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Marques, AC, Santos L, Costa MN, Dantas JM, Duarte P, Gonçalves A, Martins R, Salgueiro CA, Fortunato E.  2015.  Office Paper Platform for Bioelectrochromic Detection of Electrochemically Active Bacteria using Tungsten Trioxide Nanoprobes. Sci. Rep. 5(9910) AbstractWebsite

Electrochemically active bacteria (EAB) have the capability to transfer electrons to cell exterior, a feature that is currently explored for important applications in bioremediation and biotechnology fields. However, the number of isolated and characterized EAB species is still very limited regarding their abundance in nature. Colorimetric detection has emerged recently as an attractive mean for fast identification and characterization of analytes based on the use of electrochromic materials. In this work, WO3 nanoparticles were synthesized by microwave assisted hydrothermal synthesis and used to impregnate non-treated regular office paper substrates. This allowed the production of a paper-based colorimetric sensor able to detect EAB in a simple, rapid, reliable, inexpensive and eco-friendly method. The developed platform was then tested with Geobacter sulfurreducens, as a proof of concept. G. sulfurreducens cells were detected at latent phase with an RGB ratio of 1.10 ± 0.04, and a response time of two hours.

Morgado, L, Fernandes AP, Dantas JM, Silva MA, Salgueiro CA.  2012.  On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochemical Society transactions. 40(6):1295-1301. AbstractWebsite

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.

Morgado, L, Fernandes AP, Londer YY, Bruix M, Salgueiro CA.  2010.  One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins. Biochemical and Biophysical Research Communications. 393(3):466-470. AbstractWebsite

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural–functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (13C/15N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of 1H–13C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.

Dantas, JM, Saraiva IH, Morgado L, Silva MA, Schiffer M, Salgueiro CA, Louro RO.  2011.  Orientation of the axial ligands and magnetic properties of the hemes in the cytochromec7 family from Geobacter sulfurreducens determined by paramagnetic NMR. Dalton Transactions. 40(47):12713-12718. AbstractWebsite

Geobacter sulfurreducens is a sediment bacterium that contains a large number of multiheme cytochromes. The family of five c7 triheme periplasmic cytochromes from Geobacter sulfurreducens shows structural diversity of the heme core. Structural characterization of the relative orientation of the axial ligands of these proteins by 13C-paramagnetic NMR was carried out. The structures in solution were compared with those obtained by X-ray crystallography. For some hemes significant differences exist between the two methods such that orientation of the magnetic axes obtained from NMR data and the orientation taken from the X-ray coordinates differ. The results allowed the orientation of the magnetic axes to be defined confidently with respect to the heme frame in solution, a necessary step for the use of paramagnetic constraints to improve the complete solution structure of these proteins.

Morgado, L, Saraiva IH, Louro RO, Salgueiro CA.  2010.  Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR. FEBS Letters. 584(15):3442-3445. AbstractWebsite

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic 13C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.

Pokkuluri, PR, Londer YY, Wood SJ, Duke NEC, Morgado L, Salgueiro CA, Schiffer M.  2009.  Outer membrane cytochrome c, OmcF, from Geobacter sulfurreducens: High structural similarity to an algal cytochrome c6. Proteins: Structure, Function, and Bioinformatics. 74(1):266-270. AbstractWebsite

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