Abstract <p>This study demonstrates the impact of a Structure Maintenance of Chromosome (SMC)-like Wadjet system on the horizontal gene transfer of plasmids by conjugation to a recipient that naturally containing such a system for the first time. A <i>Clostridium cellulovorans</i> mutant with dramatically improved efficiency to receive plasmid DNA by conjugation was isolated and sequenced. Three spontaneous chromosomal deletions included a type II restriction-modification system, a putative CRISPR system, and a cluster of ORFs named <i>jetABCD</i> encoding a putative Wadjet system. Since nearly nothing is known about the role of naturally occurring Wadjet systems in their native host bacteria, markerless chromosomal deletion of <i>jetABCD</i> in the <i>C. cellulovorans</i> wildtype strain 743B was achieved and the effect on conjugative plasmid uptake was studied. The transconjugation frequency of the <i>jetABCD</i> mutant was increased by about five orders of magnitude compared to wildtype <i>C. cellulovorans</i> recipient cells. Bioinformatic analysis of genome sequences of the <i>Bacillota</i> phylum revealed near-complete mutually exclusive possession of either plasmids &lt; 40&#xa0;kb or <i>jetABCD</i> genes, indicating high efficiency of Wadjet systems in small plasmid prevention in bacteria. Importantly, the implications of this study go beyond the case of <i>C. cellulovorans</i>. Our study demonstrates that the eradication of Wadjet systems can dramatically improve the uptake of recombinant plasmids and thereby enhance genetic engineering of bacterial strains of interest for biotechnological applications.</p> Key points <p>• Native Wadjet system inhibits plasmid transfer by conjugation in <i>C. cellulovorans</i></p> <p>• Deleting <i>jetABCD</i> increased plasmid uptake by about five orders of magnitude</p> <p>• Possession of Wadjet systems efficiently block plasmid maintenance in <i>Bacillota</i></p> Graphical Abstract <p></p>