Merlin, a new superfamily of DNA transposons identified in diverse animal genomes and related to bacterial IS1016 insertion sequences

Several new families of DNA transposons were identified by computer-assisted searches in a wide range of animal species that includes nematodes, flat worms, mosquitoes, sea squirt, zebrafish, and humans. Many of these elements have coding capacity for transposases, which are related to each other and to those encoded by the IS1016 group of bacterial insertion sequences. Although these transposases display a motif similar to the DDE motif found in many transposases and integrases, they cannot be directly allied to any of the previously described eukaryotic transposases. Other common features of the new eukaryotic and bacterial transposons include similarities in their terminal inverted repeats and 8-bp or 9-bp target-site duplications. Together, these data indicate that these elements belong to a new superfamily of DNA transposons, called Merlin/IS1016, which is common in many eubacterial and animal genomes. We also present evidence that these transposons have been recently active in several animal species. This evidence is particularly strong in the parasitic blood fluke Schistosoma mansoni, in which Merlin is also the first described DNA transposon family.     

Evaluation and Allocation of Greenhouse Gas Reductions in Industrial Symbiosis

Industrial symbiosis (IS) exchanges have been recognized to reduce greenhouse gas (GHG) emission, though methods for quantification of GHG emissions in IS exchanges are varied, and no standardized methods are available. This article proposes a practical approach to quantify total and allocated GHG emissions from IS exchanges by integrating the GHG protocol and life cycle assessment. The proposed method expands the system boundaries to include all IS companies, and the functional flow is set to be the sum of the main products. The total impact of a company is allocated to the main product. Three by‐product impact allocation methods of cutoff, avoidance, and 50/50 are proposed, and the total and distributed impacts of the IS systems in an industrial park are theoretically derived. The proposed method was tested to quantify GHG reduction in a real IS exchange developed between Korea Zinc (a zinc smelter) and Hankook Paper (a paper mill company) in the Ulsan Eco‐Industrial Park initiative. The total reduction of GHG emissions in this IS exchange, 60,522 tonnes of carbon dioxide per year, was the same in the GHG protocol, whereas GHG distribution between two companies depended on the allocation method. Given that the reduction of GHG emissions from IS exchanges is the product of the collaboration of giving companies and receiving companies, the 50/50 allocation method is best from an equivalent‐responsibility and benefit‐sharing perspective. However, this study suggests a more practical implementation approach based on a flexible and negotiable method of allocating the total GHG reduction between stakeholders.     

Combined Use of Ribotyping,PFGE Typing and IS431 Typing in the Discrimination of Nosocomial Strains of Methicillin-Resistant Staphylococcus aureus

We have previously reported the phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed-field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.     

Use of Symbiosis Products from Integrated Pulp and Paper and Carbon Steel Mills: Legal Status and Environmental Burdens

This study assesses the policy/legal status of both multistream residues and potential secondary products (“symbiosis products”) and whether there could be environmental benefits associated with the utilization of residues from integrated pulp and paper and carbon steel mills as raw materials for such secondary products. Waste‐related European Union (EU) and Finnish policy and legal instruments were reviewed to identify potential constraints for, and suggested next steps in, the development of potential process industry residue‐based symbiosis products. The products were soil amendment pellets, low‐grade concrete, and mine filler. A global warming potential (GWP) assessment and an exergy analysis were applied to these potential symbiosis products. Some indicative GWP calculations of greenhouse gas emissions associating similar and/or analogous products based on virgin primary raw materials, more energy‐intensive processes, and the alternative treatment of these residues as wastes are also presented. This study addresses GWP, exergy, and legal aspects in a holistic manner to determine the potential environmental benefits of secondary products within the EU legal framework. The GWP assessment and exergy analysis indicate that the utilization of multistream residues causes very low environmental burdens in terms of GWP. The utilization option can have potential environmental benefits in terms of GWP through process replacement and avoided landfilling and waste treatment impacts, as well as potentially through emission reductions from product replacement if suitable and safe applications can be identified. Waste regulation does not define the legal requirements under which utilizing residues in such novel concepts as introduced in this study would be possible, nor how waste status could be removed and product‐based legislation be applied to the potential products instead.     

