TY  - JOUR
TI  - Plasmid Curing of Pseudoalteromonas haloplanktis TAC125 Using Homologous Recombination and PTasRNA Gene Silencing
AU  - Severino, Angelica 
AU  - Lauro, Concetta 
AU  - Calvanese, Marzia 
AU  - Parrilli, Ermenegilda 
AU  - Tutino, Maria Luisa
VL  - 16
IS  - 10
PY  - 2026
DA  - 2026/05/20
SP  - e5687
C1  - Bio-protocol 2026;16:e5687
DO  - 10.21769/BioProtoc.5687
UR  - https://doi.org/10.21769/BioProtoc.5687
AB  - Pseudoalteromonas haloplanktis TAC125 is a psychrophilic marine bacterium widely used to study cold adaptation and increasingly exploited as a non-conventional platform for biotechnological applications. The strain harbors the endogenous megaplasmid pMEGA (64.7 kb), whose presence may limit its exploitation as a cell factory, making its elimination advantageous to strain engineering. Traditional plasmid-curing approaches based on chemical and physical agents are often inefficient and unsuitable for stable endogenous replicons, such as pMEGA. Here, we describe a targeted protocol for pMEGA curing in P. haloplanktis TAC125 that combines homologous recombination with paired-termini antisense RNA (PTasRNA) gene silencing. First, a selectable marker cassette is inserted into pMEGA by homologous recombination using a suicide vector, enabling selective discrimination between plasmid-positive and plasmid-cured bacteria. Next, PTasRNA gene silencing technology is applied to target a gene essential for the replication of pMEGA, thereby transiently interfering with its replication and promoting its loss. This approach provides a specific method to cure a highly stable endogenous megaplasmid in a psychrophilic non-conventional bacterium, enabling improved functional studies and strain optimization, establishing a broadly applicable framework for targeted curing across diverse bacterial systems.
KW  - Pseudoalteromonas haloplanktis TAC125
KW  - Plasmid curing
KW  - Homologous recombination
KW  - PTasRNA technology
KW  - Marine psychrophilic bacteria
KW  - Megaplasmid
KW  - Strain engineering
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False