Improvement of Permeability to Organic Solvent in Escherichia coli for a Toxicity Biosensor

The outer membrane (OM) of gram-negative bacteria acts as an effective permeability barrier against noxious agents including several antibiotics and organic solvents, and lipopolysaccharide (LPS) is the key molecule for this function. Outer membrane modified mutants (Ml-166, M2-42, M3-21) of E. coli DH5$\alpha$/pBSl were selected through a mutation using EMS (ethyl-methane-sulfonate). Among the selected mutants, M3-21 was twice as sensitive as LumisTo $x^{ }$ to benzene and M2-41 was 8 times as sensitive as LumisTo $x^{ }$ to toluene. To identify the structural change in the membrane by mutation, the relative cell surface hydrophobicities and the absorption of the crystal violet to the organisms were measured. All the mutants absorbed more crystal violet than their parent and the absorption of crystal violet increased in cell walls as carbohydrate of lipopolysaccharide decreased. When the cell surface hydrophobicities of DH5/pBSl and its mutants were measured by the BATH, the hydrophobicities of mutants increased compared to their parent in several organic solvents. The difference of lipopolysaccharide between DH5/pBSl and its mutants was identified by various ways such as the SDS-PAGE gel, the screening of LPS molecular weights, the mass spectrometry, and MALDI-TOF.F.