A deep continental aquifer downhole sampler for microbiological studies.

To be effective, microbiological studies of deep aquifers must be free from surface microbial contaminants and from infrastructures allowing access to formation water (wellheads, well completions). Many microbiological studies are based on water samples obtained after rinsing a well without guaranteeing the absence of contaminants from the biofilm development in the pipes. The protocol described in this paper presents the adaptation, preparation, sterilization and deployment of a commercial downhole sampler (PDSshort, Leutert, Germany) for the microbiological studying of deep aquifers. The ATEX sampler (i.e., explosive atmospheres) can be deployed for geological gas storage (methane, hydrogen). To validate our procedure and confirm the need to use such a device, cell counting and bacterial taxonomic diversity based on high-throughput sequencing for different water samples taken at the wellhead or at depth using the downhole sampler were compared and discussed. The results show that even after extensive rinsing (7 bore volumes), the water collected at the wellhead was not free of microbial contaminants, as shown by beta-diversity analysis. The downhole sampler procedure was the only way to ensure the purity of the formation water samples from the microbiological point of view. In addition, the downhole sampler allowed the formation water and the autochthonous microbial community to be maintained at in situ pressure for laboratory analysis. The prevention of the contamination of the sample and the preservation of its representativeness are key to guaranteeing the best interpretations and understanding of the functioning of the deep biosphere.

Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery.

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, ß, ß' and ω are simultaneously coexpressed. Appending a 3' UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Functional dissection of the multi-domain di-heme cytochrome c(550) from Thermus thermophilus.

In bacteria, oxidation of sulfite to sulfate, the most common strategy for sulfite detoxification, is mainly accomplished by the molybdenum-containing sulfite:acceptor oxidoreductases (SORs). Bacterial SORs are very diverse proteins; they can exist as monomers or homodimers of their core subunit, as well as heterodimers with an additional cytochrome c subunit. We have previously described the homodimeric SOR from Thermus thermophilus HB8 (SOR(TTHB8)), identified its physiological electron acceptor, cytochrome c(550), and demonstrated the key role of the latter in coupling sulfite oxidation to aerobic respiration. Herein, the role of this di-heme cytochrome c was further investigated. The cytochrome was shown to be composed of two conformationally independent domains, each containing one heme moiety. Each domain was separately cloned, expressed in E. coli and purified to homogeneity. Stopped-flow experiments showed that: i) the N-terminal domain is the only one accepting electrons from SOR(TTHB8); ii) the N- and C-terminal domains are in rapid redox equilibrium and iii) both domains are able to transfer electrons further to cytochrome c(552), the physiological substrate of the ba(3) and caa(3) terminal oxidases. These findings show that cytochrome c(550) functions as a electron shuttle, without working as an electron wire with one heme acting as the electron entry and the other as the electron exit site. Although contribution of the cytochrome c(550) C-terminal domain to T. thermophilus sulfur respiration seems to be dispensable, we suggest that di-heme composition of the cytochrome physiologically enables storage of the two electrons generated from sulfite oxidation, thereof ensuring efficient contribution of sulfite detoxification to the respiratory chain-mediated energy generation.

Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

Directly created electrostatic micro-domains on hydroxyapatite: probing with a Kelvin Force probe and a protein.

Micro-domains of modified surface potential (SP) were created on hydroxyapatite films by direct patterning by mid-energy focused electron beam, typically available as a microprobe of Scanning Electron Microscopes. The SP distribution of these patterns has been studied on sub-micrometer scale by the Kelvin Probe Force Microscopy method as well as lysozyme adsorption. Since the lysozyme is positively charged at physiological pH, it allows us to track positively and negatively charged areas of the SP patterns. Distribution of the adsorbed proteins over the domains was in good agreement with the observed SP patterns.

Charge specific protein placement at submicrometer and nanometer scale by direct modification of surface potential by electron beam.

The understanding and the precise control of protein adsorption is extremely important for the development and optimization of biomaterials. The challenge resides in controlling the different surface properties, such as surface chemistry, roughness, wettability, or surface charge, independently, as modification of one property generally affects the other. We demonstrate the creation of electrically modified patterns on hydroxyapatite by using scanning electron beam to tailor the spatial regulation of protein adsorption via electrostatic interactions without affecting other surface properties of the material. We show that domains, presenting modulated surface potential, can be created to precisely promote or reduce protein adsorption.

A sulfite respiration pathway from Thermus thermophilus and the key role of newly identified cytochrome c550.

Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c550 acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c552 to the terminal ba3- and caa3-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite → SOR(HB8) → cytochrome c550 → cytochrome c552 → ba3 oxidase/caa3 oxidase → O2. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus.

Engineering of a fungal beta-galactosidase to remove product inhibition by galactose.

Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

Use of folding modulators to improve heterologous protein production in Escherichia coli.

Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed - and largely unpredictable - results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies.

Trigger factor from the psychrophilic bacterium Psychrobacter frigidicola is a monomeric chaperone.

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF(Pf)) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF(Ec)). Moreover, TF(Pf) did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF(Pf) was also significantly more effective than TF(Ec) at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF(Pf) differs from TF(Ec) in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.

Comparison of three microbial hosts for the expression of an active catalytic scFv.

Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.