Czyszczon, W. | Search bioRxiv

bioRxiv2022

Gene amplifications cause high-level resistance against albicidin in Gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public health care. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against the antibiotic albicidin. We show that this mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of the GyrI-like transcription regulator STM3175 (YgiV) which binds albicidin. X-ray crystallography and molecular docking studies reveal a conserved binding motif that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in Gram-negative bacteria and our experiments confirm that STM3175 homologs can convey resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

microbiology↗

bioRxiv2020

Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of "poisons", namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

biochemistry↗