DNA binding is rate-limiting for natural transformation

Bacteria take up environmental DNA using dynamic appendages called type IV pili (T4P) to elicit horizontal gene transfer in a process called natural transformation. Natural transformation is widespread amongst bacteria yet determining how different factors universally contribute to or limit this process across species has remained challenging. Here we show that Acinetobacter baylyi, the most naturally transformable species, is highly transformable due to its ability to robustly bind nonspecific DNA via a dedicated orphan minor pilin, FimT. We show that, compared to its homologues, A. baylyi FimT contains multiple positively charged residues that additively promote DNA binding efficiency. Expression of A. baylyi FimT in a closely related Acinetobacter pathogen is sufficient to substantially improve its capacity for natural transformation, demonstrating that T4P-DNA binding is a rate-limiting step in this process. These results demonstrate the importance of T4P-DNA binding efficiency in driving natural transformation, establishing a key factor limiting horizontal gene transfer. ImportanceNatural transformation is a multi-step, broadly conserved mechanism for horizontal gene transfer in which bacteria take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. A complete picture of the factors that limit this behavior remain unclear due to variability between bacterial systems. In this manuscript, we provide clear and direct evidence that DNA binding by type IV pili prior to DNA uptake is a rate-limiting step of natural transformation. We show that increasing DNA binding in antibiotic resistant Acinetobacter pathogens can boost their transformation rates by 100-fold. In addition to expanding our understanding of the factors that limit transformation in the environment, these results will also contribute to a deeper understanding of the spread of antibiotic resistance genes in relevant human pathogens.