Structures of an apo and a binary complex of an evolved archeal b family DNA polymerase capable of synthesising highly cy-dye labelled DNA

Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent “CyDNA” densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers

Steady-state and transient-state performance of a biotrickling filter treating chlorobenzene-containing waste gas

Biotrickling filter (BTF) technology was applied for the treatment of waste gas containing a mixture of chlorobenzene and 1,2-dichlorobenzene. An adapted microbial community was immobilised on a structured packing material. The strategy followed was to reach high removal efficiencies at initially low mass loading rates followed by an increase of the latter. This procedure was successful and resulted in a short start-up period of only 2weeks. A 3-month operation under steady-state conditions showed good performance, with >95% removal efficiency at a mass loading rate of 1,800gm−3day−1. Dimensionless concentration profiles showed that the chlorobenzenes were simultaneously degraded. Low dissolved organic carbon of 15mgl−1 and stoichiometric chloride concentrations in the trickling liquid indicated complete mineralisation of the pollutant. Transient-state experiments with five times higher mass loading rates caused a decrease in the removal efficiency that recovered rapidly once the mass loading rate returned to its original steady-state level. A progressive increase of the mass loading rate in a long-term performance experiment showed that the removal efficiency could be kept stable between 95 and 99% at loads of up to 5,200gm−3day−1 over several days. Above this mass loading rate, the elimination capacity did not increase any further. These results demonstrated that with a well-adapted inoculum and optimal operation parameters, a BTF system with excellent performance and stability that efficiently removes a mixture of cholorobenzene vapours from air can be obtaine

Reductive Transformations of Anthropogenic Chemicals in Natural and Technical Systems

Reductive transformation reactions of chemical pollutants (e.g., polyhalogenated hydrocarbons, aromatic azo and nitro compounds, chromium(VI) species) in the environment are important both from an ecotoxicological and from an environmental technology point of view. Using well-defined model reactors as well as more complex 'real world' systems, several groups at EAWAG are trying to unravel compound- and system-specific factors that control the reduction of a variety of anthropogenic chemicals under different conditions in the environment. The examples presented in this article include the reduction of nitroaromatic compounds under iron- and sulfate-reducing conditions, the reductive dehalogenation of chlorinated ethenes by cob(I)alamin and by a bacterium that uses such compounds as terminal electron acceptors, and the reduction of chromium(VI) by various reduced iron species. The link between microbial and abiotic (chemical) processes involved in reductive transformations of pollutants is emphasized. The major goal of this article is to illustrate the approaches taken to elucidate the mechanisms and kinetics of environmentally relevant reduction reactions of pollutants, and to discuss how the results of such studies can be used 1) to gain insight into what is actually happening in the environment, and 2) to develop methods for the treatment of chemical wastes or contaminated sites

Contaminated environments in the subsurface and bioremediation: organic contaminants

Due to leakages, spills, improper disposal and accidents during transport, organic compounds have become subsurface contaminants that threaten important drinking water resources. One strategy to remediate such polluted subsurface environments is to make use of the degradative capacity of bacteria. It is often sufficient to supply the subsurface with nutrients such as nitrogen and phosphorus, and aerobic treatments are still dominating. However, anaerobic processes have advantages such as low biomass production and good electron acceptor availability, and they are sometimes the only possible solution. This review will focus on three important groups of environmental organic contaminants: hydrocarbons, chlorinated and nitroaromatic compounds. Whereas hydrocarbons are oxidized and completely mineralized under anaerobic conditions in the presence of electron acceptors such as nitrate, iron, sulfate and carbon dioxide, chlorinated and nitroaromatic compounds are reductively transformed. For the aerobic often persistent polychlorinated compounds, reductive dechlorination leads to harmless products or to compounds that are aerobically degradable. The nitroaromatic compounds are first reductively transformed to the corresponding amines and can subsequently be bound to the humic fraction in an aerobic process. Such new findings and developments give hope that in the near future contaminated aquifers can efficiently be remediated, a prerequisite for a sustainable use of the precious subsurface drinking water resource

