Environmental Microbiology (2004) 6(4), 416–423 doi:10.1111/j.1462-2920.2004.00578.x © 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200464416423Original ArticleFatty acid biosynthesis and solvent toleranceA. Segura et al. Received 10 September, 2003; accepted 9 December, 2003. *For correspondence. E-mail ansegura@eez.csic.es; Tel. (+34) 958 181600; Fax (+34) 958 129600. Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E Ana Segura, 1 * Estrella Duque, 1 Antonia Rojas, 1 Patricia Godoy, 1 Antonio Delgado, 2 Ana Hurtado, 1 John E. Cronan Jr, 3 and Juan-Luis Ramos 1 1 Department of Biochemistry and Molecular and Cellular Biology of Plants, CSIC-Estación Experimental del Zaidín, E-18008 Granada, Spain. 2 Department of Earth Sciences and Environmental Chemistry, CSIC-Estación Experimental del Zaidín, E-18008 Granada, Spain. 3 Department of Microbiology, University of Illinois Urbana, Illinois 61801, USA. Summary The unusual tolerance of Pseudomonas putida DOT- T1E to toluene is based on the extrusion of this sol- vent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alter- ations. Pseudomonas putida DOT-T1E-109 is a sol- vent-sensitive mutant. Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14 C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons. This limitation could be the result of alter- ations in the outer membrane because the mutant cells released more b-lactamase to the external medium than the wild-type cells. The mutant P. putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13 CH 3 - 13 COOH incorporation into fatty acids. In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13 CH 3 - 13 COOH at a high rate into de novo synthesized lipids. The mutation in P. putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis. Introduction Aromatic hydrocarbons are extremely toxic to microorgan- isms because they dissolve in the cytoplasmic membrane, causing loss of ions, metabolites, lipids and proteins; dis- sipation of the pH gradient and electrical potential; and inhibition of membrane protein functions (Sikkema et al., 1995; Ramos et al., 1997; Segura et al., 1999). The dam- ages lead to cell death. However, independent laborato- ries have isolated Pseudomonas sp. strains able to thrive on liquid medium in the presence of very high concentra- tions of toluene, styrene and p-xylene (Inoue and Horiko- shi, 1989; Cruden et al., 1992; Weber et al., 1994; Ramos et al., 1995; Kim et al., 1998). The mechanisms underlying solvent tolerance are not yet fully understood. Several factors have been found to influence solvent-tolerance, e.g. efflux pumps and alter- ations in phospholipids (Heipieper et al., 1992; 1995; Weber et al., 1994; Aono and Kobayashi, 1997; Pinkart and White, 1997; Kieboom et al., 1998; Junker and Ramos, 1999; Kobayashi et al., 2000; Ramos et al., 2002). Efflux pumps that expel organic solvents from the cell membranes have been identified in several solvent- tolerant Pseudomonas sp. strains. The SrpABC pump is found in Pseudomonas putida S12 and Pseudomonas putida GM73 (Kieboom et al., 1998; Kim et al., 1998), and the TtgABC, TtgDEF and TtgGHI pumps are found in P. putida DOT-T1E (Ramos et al., 1998; Mosqueda and Ramos, 2000; Rojas et al., 2001). These efflux pumps belong to the Resistance-Nodulation-Cell Division (RND) family and are critical for solvent tolerance, because their inactivation leads to an increase in solvent sensitivity (Kieboom et al., 1998; Kim et al., 1998; Ramos et al., 1998; 2002; Mosqueda and Ramos, 2000; Rojas et al., 2001). Phospholipid alterations mainly involve short-term responses that take place within 1 min after solvent expo- sure, and consists of the rapid transformation of the cis fatty acids C16 : 1,9 and C18 : 1,9 into their trans isomers (Heipieper et al., 1992; Weber et al., 1994; Sikkema et al., 1995; Junker and Ramos, 1999). The cti gene that encodes the P. putida cis/trans-isomerase has been cloned (Holwick et al., 1997; Junker and Ramos, 1999), and a knock-out cti mutant of P. putida DOT-T1E was isolated and characterized (Junker and Ramos, 1999). Growth of this mutant was delayed in the presence of solvents. emi_578.fm Page 416 Thursday, March 4, 2004 4:25 PM