Rapid Preparation of Electrocompetent Listeria monocytogenes and Enhancement of Transformation Efficiency with cAMP Supplementation

L. monocytogenes is a significant foodborne pathogen associated with serious health risks. The ability to genetically manipulate this bacterium is critical for understanding its pathogenic mechanisms and developing new interventions. However, low transformation efficiency and the absence of natural competence in L. monocytogenes present challenges for genetic studies. We optimized transformation by testing two isolates closely related to widely used laboratory reference strains F2365 and EGD-e (BL87-016 and BL91-025, respectively), and one CC121 isolate (BL87-028-B) that is ecologically relevant to food environments. Increasing the electroporation voltage from 10 to 11 kV/cm resulted in an increase in efficiency. In addition, supplementation with cyclic adenosine monophosphate (cAMP) prior to electroporation increased transformation efficiency in a dose-dependent manner, with an optimal concentration of 32 mM cAMP yielding increases of up to 377-fold. Recognizing the time constraints associated with liquid-based protocols, we developed a rapid agar-lawn method that simplified the workflow and reduced preparation time from approximately 10 h to under 3 h, though efficiencies remained lower than the gold standard method by Monk et al. (2008). The rapid agar-lawn protocol was tested in a panel of 66 L. monocytogenes isolates, with 83% of isolates showing transformability. Transformability showed no statistical correlation between lineage or mobile genetic elements (MGEs). Cooccurrence network analysis of defense and antidefense systems uncovered a higher incidence of interconnected defense repertoires in transformable isolates, suggesting adaptive immune architectures that facilitate DNA uptake. These findings establish optimized protocols and identify potential genomic determinants of transformability under the rapid agar-lawn protocol, broadening genetic accessibility for functional genomics and pathogenesis studies in L. monocytogenes.