A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs

What is it about?

To understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition

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Why is it important?

Biological membranes play essential roles in cell physiology because they create a permeability barrier and a matrix containing proteins that perform multiple functions. Some of these functions are the generation of proton motive force, the exchange of solutes between the cell and the external environment, and adherence to biotic and abiotic surfaces (Sikkema et al., 1995; Hancok, 1997; Nikaido, 1999). Bacterial membranes constitute the first contact point between microbes and the environment, and for this reason bacteria have developed different mechanisms to defend themselves from the action of different membrane-disturbing agents such as changes in temperature or pH, and deleterious chemicals. Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called ‘homeoviscous adaptation’. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. In an attempt to elucidate the role of CL in the fluidity of P. putida DOT-T1E membranes and to understand the role of this phospholipid head group in stress responses, we generated a mutant in the cls gene that catalyses the transfer of a phosphatidyl group from one PG molecule to another. We found that the decrease in CL content in the membranes of P. putida influences the size of the cells, membrane fluidity and the efficiency of the multidrug efflux pumps involved in antibiotic and organic solvent resistance.

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