Biology subjects

Silva-Rocha, R.

Publications and source records attributed to Silva-Rocha, R..

5 recordsLinked to original sources

bioRxiv2018

Reverse engineering of an aspirin-responsive regulator in bacteria

Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work 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 these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, 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 enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.

synthetic biology↗

bioRxiv2017

Assessing and characterising the repertoire of constitutive promoter elements in soil metagenomic libraries in Escherichia coli

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we have developed a novel framework for prospecting, characterising and estimating the accessibility of promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Using the expression profiling of fluorescent clones from two independent libraries from soil samples, we directly analysed the regulatory dynamics of novel promoter elements, addressing the relationship between the \"metaconstitutome\" of a bacterial community and its environmental context. Through the construction and screening of plasmid-based metagenomic libraries followed by in silico analyses, we were able to provide both (i) a consensus exogenous promoter elements recognizable by Escherichia coli and (ii) an estimation of the accessible promoter sequences in a metagenomic library, which was close to 1% of the whole set of available promoters. The results presented here should provide new directions for the exploration through functional metagenomics of novel regulatory sequences in bacteria, which could expand the Synthetic Biology toolbox for novel biotechnological and biomedical applications.

microbiology↗

bioRxiv2017

Big trouble with little inserts: boundaries in metagenomic screenings using lacZα based vectors

The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from microbial communities inhabiting a soil sample by using the lacZ-based plasmid pSEVA232 in the generation of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ alpha gene present in the original vector. We concluded that the current method has a higher tendency for false positive recovery of clones, when used in combination with a lacZ-based vector. Finally, we discuss the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in metagenomic screenings methodologies.

molecular biology↗

bioRxiv2017

Systematic identification of novel regulatory interactions controlling biofilm formation in the bacterium Escherichia coli

Here, we investigated novel interactions of three global regulators of the network that controls biofilm formation in the model bacterium Escherichia coli using computational network analysis, an in vivo reporter assay and physiological validation experiments. We were able to map critical nodes that govern planktonic to biofilm transition and identify 8 new regulatory interactions for CRP, IHF or Fis responsible for the control of the promoters of rpoS, rpoE, flhD, fliA, csgD and yeaJ. Additionally, an in vivo promoter reporter assay and motility analysis revealed a key role for IHF as a repressor of cell motility through the control of FliA sigma factor expression. This investigation of first stage and mature biofilm formation indicates that biofilm structure is strongly affected by IHF and Fis, while CRP seems to provide a fine-tuning mechanism. Taken together, the analysis presented here shows the utility of combining computational and experimental approaches to generate a deeper understanding of the biofilm formation process in bacteria.

microbiology↗

bioRxiv2017

Emergent properties in complex synthetic bacterial promoters

Regulation of gene expression in bacteria results from the interplay between transcriptional factors (TFs) at target promoters, and how the arrangement of binding sites determines the regulatory logic of promoters is not well known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these TFs. We found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as \"UP\" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

synthetic biology↗