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

Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.

Markerless gene deletion in Porphyromonas gingivalis using a pheS*-based counterselection system

ABSTRACT Porphyromonas gingivalis is an oral pathobiont implicated in periodontitis and several systemic diseases and serves as an important model organism. However, the routine generation of markerless mutants in P. gingivalis has remained challenging due to the lack of an efficient counterselection system for the double cross-over approach. Markerless gene deletion is crucial for bacterial genetic manipulations, in particular for generating multiple gene deletions or introducing point mutations. In this study, a counterselection system for P. gingivalis was established by placing the pheS* gene under the control of a P. gingivalis promoter enabling efficient expression. The construct was delivered to P. gingivalis via a suicide plasmid by conjugation. Using PG0719 as a representative target gene, first cross-over recombinants were selected using erythromycin resistance encoded on the suicide plasmid. Cells were then subjected to counterselection in the presence of p-chloro-phenylalanine (p-Cl-Phe). Retention of pheS* in the genome reduced viability, thereby enriching recombinants that had undergone a second recombination event and loss of the plasmid sequence. Candidate clones were screened by colony PCR analysis to confirm the loss of the gene of interest. A markerless PG0719 mutant was generated and further validated by Sanger sequencing, demonstrating a practical approach for markerless gene deletion in P. gingivalis and providing a framework for further genome modifications in the organism. IMPORTANCE Although Porphyromonas gingivalis is a widely studied model organism, the genetic manipulation of this bacterium has remained limited by the lack of efficient tools for markerless genome editing. Here, we established a counterselection system based on pheS∗ that enables markerless gene deletion in P. gingivalis. This approach addresses a technical limitation in the field and provides a practical and broadly applicable framework for advanced genetic manipulation in this important oral pathobiont. Although Porphyromonas gingivalis is a widely studied model organism, the genetic manipulation of this bacterium has remained limited by the lack of efficient tools for markerless genome editing. Here, we established a counterselection system based on pheS∗ that enables markerless gene deletion in P. gingivalis. This approach addresses a technical limitation in the field and provides a practical and broadly applicable framework for advanced genetic manipulation in this important oral pathobiont.

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.

Leucine auxotrophic marker for genetic manipulation of Mycobacterium abscessus

ABSTRACT Mycobacterium abscessus is a clinically important multidrug-resistant pathogen for which genetic manipulation remains challenging. Here, we developed a second-generation genetic toolbox based on leucine auxotrophy that enables antibiotic-free positive selection. A ΔleuB mutant, lacking the gene encoding isopropyl malate dehydrogenase in the leucine biosynthesis pathway, was generated by targeted gene deletion. This mutant requires exogenous leucine for growth and can be complemented by plasmid-borne leuB, establishing a robust auxotrophy-based selection system. To support genetic manipulation, we constructed a suite of second-generation vectors, including a multicopy replicative vector (pRep-amp-leuB), a single-copy integrative vector (pInt-amp-leuB), and a suicide vector (pSuc-amp-leuB) for allelic replacement. These vectors enable gene overexpression, complementation, and targeted gene deletion, respectively, without reliance on aminoglycoside resistance markers. Using this system, we demonstrate efficient transformation and functional complementation of the ΔleuB mutant, achieving high transformation efficiencies and near-zero background growth under leucine selection. In contrast to antibiotic-based systems, this approach eliminates nonspecific background and avoids activation of stress response pathways, such as the whiB7 regulon. Overall, this auxotrophy-based toolbox provides a versatile platform for precise genetic manipulation in M. abscessus, improving selection stringency and enabling antibiotic-free functional genomics approaches, as demonstrated by deleting aac(2′), eis2, and a 19-kb fragment of the gpl locus. IMPORTANCE The development of a leucine auxotroph-based genetic system for Mycobacterium abscessus addresses critical challenges in mycobacterial genetics. By avoiding established antibiotic resistance markers, this approach reduces selective pressure for antibiotic-resistant transformants, supports antibiotic stewardship, and minimizes costly disposal from laboratory waste. It also avoids unintended activation of whiB7, a master regulator of approximately 100 genes, particularly those involved in antibiotic stress responses, thereby improving the accuracy of phenotypic drug susceptibility testing. The versatile genetic toolbox developed here, including novel replicative, integrative, and suicide plasmids, provides precise control over functional studies, overexpression, complementation, and gene deletion. It significantly reduces dependency on antibiotics for genetic manipulation, aligning with the goals of sustainable research and offering new opportunities for studying this clinically significant pathogen. This approach represents a critical advance in microbial genetics, enhancing our capacity to explore the molecular basis of pathogenesis and drug resistance in M. abscessus. The development of a leucine auxotroph-based genetic system for Mycobacterium abscessus addresses critical challenges in mycobacterial genetics. By avoiding established antibiotic resistance markers, this approach reduces selective pressure for antibiotic-resistant transformants, supports antibiotic stewardship, and minimizes costly disposal from laboratory waste. It also avoids unintended activation of whiB7, a master regulator of approximately 100 genes, particularly those involved in antibiotic stress responses, thereby improving the accuracy of phenotypic drug susceptibility testing. The versatile genetic toolbox developed here, including novel replicative, integrative, and suicide plasmids, provides precise control over functional studies, overexpression, complementation, and gene deletion. It significantly reduces dependency on antibiotics for genetic manipulation, aligning with the goals of sustainable research and offering new opportunities for studying this clinically significant pathogen. This approach represents a critical advance in microbial genetics, enhancing our capacity to explore the molecular basis of pathogenesis and drug resistance in M. abscessus.

Methods for the Evasion of Restriction-Modification Systems During Genetic Engineering of Fusobacterium Species.

Optimization of a Genome Editing System for Lactococci Based on a CRISPR-Associated Transposon