Engineering the MarinePseudoalteromonas haloplanktisTAC125 via pMEGA Plasmid Targeted Curing Using PTasRNA Technology

Abstract Marine bacteria that have adapted to thrive in extreme environments, such as Pseudoalteromonas haloplanktis TAC125 (PhTAC125), offer a unique biotechnological potential. The discovery of an endogenous megaplasmid (pMEGA) raised questions about its metabolic impact and functional role in this strain. This study aimed at streamlining the host genetic background by curing PhTAC125 from the pMEGA plasmid using a sequential genetic approach. We combined homologous recombination by exploiting a suicide vector with the PTasRNA gene silencing technology to interfere with pMEGA replication machinery. This approach led to the construction of the novel PhTAC125 KrPL2 strain, cured from the pMEGA plasmid, which exhibited no significant differences in the growth behaviour, though showcasing enhanced resistance to oxidative stress and a reduced capability of biofilm formation. These findings represent a significant achievement for understanding of the role of pMEGA plasmid and for the biotechnological applications of PhTAC125 in recombinant protein production. This opens up the possibility to exploit pMEGA valuable genetic elements and further advancing the genetic tools for PhTAC125. Competing Interest Statement The authors have declared no competing interest. Footnotes angelica.severino{at}unina.it; marzia.calvanese{at}unina.it; ermenegilda.parrilli{at}unina.it; christopher.riccardi{at}unifi.it; marco.fondi{at}unifi.it acolarusso{at}scripps.edu