Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.Fil: Monteiro, Lummy Maria Oliveira. Universidade de Sao Paulo; BrasilFil: Arruda, Leticia Magalhães. Universidade de Sao Paulo; BrasilFil: Sanches Medeiros, Ananda. Universidade de Sao Paulo; BrasilFil: Martins Santana, Leonardo. Universidade de Sao Paulo; BrasilFil: Alves, Luana de Fátima. Universidade de Sao Paulo; BrasilFil: Defelipe, Lucas Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Turjanski, Adrian Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guazzaroni, Mara Eugenia. Universidade de Sao Paulo; BrasilFil: de Lorenzo, Victor. Consejo Superior de Investigaciones Científicas. Centro Nacional de Biotecnología; EspañaFil: Silva Rocha, Rafael. Universidade de Sao Paulo; Brasi

Geobiology of Andean Microbial Ecosystems Discovered in Salar de Atacama, Chile

The Salar de Atacama in the Chilean Central Andes harbors unique microbial ecosystems due to extreme environmental conditions, such as high altitude, low oxygen pressure, high solar radiation, and high salinity. Combining X-ray diffraction analyses, scanning electron microscopy and molecular diversity studies, we have characterized twenty previously unexplored Andean microbial ecosystems in eight different lakes and wetlands from the middle-east and south-east regions of this salt flat. The mats and microbialites studied are mainly formed by calcium carbonate (aragonite and calcite) and halite, whereas the endoevaporites are composed predominantly of gypsum and halite. The carbonate-rich mats and microbialites are dominated by Bacteroidetes and Proteobacteria phyla. Within the phylum Proteobacteria, the most abundant classes are Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. While in the phylum Bacteroidetes, the most abundant classes are Bacteroidia and Rhodothermia. Cyanobacteria, Chloroflexi, Planctomycetes, and Verrucomicrobia phyla are also wellrepresented in the majority of these systems. Gypsum endoevaporites, on the contrary, are dominated by Proteobacteria, Bacteroidetes, and Euryarchaeota phyla. The Cyanobacteria phylum is also abundant in these systems, but it is less represented in comparison to mats and microbialites. Regarding the eukaryotic taxa, diatoms are key structural components in most of the microbial ecosystems studied. The genera of diatoms identified were Achnanthes, Fallacia, Halamphora, Mastogloia, Navicula, Nitzschia, and Surirella. Normally, in the mats and microbialites, diatoms form nano-globular carbonate aggregates with filamentous cyanobacteria and other prokaryotic cells, suggesting their participation in the mineral precipitation process. This work expands our knowledge of the microbial ecosystems inhabiting the extreme environments from the Central Andes region, which is important to ensure their protection and conservation.Centro de Investigaciones Geológica

Changes in structure upon phosphorylation.

<p>(A) and (B) Structure of the Wild Type showing polar and charged inter protein interactions respectively. (C) and (D) depict the interactions of the15pSer and charged interactions respectively. (E) and (F) depict the interactions of the 18pThr and charged interactions respectively. (G and H) depict the interactions of the 20pSer and charged interactions respectively.</p

Dynamics and interactions in the Arg1737Ala mutant of p53 15pSer state.

<p>(A) Structure of Arg1737Ala mutant of p53 15pSer state, showing interactions between some residues around the phosphate group. (B) Distance of phosphorus atom of Sep15 and Nζ of Lys1740 versus time in the Arg1737Ala mutant of p53 15pSer state molecular dynamics.</p

Structural comparison between different phosphorylation states.

<p>Structural comparison between different phosphorylation states.</p

Backbone RMSD vs time and RMSF plots.

<p>(A) Plot of the Backbone RMSD of TAZ2 along the simulation. The MD was fitted against the average structure and only 30 ns-130 ns of the dynamics was used. (B) Backbone RMSD of p53TAD fitted against the average structure of the 30 ns-130 ns MD. (C) Backbone RMSF vs residue plot of TAZ2. The structure was fitted against the average structure of 30ns-130ns MD siulation. (D) Backbone RMSF vs residue plot of p53TAD. Again the structure was fitted against the average structure. In all cases p53 WT is black, p53 15pSer is green, p53 18pThr is red and p53 20pSer is blue.</p

Percentage of time spent in a helix secondary structure versus number of p53TAD residues.

<p>Percentage of time spent in a helix secondary structure versus number of p53TAD residues.</p

Structure of CBP and p53.

<p>(A) Domain structure of p53. (B) Domain structure of CREB Binding Protein (CBP). (C) The structure of the TAZ2 (green) domain of CBP with Zn²⁺ ions (cyan balls) bound to the p53TAD peptide (red). Ribbon representation is used for both domains.</p

ΔΔG of binding of TAZ2 and p53TAD for several mutations (kJ/mol).

<p>ΔΔG of binding of TAZ2 and p53TAD for several mutations (kJ/mol).</p

Genome comparison of two Exiguobacterium strains from high altitude andean lakes with different arsenic resistance: Identification and 3D modeling of the Acr3 efflux pump.

Arsenic exists in natural systems in a variety of chemical forms, including inorganic arsenite (As [III]) and arsenate (As [V]). The majority of living organisms have evolved various mechanisms to avoid occurrence of arsenic inside the cell due to its toxicity. Common core genes include a transcriptional repressor ArsR, an arsenate reductase ArsC, and arsenite efflux pumps ArsB and Acr3. To understand arsenic resistance we have performed arsenic tolerance studies, genomic and bioinformatic analysis of two Exiguobacterium strains, S17 and N139, from the high-altitude Andean Lakes. In these environments high concentrations of arsenic were described in the water due to a natural geochemical phenomenon, therefore, these strains represent an attractive model system for the study of environmental stress and can be readily cultivated. Our experiments show that S17 has a greater tolerance to arsenite (10nM) than N139, but similar growth in arsenate (150nM). We sequenced the genome of the two Exiguobacterium and identified an acr3 gene in S17 as the only difference between both species regarding known arsenic resistance genes. To further understand the Acr3 we modeled the 3D structure and identified the location of relevant residues of this protein. Our model is in agreement with previous experiments and allowed us to identify a region where a relevant cysteine lies. This Acr3 membrane efflux pump, present only in S17, may explain its increased tolerance to As(III) and is the first Acr3-family protein described in Exiguobacterium genus