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

ABSTRACT Despite substantial advances in bacterial genome engineering, functional genetic analysis remains challenging in many non-model bacterial species, particularly among host-associated gram-positive bacteria. The fructophilic species Apilactobacillus kunkeei has been investigated for more than two decades and is a dominant member of the honeybee microbiome, where it contributes to pathogen resistance and colony fitness. Nevertheless, the mechanistic investigation of this ecologically important species has remained limited despite its growing probiotic relevance. To enable functional genomics in this organism, we developed an inducible genome-engineering platform that leverages its endogenous Type II-A CRISPR-Cas9 system. The system uses a sakacin-responsive dual-plasmid initiator–effector design in which phage-derived recombineering genes and a single-guide RNA are coordinately expressed, while DNA cleavage is mediated by natively expressed Cas9. Using this approach, we achieved scarless deletion of individual genes, including targets as large as ~25 kb, gene replacement with a fluorescent reporter, C-terminal epitope tagging, and precise nucleotide substitutions, with editing efficiencies approaching 100%. Both plasmids can be readily cured following modification, allowing recovery of clean mutant genotypes. We further demonstrate that endogenous Cas9 can be repurposed for CRISPR interference using a single, self-contained plasmid to enable targeted transcriptional repression. Together, this work establishes a robust strategy for genetic manipulation of A. kunkeei and expands the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria. IMPORTANCE Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria. Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria.

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

Genetic engineering of non-conventional yeasts is frequently limited by the lack of robust dominant selection systems that function across phylogenetically diverse hosts. These organisms are increasingly important platforms for sustainable bioproduction due to their unique metabolic capabilities. Candida famata , an industrial riboflavin overproducer belonging to the CTG clade with alternative codon decoding, represents a particularly relevant model for evaluating cross-species selection strategies. Many commonly used antibiotic resistance markers exhibit strong host dependence, especially in yeasts with non-standard genetic codes, thereby restricting strain construction and metabolic engineering. Aureobasidin A (AbA) is a potent antifungal compound that inhibits inositol phosphorylceramide (IPC) synthase, a key enzyme in sphingolipid biosynthesis, making it an attractive candidate for dominant selection. In this study, we evaluated the sensitivity to AbA across phylogenetically diverse yeast species and developed a resistance marker based on a codon-optimised variant of the Saccharomyces cerevisiae AUR1 gene, designed to ensure correct translation in CTG-clade yeasts. Sensitivity assays confirmed that AbA efficiently inhibited the growth of multiple yeast species at low concentrations, supporting its use as a selective agent. The native C. albicans AUR1 gene did not confer functional resistance in C. famata despite successful transformation, highlighting limitations imposed by host-dependent gene expression. In contrast, the codon-optimized S. cerevisiae AUR1 construct restored robust resistance across the tested yeast species. Transformation yielded stable mutants with reproducible resistance phenotypes, confirmed by molecular validation and maintained through serial passaging. Importantly, the construct remained functional not only in CTG-clade yeasts but also in species using the standard genetic code. Codon usage analysis showed that replacement of CTG codons eliminated ambiguous decoding while introducing leucine codons broadly preferred among yeast species, providing a plausible explanation for the observed cross-species functionality of the AUR1 marker. Fusion with GFP confirmed correct expression and intracellular localisation without detectable impact on host physiology, while fermentation experiments demonstrated that the system did not significantly affect riboflavin production under the tested conditions. Collectively, these results establish AbA as a highly effective selective agent and demonstrate that rational recoding of AUR1* enables the development of an efficient dominant selection marker across the yeast species evaluated in this study. This platform expands the genetic toolkit available for non-conventional yeasts and provides a versatile solution for strain engineering across multiple yeast hosts.