Biology subjects

Garcia-Villada, L.

Publications and source records attributed to Garcia-Villada, L..

4 recordsLinked to original sources

bioRxiv2026

Redox stress agents strongly enhance mutagenesis during horizontal gene transfer in bacteria and leave distinct mutational and metabolic footprints

Redox stress induces DNA mutations that contribute to chronic conditions affecting human health and to the emergence of antibiotic resistance. Yet, the impact of redox stress-induced mutagenesis remains difficult to decipher because redox agents are diverse and produce hard-to-detect mutational outcomes. Single-stranded DNA (ssDNA) provides a useful tool for studying mutagenic effects of redox agents, as it is particularly susceptible to damage and cannot be repaired by most DNA repair pathways. Here, we established a protocol to investigate redox stress-induced mutagenesis based on the Escherichia coli conjugative ssDNA that is transferred from donor to recipient cells. Using the environmentally relevant redox agents, potassium bromate and hydrogen peroxide, we show that the F episome is remarkably sensitive to weak mutagens during conjugation, enabling the detection of significant differences in mutational spectra induced by these agents. We support our findings with metabolomic analysis, which reveals agent-specific responses in E. coli. We compare these results with those obtained using a yeast ssDNA reporter and conclude that redox-induced mutagenesis depends, among other factors, on the metabolic context of the analysed system. These findings have important implications because the high sensitivity of conjugation-associated ssDNA to environmental mutagens may contribute to the evolution of antibiotic resistance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/730102v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16a2a1eorg.highwire.dtl.DTLVardef@f329bcorg.highwire.dtl.DTLVardef@38fd4eorg.highwire.dtl.DTLVardef@17e4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Structural and Computational Analysis of Pseudomonas Aeruginosa DNA Gyrase Reveals Molecular Characteristics That May Contribute to Ciprofloxacin Resistance

Pseudomonas aeruginosa is considered a priority pathogen by the World Health Organization due to its resistance to antibiotics. Isolates resistant to ciprofloxacin (CPFX), a bactericide commonly used against P. aeruginosa, usually carry the mutations T83I or D87N in the GyrA subunit of the DNA gyrase. Yet, the molecular mechanisms by which these mutations confer CPFX-resistance to P. aeruginosa are unknown. Here we solved the crystal structure of the P. aeruginosa gyrase catalytic cleavage core and used it to carry out molecular dynamic (MD) simulations of CPFX-gyrase binding in the wild-type as well as the T83I and the D87N mutant systems. Our results show that DNA plays the most prominent role in maintaining the CPFX-bound conformation, with no appreciable contributions from Thr83 or Asp87. Interestingly, we found a solvent cavity adjacent to these residues that may provide CPFX access to the active site. Interaction energy analysis using Umbrella Sampling indicates that Thr83 and Asp87 may influence CPFX trajectory during binding. In the mutant systems, the attractive potential decreases, which may hinder CPFX accessing the binding site. These results shed light on P. aeruginosa resistance to CPFX and may help provide a methodology to identify new therapeutic agents to target fluoroquinolone resistant bacteria. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/695517v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c203f0org.highwire.dtl.DTLVardef@90aa78org.highwire.dtl.DTLVardef@1a7d286org.highwire.dtl.DTLVardef@ee8c7a_HPS_FORMAT_FIGEXP M_FIG C_FIG Molecular dynamic positioning of ciprofloxacin (CPFX) from the cavity site (green) to the bound inhibition site (magenta) suggests that mutations T83I and D87N, conferring CPFX resistance to the P. aeruginosa gyrase, may have an influence on the ability of CPFX to access the binding site. Protein is shown in cyan, DNA backbone in orange, and DNA bases in gradient from cyan to blue.

molecular biology↗

bioRxiv2022

Starvation Response and RNA Polymerase Guide Antibiotic-induced Mutable Gambler Cells

Antibiotics can induce mutations that cause antibiotic resistance, triggered by stress responses. The identities of the stress-response activators reveal environmental cues that elicit mutagenesis, and are weak links in mutagenesis networks, inhibition of which could slow evolution of resistance during antibiotic therapies. Despite pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin (stress)-induced mutagenesis. We show its role in transient differentiation of a mutable "gambler" cell subpopulation. Stringent activity is induced by reactive oxygen and upregulates two small (s)RNAs that induce the general stress response, which allows mutagenic DNA-break repair. Surprisingly, RNA polymerase and response-activator nucleotide (p)ppGpp promote ciprofloxacin-induced DNA-damage signaling. We propose a model for their regulation of DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNA-polymerase regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.

microbiology↗

bioRxiv2018

Gamblers: an Antibiotic-induced Evolvable Cell Subpopulation Differentiated by Reactive-oxygen-induced General Stress Response

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations that cause drug resistance by triggering differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS) to signal the sigma-S ({sigma}S) general-stress response. Cipro-generated DNA breaks activate the SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is restricted to a cell subpopulation in which electron transfer and SOS induce ROS, which activate the {sigma}S response, allowing mutagenesis during DNA-break repair. When sorted, this small {sigma}S-response-\"on\" subpopulation produces most antibiotic cross-resistant mutants. An FDA-approved drug prevents {sigma}S induction specifically inhibiting antibiotic-promoted mutagenesis. Furthermore, SOS-inhibited cell division, causing multi-chromosome cells, is required for mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a \"gambler\" cell subpopulation promote resistance evolution without risking most cells.

microbiology↗