Content of cardiolipin of the membrane and sensitivity to cationic surfactants in Pseudomonas putida.

AIMS: To establish the role of cardiolipin (CL) of the membrane in response to the presence of tetradecyltrimethylammonium in Pseudomonas putida A (ATCC 12633). METHODS AND RESULTS: Two ORFs of Ps. putida A (ATCC 12633), which in Ps. putida KT2440 encode the putative CL synthase genes cls and cls2, were cloned, sequenced and mutated. Only the double mutant lacking cls and cls2 showed a reduction of the CL content, 83% lower than the amount produced by the wild-type. Accompanying this change was a 40% decrease in the content of unsaturated fatty acid. Consequently, the membrane of the mutant was more rigid than the one of the parental strain, as observed using fluorescence polarization techniques. The mutant strain showed reduced viability in the presence of tetradecyltrimethylammonium. The incorporation of exogenous CL into its membrane relieved sensitivity to the cationic detergent. CONCLUSIONS: Pseudomonas Putida cells with low levels of CL die in the presence of tetradecyltrimethylammonium, because they cannot counter the fluidizing effect of the cationic surfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: The modification in the membrane phospholipids composition allows knowing the adaptation strategy of Ps. putida when these bacteria are exposed to cationic surfactant.

A fluorescence assay for tetradecyltrimethylammonium mono-oxygenase activity that catalyzes the cleavage of the C-N bond with the production of trimethylamine.

This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminum (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB mono-oxygenase reaction and the amount of protein used in the reaction. The kinetic behavior is characterized by K0.5=4.26x10(-4) M, and the apparent Hill coefficient (napp)=2.24. These values are both comparable to those determined by GC-MS (K0.5=4.41x10(-4) M and napp=2.35). The advantages of this assay include rapid and efficient implementation and potential employment for routine accurate determinations of TTAB mono-oxygenase activity over a wide range of substrate concentrations.

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium.

AIMS: To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida were grown with TTAB in the presence or absence of AlCl(3), and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl(3), phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl(3), indicating that the PC level is dependent upon activation of this enzyme. CONCLUSIONS: The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al(3+) and should be viewed as a temporary reservoir of available Al(3+) to allow a more efficient utilization of TTAB by Ps. putida. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in the PL of Ps. putida in the presence of TTAB and AlCl(3) indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.

Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium.

AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.