Importance of Illegitimate Recombination and Transposition in IS30-Associated Excision Events

In the present study we report on the excision of IS30 elements and IS30-derived composite transposons. Frequent loss of IS30 was observed during dissolution of dimeric IS30 structures, containing IR–IR junctions, leading to resealed donor molecules. In contrast, unambiguous transpositional excision resulting in resealed remainder products could not be identified in the case of a monomeric element. The bias in the excision of monomeric and dimeric IS30 structures indicates a difference in the molecular mechanism of transposition of IS30 monomers and dimers. Sequence data on the rarely detected plasmids missing full IS or Tn copies rather suggest that all products were derived from illegitimate recombination. The reaction occurred between short homologies and was independent of the transposase activity. Similar IS30 excision events accompanied by multiple plasmid or genome rearrangements were detected in Pseudomonas putida and Rhizobium meliloti, yielding stable replicons that retained the selective marker gene of the transposon. We provide evidence that both transposition and illegitimate recombination can contribute to the stabilization of replicons through the elimination of IS elements, which emphasizes the evolutionary significance of these events.     

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0 mm × 2.0 mm, 5 μm) with a mobile phase of acetonitrile (buffer B) and 5 mM ammonium formate (buffer A) at a flow-rate of 0.3 mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to −4.1 kV and ion spray temperature to 400 °C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4 m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25 ng/mL and 5 μg/mL and the achieved correlation coefficient (r²) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.     

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 IS Pre1 -like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 IS Pre2 -like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer – we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of IS Pre2 -like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.     

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.     

Comparative Analysis of the Mosaic Genomes of Tailed Archaeal Viruses and Proviruses Suggests Common Themes for Virion Architecture and Assembly with Tailed Viruses of Bacteria

Tailed double-stranded DNA viruses (order Caudovirales) represent the dominant morphotype among viruses infecting bacteria. Analysis and comparison of complete genome sequences of tailed bacterial viruses provided insights into their origin and evolution. Structural and genomic studies have unexpectedly revealed that tailed bacterial viruses are evolutionarily related to eukaryotic herpesviruses. Organisms from the third domain of life, Archaea, are also infected by viruses that, in their overall morphology, resemble tailed viruses of bacteria. However, high-resolution structural information is currently unavailable for any of these viruses, and only a few complete genomes have been sequenced so far. Here we identified nine proviruses that are clearly related to tailed bacterial viruses and integrated into chromosomes of species belonging to four different taxonomic orders of the Archaea. This more than doubled the number of genome sequences available for comparative studies. Our analyses indicate that highly mosaic tailed archaeal virus genomes evolve by homologous and illegitimate recombination with genomes of other viruses, by diversification, and by acquisition of cellular genes. Comparative genomics of these viruses and related proviruses revealed a set of conserved genes encoding putative proteins similar to virion assembly and maturation, as well as genome packaging proteins of tailed bacterial viruses and herpesviruses. Furthermore, fold prediction and structural modeling experiments suggest that the major capsid proteins of tailed archaeal viruses adopt the same topology as the corresponding proteins of tailed bacterial viruses and eukaryotic herpesviruses. Data presented in this study strongly support the hypothesis that tailed viruses infecting archaea share a common ancestry with tailed bacterial viruses and herpesviruses.     

Optimization of variable number tandem repeat typing set for differentiating Mycobacterium tuberculosis strains in the Beijing family

Variable number tandem repeat (VNTR) typing of Mycobacterium tuberculosis, as presently used, has often failed to differentiate clonal strains especially in areas where the Beijing family genotype is predominant. We have evaluated mycobacterial interspersed repetitive units (MIRUs), Queen's University of Belfast (QUB) loci and exact tandem repeat (ETR) loci individually for their abilities to differentiate the Beijing and non-Beijing genotype families of M. tuberculosis. The best locus was QUB 3232 followed by QUB 11b. Available MIRU, QUB and ETR loci were then consecutively arranged in declining order of discriminatory power, and combined to form an optimal set of 12-loci VNTR typing (MIRU 10, 26, 39, 40; QUB 11a, 11b, 15, 18, 26, 3232, 3336; ETR A) that possessed comparable discriminatory ability to IS6110 restriction fragment length polymorphism. This optimized 12-loci VNTR typing set can become an important tool for tracking M. tuberculosis of the Beijing family.