Lysinibacillus sphaericus III(3)7 and Plasmid Vector pMK4: New Challenges in Cloning Platforms

Abstract

The acquisition and especially the maintenance of a plasmid usually brings a fitness cost that reduces the reproductive rate of the bacterial host; for strains like Lysinibacillus sphaericus III(3)7, which possesses important environmental properties, this alteration along with morphological changes and reduced sporulation rates may exert a negative effect on metabolic studies using plasmids as cloning platforms. The aim of this study is to approach the metabolic behavior of pMK4-bearing cells of L. sphaericus III(3)7 through the use of bioinformatic and in vitro analyses. An incompatibility model between the pMK4 vector and a predicted megaplasmid, pBsph, inside III(3)7 cells was constructed based on an incA region. Additionally, in vitro long-term plasmid stability was not found in plasmid-bearing cells. Alignments between replicons, mobile genetic elements and RNA-RNA interactions were assessed, pairwise alignment visualization, graphic models and morphological changes were evaluated by SEM. Metabolite analysis was done through HPLC coupled to a Q-TOF 6545, and electrospray ionization was used, finally, Aedes aegypti and Culex quinquefasciatus larvae were used for larvicidal activity assessment. Results found, a decreased growth rate, spore formation reduction and morphological changes, which supported the idea of metabolic cost exerted by pMK4. An incompatibility between pMK4 and pBsph appears to take place inside L. sphaericus III(3)7 cells, however, further in vitro studies are needed to confirm it.