Compensatory role of the cis-trans-isomerase and cardiolipin synthase in the membrane fluidity of Pseudomonas putida DOT-T1E.

In Gram-negative bacteria, cell membrane fluidity is influenced by phospholipid head group composition and linked fatty acids. Exposure of Pseudomonas putida to stressing agents results in short- and long-term modifications in membrane lipids. The main adaptive change observed in response to organic solvents in the short term is the cis- to trans-isomerization of unsaturated fatty acids in a reaction mediated by cis/trans-isomerase (CTI); whereas in the long term an increase in cardiolipin content takes place. Despite the interest of these genes in the context of stress responses, the transcriptional regulation of the cti and cls genes has not been studied before. The cti and cls (cardiolipin synthase) genes in the solvent-tolerant P. putida DOT-T1E strain form monocistronic units and are expressed from sigma-70 promoters. Expression from the cls promoter is sixfold higher in the stationary phase than in the log phase, and expression of the cls gene is not influenced by solvents. The cti gene is expressed at fairly constant levels in the log and stationary phase, but its level of expression is moderately upregulated in response to toluene. We used fluorescence polarization assays to show that mutants deficient in the cti gene exhibit less rigid membranes than the wild-type strain, whereas mutants with a knockout in the cls gene exhibit increased membrane rigidity. A double cti/cls mutant has similar membrane rigidity as the wild-type strain, which points towards a compensatory effect of the mutations with regard to membrane fluidity. However, the cls and cls/cti mutants were more sensitive to solvents than the wild-type and the cti mutant because of the impaired functioning of efflux drug transporters.