Biology subjects

Medeiros, A. S.

Publications and source records attributed to Medeiros, A. S..

4 recordsLinked to original sources

bioRxiv2026

Repurposing a chromosome segregation ParB-CTPase fold into an ATPase toxin for contact-dependent growth inhibition in plant and animal pathogens

Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation.

microbiology↗

bioRxiv2024

A toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of the second messenger regulatory network

C-di-GMP is a widespread second messenger, coordinating various cellular functions in bacteria. Levels of c-di-GMP can be highly dynamic and vary over a wide range of concentrations. Here we constructed a large set of FRET-based c-di-GMP biosensors, using homologues of c-di-GMP-binding effector YcgR from different bacterial species. This biosensor library was characterized using a newly established protocol to quantify FRET efficiency using flow cytometry. The resulting toolbox of 18 selected biosensors that undergo large FRET signal change upon c-di-GMP binding displays a [~]100-fold range of c-di-GMP binding affinities. We combined this toolbox with a barcoded Tn5 transposon library and cytometry-based cell sorting to develop FRET-To-Sort, a new application for systematic characterization of gene networks regulating levels of FRET-detected small molecules. Applied to planktonic E. coli cells, FRET-To-Sort identified both known and novel regulatory modules controlling c-di-GMP levels, including flagellum and fimbria biogenesis, lipid metabolism and stress response genes.

microbiology↗

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

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↗