantly disrupt operons and functionally related genes. Thermus species appeared to have a developed capability for acquiring DNA through horizontal gene transfer as shown by the donor-recipient stratigraphic analysis.

Analysis of protein thermostability enhancing factors in industrially important thermus bacteria species.

Elucidation of evolutionary factors that enhance protein thermostability is a critical problem and was the focus of this work on Thermus species. Pairs of orthologous sequences of T. scotoductus SA-01 and T. thermophilus HB27, with the largest negative minimum folding energy (MFE) as predicted by the UNAFold algorithm, were statistically analyzed. Favored substitutions of amino acids residues and their properties were determined. Substitutions were analyzed in modeled protein structures to determine their locations and contribution to energy differences using PyMOL and FoldX programs respectively. Dominant trends in amino acid substitutions consistent with differences in thermostability between orthologous sequences were observed. T. thermophilus thermophilic proteins showed an increase in non-polar, tiny, and charged amino acids. An abundance of alanine substituted by serine and threonine, as well as arginine substituted by glutamine and lysine was observed in T. thermophilus HB27. Structural comparison showed that stabilizing mutations occurred on surfaces and loops in protein structures.

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01.

BACKGROUND: Many strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different Thermus thermophilus strains have been completed. This paper represents the completed genome from a second Thermus species - T. scotoductus. RESULTS: The genome of Thermus scotoductus SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the Thermus thermophilus genomes. The T. thermophilus megaplasmid genes are part of the T. scotoductus chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of T. scotoductus and T. thermophilus. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from Meiothermus ruber. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of Thermus scotoductus was confirmed experimentally as expected as most of the proteins of the natural transformation system of Thermus thermophilus are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle. CONCLUSIONS: The genome of Thermus scotoductus SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to Thermus thermophilus. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of Thermus scotoductus illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.

A thioredoxin reductase-like protein from the thermophile, Thermus scotoductus SA-01, displaying iron reductase activity.

The transition metal iron is an important element for the sustenance of life--it can function either as an electron acceptor or as a donor and serves as a cofactor in many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here we report the sequence of the ferric reductase (which is typically annotated as a thioredoxin reductase-like protein) and a comparative kinetic study with the thioredoxin reductase from SA-01. Structurally, the most noteworthy difference, immediately apparent from the protein sequence, is the absence of the disulphide redox centre in the ferric reductase. This is the first report relating the attributes of such a redox protein to its ability to reduce a ferric substrate.

Crystal structure of a thermostable old yellow enzyme from Thermus scotoductus SA-01.

Recent characterization of the chromate reductase (CrS) from the thermophile Thermus scotoductus SA-01 revealed this enzyme to be related to the Old Yellow Enzyme (OYE) family. Here, we report the structure of a thermostable OYE homolog in its holoform at 2.2A as well as its complex with p-hydroxybenzaldehyde (pHBA). The enzyme crystallized as octamers with the monomers showing a classical TIM barrel fold which upon dimerization yields the biologically active form of the protein. A sulfate ion is bound above the si-side of the non-covalently bound FMN cofactor in the oxidized solved structure but is displaced upon pHBA binding. The active-site architecture is highly conserved as with other members of this enzyme family. The pHBA in the CrS complex is positioned by hydrogen bonding to the two conserved catalytic-site histidines. The most prominent structural difference between CrS and other OYE homologs is the size of the "capping domain". Thermostabilization of the enzyme is achieved in part through increased proline content within loops and turns as well as increased intersubunit interactions through hydrogen bonding and complex salt bridge networks. CrS is able to reduce the C=C bonds of alpha,beta-unsaturated carbonyl compounds with a preference towards cyclic substrates however no activity was observed towards beta-substituted substrates. Mutational studies have confirmed the role of Tyr177 as the proposed proton donor although reduction could still occur at a reduced rate when this residue was mutated to phenylalanine.

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species.

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.

In vitro effect of a thrombin inhibition peptide selected by phage display technology.

A repeated selection of phages from a cyclic heptapeptide phage display library resulted in the enrichment of phages that bind to human alpha-thrombin. One clone of the binding phages that competed with PPACK for binding to thrombin and that had the best binding characteristics was chosen. The amino acid sequence of the peptide displayed on this phage was determined and a peptide with the sequence, Cys-Asn-Arg-Pro-Phe-Ile-Pro-Thr-Cys was synthesised. This peptide, thrombin-inhibiting peptide (TIP), is a full competitive inhibitor of thrombin with an inhibition constant (K(i)) of 0.4974 mM. It lengthened the thrombin time and inhibited thrombin-induced platelet activation and the platelet release reaction, both in a dose-dependent manner. It also reduced platelet adhesion onto a human microvascular endothelial matrix in the parallel plate flow chamber under both arterial and venous shear conditions. Thus, we have selected and synthesised a cyclic heptapeptide that competes with PPACK to bind to thrombin and that can be developed as a direct antithrombin.