MOBILE GENETIC ELEMENTS IN MICROBIAL GENETICS: MECHANISMS OF HORIZONTAL GENE TRANSFER AND THEIR ROLE IN THE EVOLUTION OF ANTIMICROBIAL RESISTANCE: A MINI REVIEW

Advances in understanding of plasmid biology through the application of transposon insertion sequencing

Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.

Stage-Specific Strategies to Limit the Spread of Antibiotic Resistance in Bacteria with a Focus on Conjugation

Horizontal gene transfer (HGT) is the main reason for antibiotic resistance evolution, enabling bacteria to obtain resistance determinants far more rapidly than through point mutations alone. Among HGT mechanisms, conjugation plays a particularly critical role for its efficiency, broad host range, and capacity in disseminating complex resistance plasmids across microbial communities. Despite its clinical significance, conjugation has remained an under-explored target for antimicrobial intervention. This research aims to provide an integrated analysis of bacterial conjugation within the broader context of HGT, emphasizing its evolutionary persistence and mechanistic vulnerabilities. Evidence is synthesized from molecular biology, experimental evolution, and population genetics to uphold the argument for the maintenance of plasmid-mediated resistance through a balance of transfer efficiency, compensatory adaptations, and post-transfer stabilization mechanisms. Building on this foundation, a stage-specific framework is proposed for disrupting conjugation by targeting pre-transfer cell-cell contact, peri-transfer DNA processing, and post-transfer plasmid maintenance. Multiple intervention strategies are reviewed and evaluated in this study, including conjugation inhibitors, relaxase-targeting compounds, CRISPR-based barriers, plasmid curing compounds, restriction-modification systems, as well as the plasmid addiction modules. Collectively, these approaches demonstrate that plasmid persistence is an evolvable trait that can be subjected to targeted interference. Overall, this study highlights conjugation-specific interfering strategies as a potential opportunity in slowing down resistance dissemination and preserving the efficacy of existing antibiotics.

From Selfish Transposon Enzymes to Programmable Miniature Genome Editors: The Biology, Evolution, and Engineering of TnpB Nucleases

TnpB proteins are among the most abundant genes encoded in bacterial and archaeal genomes, yet their function in the transposon life cycle remained unclear for decades. Recognition that TnpB is the likely evolutionary ancestor of type V CRISPR-Cas12 effector nucleases, followed by experimental demonstration that TnpB is itself a compact RNA-guided DNA endonuclease, has transformed these previously regarded accessory transposon proteins into a promising frontier in genome engineering. At roughly 350-410 amino acids, TnpB is less than half the size of Cas12a and approximately one-third the size of Cas9, making it well suited to delivery vehicles with constrained cargo capacity, such as adeno-associated virus (AAV). This review discusses recent progress in TnpB biology and technology. It first describes the biochemical and structural basis of RNA-guided DNA cleavage by TnpB and its role in transposon homing. It then summarises comparative genomic analyses that reveal the diversity of TnpB, repeated independent evolutionary transitions from TnpB to Cas12, and recurrent exaptation of TnpB for cellular functions unrelated to transposition. The review also considers how mining natural TnpB diversity and high-throughput protein engineering have produced compact editors with activity and specificity approaching established CRISPR-Cas tools in selected contexts, including early demonstrations of TnpB-mediated editing in animals and crop plants. It concludes by outlining the principal challenges, including transposon-associated motif (TAM) restriction, off-target activity, delivery and mechanistic understanding, that must be addressed before the therapeutic and agricultural potential of TnpB-derived technologies can be fully realised.

Bacterial DNA invasion triggers transposable element proliferation and genome expansion

Genome size variation in eukaryotes is driven largely by transposable elements (TEs), yet the biological mechanisms that initiate their proliferation remain understudied. Here, we identify a recurrent association between bacterial horizontal gene transfer (HGT) and bursts of TE activity that contribute to genome expansion. By leveraging comparative genomics and genus-level pangenome analyses across three species of the nut weevil, Curculio, we detect extensive bacterially derived DNA sequences embedded within structurally dynamic genomic regions. These HGT-associated regions are dominated by a small number of young, proliferating TE families, particularly DNA type II Mavericks, which encapsulate transferred bacterial sequences and comprise a substantial fraction of recent genomic DNA in derived lineages. Analyses of codon usage bias, intron length, and functional enrichment suggest that most transferred genes undergo progressive pseudogenization over evolutionary time, whereas a subset of selectively advantageous HGTs persist. Together, our findings support a model linking foreign DNA invasion with TE proliferation, genome size variation, and molecular innovation.

Control of foreign DNA: emerging roles of xenogeneic silencers.

Bacteria continuously acquire foreign DNA through horizontal gene transfer, yet its successful integration depends on regulatory mechanisms that balance genome protection with evolutionary innovation. Xenogeneic silencers are central to this process: they preferentially bind AT-rich DNA, a common feature of many horizontally acquired genetic elements, and repress its transcription. Recent studies, however, reveal a much broader regulatory repertoire. Beyond transcriptional repression, these proteins contribute to chromosome organization by forming higher-order nucleoprotein complexes and phase-separated condensates that shape bacterial nucleoid architecture. Furthermore, they play roles in regulating bacteriophage infection cycles, including mechanisms by which phages hijack host silencing activities for their own benefit. Their extensive regulatory reach, spanning virulence genes, biofilm formation, specialized metabolite production, and mobile genetic elements (MGEs), underscores their central role in connecting environmental signals, including fluctuations in the second messenger c-di-GMP, with gene expression, and genome organization. The diversification of xenogeneic silencers across bacterial chromosomes, plasmids, phages, and other MGEs highlights their evolutionary significance. Together, these recent findings position xenogeneic silencers as dynamic regulatory modules that shape the fate of foreign DNA across the horizontal gene transfer network.

Genetic architecture and functional dynamics of integrons in Staphylococcus aureus resistance