Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

Abstract

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.

Analysis of solvent tolerance in Pseudomonas putida DOT‐T1E based on its genome sequence and a collection of mutants

“…One of the most relevant features observed with a Δ cyo B mutant of T1E is that it exhibited a marked reduction in global metabolism suggesting that the limitation in energy generation associated with this terminal oxidase has a general effect on cell metabolism in the presence of toluene. For example, fatty acid biosynthesis is greatly limited, which agrees with previous observations that CyoB mutant cells produce membrane invaginations that affect cell membrane structure and consequently result in extreme sensitivity to solvent shocks [55].…”

A two‐partner secretion system is involved in seed and root colonization and iron uptake by Pseudomonas putida KT2440

“…Proteins present in the outer membrane or periplasm were extracted after obtaining spheroplasts as previously described (Duque et al ., 2004). Proteins present in the supernatant of grown cultures were extracted as follows.…”

Modification of phospholipid composition inPseudomonas putidaA ATCC 12633 induced by contact with tetradecyltrimethylammonium

“…At this point, the culture was divided into five parts: one part was kept as a control and to the rest, 10, 50, 100 and 150 mg l )1 TDTMA was added. The number of viable cells (CFU ml )1 ) was determined by plating the serially diluted cell suspension on LB plates before TDTMA was added and at 10, 30, 60 and 120 min after TDTMA addition (Duque et al 2004). All experiments were conducted in triplicate, repeated independently and the standard deviations (SDs) are shown as error bars in the figures.…”