Regional-scale integration of multi-scale hydrological and geophysical data using a two-step Bayesian sequential simulation approach

Significant progress has been made with regard to the quantitative integration of geophysical and hydrological data at the local scale for the purpose of improving predictions of groundwater flow and solute transport. However, extending corresponding approaches to the regional scale still represents one of the major challenges in the domain of hydrogeophysics. To address this problem, we have developed a regional-scale data integration methodology based on a two-step Bayesian sequential simulation approach. Our objective is to generate high-resolution stochastic realizations of the regional-scale hydraulic conductivity field in the common case where there exist spatially exhaustive but poorly resolved measurements of a related geophysical parameter, as well as highly resolved but spatially sparse collocated measurements of this geophysical parameter and the hydraulic conductivity. To integrate this multi-scale, multi-parameter database, we first link the low- and high-resolution geophysical data via a stochastic downscaling procedure. This is followed by relating the downscaled geophysical data to the high-resolution hydraulic conductivity distribution. After outlining the general methodology of the approach, we demonstrate its application to a realistic synthetic example where we consider as data high-resolution measurements of the hydraulic and electrical conductivities at a small number of borehole locations, as well as spatially exhaustive, low-resolution estimates of the electrical conductivity obtained from surface-based electrical resistivity tomography. The different stochastic realizations of the hydraulic conductivity field obtained using our procedure are validated by comparing their solute transport behaviour with that of the underlying ?true? hydraulic conductivity field. We find that, even in the presence of strong subsurface heterogeneity, our proposed procedure allows for the generation of faithful representations of the regional-scale hydraulic conductivity structure and reliable predictions of solute transport over long, regional-scale distances

Nanostructures from Synthetic Genetic Polymers.

Nanoscale objects of increasing complexity can be constructed from DNA or RNA. However, the scope of potential applications could be enhanced by expanding beyond the moderate chemical diversity of natural nucleic acids. Here, we explore the construction of nano-objects made entirely from alternative building blocks: synthetic genetic polymers not found in nature, also called xeno nucleic acids (XNAs). Specifically, we describe assembly of 70 kDa tetrahedra elaborated in four different XNA chemistries (2'-fluro-2'-deoxy-ribofuranose nucleic acid (2'F-RNA), 2'-fluoroarabino nucleic acids (FANA), hexitol nucleic acids (HNA), and cyclohexene nucleic acids (CeNA)), as well as mixed designs, and a ∼600 kDa all-FANA octahedron, visualised by electron microscopy. Our results extend the chemical scope for programmable nanostructure assembly, with implications for the design of nano-objects and materials with an expanded range of structural and physicochemical properties, including enhanced biostability

Steady-state and transient-state performance of a biotrickling filter treating chlorobenzene-containing waste gas

Biotrickling filter (BTF) technology was applied for the treatment of waste gas containing a mixture of chlorobenzene and 1,2-dichlorobenzene. An adapted microbial community was immobilised on a structured packing material. The strategy followed was to reach high removal efficiencies at initially low mass loading rates followed by an increase of the latter. This procedure was successful and resulted in a short start-up period of only 2 weeks. A 3-month operation under steady-state conditions showed good performance, with >95% removal efficiency at a mass loading rate of 1,800 g m(-3) day(-1). Dimensionless concentration profiles showed that the chlorobenzenes were simultaneously degraded. Low dissolved organic carbon of 15 mg l(-1) and stoichiometric chloride concentrations in the trickling liquid indicated complete mineralisation of the pollutant. Transient-state experiments with five times higher mass loading rates caused a decrease in the removal efficiency that recovered rapidly once the mass loading rate returned to its original steady-state level. A progressive increase of the mass loading rate in a long-term performance experiment showed that the removal efficiency could be kept stable between 95 and 99% at loads of up to 5,200 g m(-3) day(-1) over several days. Above this mass loading rate, the elimination capacity did not increase any further. These results demonstrated that with a well-adapted inoculum and optimal operation parameters, a BTF system with excellent performance and stability that efficiently removes a mixture of cholorobenzene vapours from air can be obtained

Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes

In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting