Environmental Microbiology (2004) 6(10), 1021–1031 doi:10.1111/j.1462-2920.2004.00621.x © 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 20046 1010211031Original ArticleSolvent sensitivity in cyoB mutantE. Duque et al. Received 1 December, 2003; accepted 18 February, 2004. *For cor- respondence. E-mail jlramos@eez.csic.es; Tel. (+34) 958181608; Fax (+34) 958135740. Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida Estrella Duque, Vanina García, Jesús de la Torre, Patricia Godoy, Patricia Bernal and Juan-Luis Ramos* Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Plant Biochemistry and Molecular and Cellular Biology, Calle Profesor Albareda number 1, E-18008 Granada, Spain. Summary The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp ‘cyoB’ DNA fragment was cloned in pCHESI- WKm and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the gen- eration 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 mem- branes, which eventually lead to cell death. Introduction Aerobic bacteria generate energy through respiratory pro- cesses in which electrons flow through a series of electron carriers until they reach terminal oxidases that reduce oxygen to water. In Pseudomonas sp. two main terminal cytochrome oxidases are known, namely the cytochrome ubiquinol o oxidase (Cyo) and the cyanide insensitive oxidase (Cio) (Cunningham and Williams, 1995; Cooper et al., 2003). The cyoABCDE gene cluster encodes the cytochrome o oxidase complex (CyoA,B,C and D pro- teins), and the heme o synthase (CyoE) (Hirayama et al., 1998; Dinamarca et al., 2002). This cytochrome receives electrons from the ubiquinone pools (Nakamura et al., 1997). The CyoABCD terminal oxidase seems to play a major role in cells growing in conditions of high oxygen tension (Sweet and Peterson, 1978; Cotter et al., 1990; 1997), and is expressed preferentially in cells in the expo- nential phase although expression in the stationary phase also takes place but at a lower level (Dinamarca et al., 2003). In contrast, at least in Escherichi coli the CydAB terminal oxidase (CioAB in P. aeruginosa) is preferentially expressed in cells growing under low oxygen tension and in the stationary phase (Taylor and Zhulin, 1999). None- theless, in Escherichia coli each of these terminal oxi- dases replaces the other one in isogenic mutants deficient in their synthesis. Mutants in the cyo genes in different strains of Pseudomonas putida have been isolated and the effects on gene regulation and solvent tolerance have been anal- ysed. Mutants in the cyoABCDE cluster in P. putida KT2440 (Dinamarca et al., 2002; 2003) and P. putida strain H (Petruschka et al., 2001) escape from catabolite control, in the case of the alkane degradation pathway in the KT2440 strain, and the phenol degradation pathway, in the H strain. It was hypothesized that the catabolic repression system could monitor the physiological status of the cells by sensing the redox state of the elements of the electron transport chain, and that this could be used as a signalling system. However, the authors could not ascertain whether the Cyo terminal oxidase plays a direct or an indirect role in signal transmission. A mutant in the cytochrome ubiquinol oxidase system was also described in the solvent-tolerant P. putida IH2000 strain. This muta- tion lead to increased solvent sensitivity in comparison with wild-type strain (Hirayama et al., 1998). The reason for this increased tolerance was believed to be changes in the membrane hydrophobicity, although the intimate reasons remain unknown (Kobayashi et al., 1999). Pseudomonas putida DOT-T1E is a solvent-tolerant strain (Ramos et al., 1995) that exhibits physical and bio- chemical barriers to decrease solvent toxicity (Ramos et al., 1997; 2002). In response to toluene in the culture medium, the cell membrane is packaged by transforming