| Abstract |
Mutations that shorten the lipopolysaccharide (LPS) in Escherichia coli were found to significantly increase the number of transformants after electroporation. The loss of the LPS outer core increased the number of transformants with plasmid pAmCyan (3.3 kb) from 5.0 × 105 colony-forming units (CFU)/μg in the wild-type E. coli BW25113 to 3.3 × 107 CFU/μg in a ΔwaaG background, a 66.2-fold increase in efficiency. Truncation of the inner core improved this even further, with the ΔwaaF mutant exhibiting the best transformation efficiencies obtained, i.e., a 454.7-fold increase in the number of colonies over the wild-type strain. Similar results were obtained when a larger plasmid (pDA1; 11.3 kb) was used, with the ΔwaaF mutant once more giving the best transformation rates, i.e., a 73.7-fold increase. Subsequent tests proved that the enhanced transformabilities of these mutants were not due to a better survival or their surface charge properties, nor from preferential binding of these strains to the plasmid. Using N-phenyl-1-naphthylamine (NPN), we confirmed that the outer membranes of these mutant strains were more permeable. We also found that they leaked more ATP (3.4- and 2.0-fold higher for the ΔwaaF and ΔwaaG mutants, respectively, than wild-type E. coli BW25113), suggesting that the inner membrane stability is also reduced, helping to explain how the DNA enters these cells more easily. KEY POINTS: • LPS inner core gene knockouts increase the electrocompetence of E. coli. • No significant difference in survival, surface charge, or DNA binding was evident. • The LPS inner core mutants, however, exhibited higher outer membrane permeability. • Their inner membranes were also leaky, based on supernatant ATP concentrations.
|
