A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality

Abstract

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. To further 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.

TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12

“…Strain S12DtrgI lost the ability to grow in mineral salt medium on glucose or fructose. As the functioning of transport proteins (Bernal et al 2007) and membrane protein topology (Bogdanov et al 2002) are known to be affected by membrane composition, the inability to utilize these sugars may be connected to changes in the outer cell structure preventing the sugars from being transported into the cells. The effect appears to be specific as the ability of S12DtrgI to utilize glycerol, succinate or decanol was not affected.…”

“…The inability of the trgI-knock-out strain to grow on glucose and fructose can be explained by trgI not being expressed at all in this strain, whereas in the wild-type it is only down-regulated to 35% of the non-stressed expression level in 3 mM toluene and to 28% in 5 mM toluene. Also, changes in the outer cell structure of S12DtrgI may influence the functioning of the proteins involved in transport of glucose and fructose (Bogdanov et al 2002;Bernal et al 2007).…”

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

“…Staining of Anionic Lipid Membrane Domains with NAOTo study possible formation of anionic lipid membrane domains in ⌬pgsA cells, we used a visualization technique developed previously for staining CL domains in bacteria with the fluorescent dye NAO (2) and successfully used by other groups (3,4,23). Association of NAO with CL results in the appearance of a red emission maximum in the dye fluorescence spectrum, and CL membrane domains emitting both green and red fluorescence were previously demonstrated in E. coli and Bacillus subtilis with wild-type phospholipid composition (2,3,23,24).…”

“…Using the cardiolipin (CL) 4 -specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2). Subsequently others reported similar CL domains in Bacillus subtilis (3) and Pseudomonas putida (4). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ⌬minCDE mutant (5).…”

Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes

“…E. coli membranes consist of ∼5% cardiolipin (CL, also referred to as "diphosphatidylglycerol"), 20-25% phosphatidylglycerol (PG), and 70-80% phosphatidylethanolamine (PE) (16). CL is distributed between the two leaflets of the bilayers and is located preferentially at the poles and septa in rod-shaped cells of E. coli, B. subtilis, and Pseudomonas putida (17)(18)(19). The polar positioning of the proline transporter ProP and the mechanosensitive ion channel of small conductance MscS in E. coli is dependent on CL (20).…”