A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks

Lipopolysaccharides (LPS) are major components of the outer membrane of gra m-negative bacteria and are considered a defense barrier. To determine if L PS play a role in resistance to solvents in the solvent-tolerant Pseudomona s putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begin s the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette enc oding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was co mpared with the wild-type strain with regard to tolerance to a number of to xic compounds, including chelating agents, organic acids, detergents, and a romatic hydrocarbons. It was found that the mutant was as tolerant as the w ild-type strain to organic acids and aromatic hydrocarbons and more sensiti ve to ethylenediaminetetraacetic acid and deoxycholate.