The leading region of many plasmids is adapted for translational efficiency.

To successfully transfer, conjugative plasmids must overcome a recipient cell's defence mechanisms. The leading region of many plasmids-the first DNA to enter-is known to be enriched in anti-defence genes. Here, we investigate whether selective pressure from defence systems has also affected its sequence composition. We consider two possibilities: greater target depletion (to avoid triggering defence systems) or greater codon adaptation (to rapidly express anti-defence genes). We analyse 1751 conjugative plasmids in 13 plasmid taxonomic units in Enterobacterales. First, we test GC content and depletion of short palindromic motifs (4-8 bp), a potential signature of selective pressure from Type II restriction-modification systems. The leading region is GC-rich but not consistently depleted in these motifs. Then, we use the codon adaptation index (CAI) to assess codon usage. Leading regions in plasmids with MOB-F and MOB-P relaxases are more adapted to optimal host codon usage, consistent with higher translational efficiency, with anti-restriction genes showing particular high CAI. We demonstrate the potential of codon adaptation to uncover new plasmid biology by identifying a single-stranded promoter in the F plasmid, consistent with independent experimental findings. Our findings emphasize the intensity of the selective pressure that plasmids face from defence systems.