Novel toluene elimination system in a toluene-tolerant microorganism

Abstract

In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 +/- 0. 012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

FIG. 1

The formation of MVs (indicated by arrowheads) from the outer membrane of IH-2000 was observed when cells were grown in a medium containing toluene. MV formation would start with the swelling of the outer membrane (A). The outer membrane was not damaged by MV formation (B). The MVs released were also detected, and there were no electron-dense materials enclosed (C). Incomplete MVs (indicated by arrowheads) were produced by a toluene-sensitive mutant, strain 32 (D and E). The MVs of strain 32 were smaller than those of IH-2000 and differed in morphology. Bar = 100 nm.

FIG. 1

The formation of MVs (indicated by arrowheads) from the outer membrane of IH-2000 was observed when cells were grown in a medium containing toluene. MV formation would start with the swelling of the outer membrane (A). The outer membrane was not damaged by MV formation (B). The MVs released were also detected, and there were no electron-dense materials enclosed (C). Incomplete MVs (indicated by arrowheads) were produced by a toluene-sensitive mutant, strain 32 (D and E). The MVs of strain 32 were smaller than those of IH-2000 and differed in morphology. Bar = 100 nm.

FIG. 2

Toluene molecules adhering to IH-2000 (●) or strain 32 (▴) cells or MVs (■) and the amount of fatty acids in MVs (□) in the culture. Toluene was added to a culture of strain IH-2000 or 32 (time = 0). Then the amount of toluene molecules adhering to the cells was measured, and the amount per fatty acid chain was calculated because the MVs were found to be composed of phospholipids and LPS. Error bars show means ± SDs (n = 3). The MV fractions were periodically prepared as described in Materials and Methods from the culture of IH-2000.

FIG. 3

Model of toluene elimination by production of MVs in strain IH-2000. Toluene molecules rapidly adhere to the outer membrane. The toluene molecules are stable in the hydrophobic region of the membrane. The MVs are formed from the outer membrane, and these serve to eliminate toluene molecules from the cell. The MVs released are not likely to fuse to other cells because the surface of the MVs is covered with the polysaccharide chains of LPS.