Leucine auxotrophic marker for genetic manipulation of Mycobacterium abscessus

A Conditional Plasmid System for Markerless Gene Deletion in Genetically Recalcitrant Fusobacterium nucleatum subsp. animalis.

This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.

Development of an arabinose-inducible gene expression system for nontuberculous mycobacteria.

Nontuberculous mycobacteria (NTM) are emerging pathogens for which genetic tools remain limited. Here, we developed an arabinose-inducible gene expression system based on a modified pBAD24 vector adapted for mycobacterial hosts. The vector carries replication origins for mycobacteria and Escherichia coli, as well as selectable markers compatible with NTM. In Mycobacterium abscessus (Mycobacteroides abscessus), the system enabled dose-dependent induction of target gene expression by arabinose, as demonstrated by increased antibiotic resistance and quantitative RT-PCR analysis. Although basal expression was observed in the absence of arabinose, expression levels were tunable across arabinose concentrations. The system was also functional in Mycobacterium smegmatis (Mycolicibacterium smegmatis) and Mycobacterium bovis BCG, although the degree of basal expression varied among host species. These results establish a tunable inducible expression system for mycobacteria and provide a useful genetic tool for studies of NTM biology.

R-pyocin-mediated selection reverses pan-drug resistance in Pseudomonas aeruginosa

The global escalation of antibiotic resistance is a critical threat necessitating the development of innovative strategies to provide new therapeutic options and restore the efficacy of conventional drugs. Pseudomonas aeruginosa exemplifies this challenge by utilizing a robust genomic resistome to persist in clinical settings. Here, we demonstrate that R-pyocins (phage-like bactericidal particles) can be leveraged not merely as conventional biocides, but as precise selective forces to drive an evolutionary “checkmate” strategy. We subjected the laboratory strains PAO1 and PAK and the clinical pan-drug-resistant (PDR) wound isolate MRSN 6220 to R-pyocin selective pressure. To evade R-pyocins targeting the host lipopolysaccharide (LPS) core, resistance consistently emerges through large-scale chromosomal deletions spanning 250-388 kbp. Crucially, these deletions encompass a conserved region harboring the galU gene (essential for LPS synthesis), the hmgA gene (yielding a pyomelanogenic ‘brown’ phenotype), and the mexXYZ multidrug efflux operon. While the loss of galU confers broad cross-resistance to R-pyocins by likely truncating the LPS receptor, the concurrent excision of mexXY induces profound collateral sensitivity to aminoglycosides. Furthermore, these large deletions systematically eliminate critical virulence factors and biofilm clusters, including the hcnABC, exoY, phzABCDEFG, and cup operons. In Galleria mellonella and murine chronic wound models, the resulting brown mutants were rendered non-lethal and exhibited a significant 3-log reduction in bacterial load following gentamicin treatment. Ultimately, this work establishes a framework for utilizing R-pyocins as potent evolutionary steering agents to force the predictable reversion of multidrug resistance into an attenuated, biofilm-deficient, and clinically manageable state. Significance Statement Pan-drug-resistant (PDR) pathogens demand novel strategies that both kill and restore antibiotic efficacy. Here, we describe an evolutionary ‘checkmate’ for Pseudomonas aeruginosa, where selection for R-pyocin resistance drives large-scale (∼300 kb) chromosomal remodeling. Although these deletions confer R-pyocin immunity via loss of the galU gene, they simultaneously collapse the pathogen’s virulence and defense. Crucially, the excision of the mexXY efflux operon resensitizes PDR strains to conventional aminoglycosides, while the collateral loss of critical virulence and biofilm clusters abrogates pathogenesis. By coupling resistance acquisition to substantial fitness costs, our work establishes a framework for using R-pyocins to force predictable evolutionary trade-offs, driving the reversion of multidrug resistance to an attenuated, biofilm-deficient, and clinically manageable state.

Harnessing endogenous CRISPR-Cas9 for inducible genetic engineering of Apilactobacillus kunkeei

An inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota, is established and the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria is expanded.

Adaptive Evolution in Aminoacyl-tRNA Synthetases Drives Antibiotic Tolerance and Resistance in Clinical Klebsiella pneumoniae Isolates.

Development of a codon-optimised AUR1 selectable marker for genetic engineering of diverse yeast species

AbA is established as a highly effective selective agent and rational recoding of AUR1* enables the development of an efficient dominant selection marker across the yeast species evaluated in this study, expanding the genetic toolkit available for non-conventional yeasts and provides a versatile solution for strain engineering across multiple yeast hosts.