Involvement of Cyclopropane Fatty Acids in the Response of <i>Pseudomonas putida</i> KT2440 to Freeze-Drying

Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.