Outer membrane changes in a toluene-sensitive mutant of toluene-tolerant Pseudomonas putida IH-2000

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed u nchanged antibiotic resistance levels, from toluene-tolerant Pseudomonas pu tida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by inse rtion of Tn5 was identified as cyoC, which is one of the subunits of cytoch rome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic aci d, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in out er membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydro phobicity, and mutant No. 32 is considered to be sensitive to toluene.