Wadjet System Inactivation Boosts Plasmid Acquisition in Phylogenetically Distant Gram-positive Bacteria Clostridium cellulovorans and Micrococcus luteus

Innate defense mechanisms heavily influence the ability of some bacteria to take up and maintain plasmid DNA. The Wadjet system is a recently characterized defense system that targets and cleaves circular plasmid DNA. In this study, we report the consequences of the knockout of Wadjet system-encoding genes in two Gram-positive model bacteria, Clostridium cellulovorans (phylum Bacillota) and Micrococcus luteus (phylum Actinomycetota), for the uptake of plasmid DNA via transconjugation and natural transformation, respectively.

Genome sequencing of a spontaneous C. cellulovorans mutant with a dramatically enhanced frequency of plasmid DNA acquisition through conjugation revealed three chromosomal deletions, one of them including a putative Wadjet system-encoding jetABCD gene cluster. After deletion of only this gene cluster in the wildtype, the efficiency of conjugative plasmid transfer from E. coli to C. cellulovorans ΔjetABCD increased by about five orders of magnitude, confirming that the Wadjet system represents an efficient barrier to plasmid transfer in C. cellulovorans. Additional in vivo methylation of the plasmid in the E. coli donor to protect it from restriction by a type II RM endonuclease present in C. cellulovorans further improved uptake by conjugation.

In M. luteus strain TrpE16, a model organism for the study of DNA uptake by natural transformation in high-GC Gram-positive bacteria, deletion of the chromosomal jetABCD gene cluster likewise substantially increased the frequency of plasmid uptake, in this case by natural transformation. Via introduction of the ΔjetABCD deletion it was possible to overcome a known limitation of M. luteus TrpE16 to be transformed with circular plasmid DNA. Notably, reisolation and analysis of plasmids from the Wadjet-deficient and the parental M. luteus strains seemed to yield different topologies or concatemeric states of the plasmid.

These findings collectively underscore the restrictive role of Wadjet systems in two phylogenetically distant Gram-positive bacteria from the Actinomycetota (high-GC) and Bacillota (low-GC) on plasmid stability and consequently on the efficiency of gene transfer via natural transformation and conjugation. This demonstrates the importance of considering Wadjet systems in addition to RM and CRISPR-Cas systems as defense systems that can seriously hinder the introduction of recombinant plasmids for genetic engineering purposes.