RESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Pseudomonas putida/efectos de los fármacos , Tensoactivos/farmacología , Compuestos de Trimetilamonio/farmacología , Cardiolipinas/análisis , Cardiolipinas/metabolismo , Clonación Molecular , Polarización de Fluorescencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Sistemas de Lectura Abierta , Fosfolípidos/metabolismo , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
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.
Asunto(s)
Compuestos de Aluminio/farmacología , Fosfatidilcolinas/metabolismo , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/metabolismo , Compuestos de Trimetilamonio/farmacología , Proteínas Bacterianas/metabolismo , Fosfatidilcolinas/química , Pseudomonas putida/química , Pseudomonas putida/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
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.
Asunto(s)
Metilaminas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Espectrometría de Fluorescencia/métodos , Tensoactivos/análisis , Tensoactivos/química , Compuestos de Trimetilamonio/análisis , Compuestos de Trimetilamonio/química , Aluminio/química , Carbono/química , Catálisis , Flavonoides/química , Metilaminas/química , Nitrógeno/química , Tensoactivos/metabolismo , Compuestos de Trimetilamonio/metabolismoRESUMEN
AIMS: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis' acid in the medium influences its biodegradability. METHODS AND RESULTS: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l(-1) of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0.1 mmol l(-1) AlCl(3) added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al(3+) : TMA complex. CONCLUSIONS: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl(3). SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Lewis' acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.
Asunto(s)
Microbiología Ambiental , Pseudomonas putida/metabolismo , Tensoactivos , Compuestos de Trimetilamonio , Aluminio/farmacología , Técnicas Bacteriológicas , Biodegradación Ambiental , Cationes , Concentración de Iones de Hidrógeno , Metilaminas/análisis , Metilaminas/metabolismo , Especificidad de la Especie , Tensoactivos/análisis , Compuestos de Trimetilamonio/análisisRESUMEN
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.
Asunto(s)
Desinfectantes/farmacología , Fosfolípidos/metabolismo , Pseudomonas putida/efectos de los fármacos , Compuestos de Trimetilamonio/farmacología , Cardiolipinas/metabolismo , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Ácidos Fosfatidicos/metabolismo , Fosfatidilgliceroles/metabolismo , Pseudomonas putida/crecimiento & desarrollo , Pseudomonas putida/metabolismoRESUMEN
Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent Km value of 53 microM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.
Asunto(s)
Colina/metabolismo , Pseudomonas aeruginosa/metabolismo , Cloruro de Amonio/farmacología , Betaína/farmacología , Transporte Biológico , Colina/farmacología , Cinética , Concentración Osmolar , Sarcosina/análogos & derivados , Sarcosina/farmacología , Cloruro de Sodio/farmacología , Succinatos/farmacologíaRESUMEN
The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.
Asunto(s)
Carnitina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Fosfatasa Ácida/biosíntesis , Betaína/metabolismo , Carbono/metabolismo , Carnitina/metabolismo , Colina/farmacología , Colinesterasas/biosíntesis , Medios de Cultivo , Inducción Enzimática/efectos de los fármacos , Nitrógeno/metabolismo , Fosfatos/metabolismo , Fosfolipasas de Tipo C/biosíntesis , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-Pi basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite. 2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate. 3. The peak containing the PLC activity revealed a single protein after SDS-PAGE. 4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous. 5. The molecular weight for both PLC preparations was about 70 kDa. 6. Both PLC used phosphatidylcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine. 7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100. 8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP1-BSM plus choline but not the enzyme from the LP1-CM.
Asunto(s)
Pseudomonas aeruginosa/enzimología , Fosfolipasas de Tipo C/aislamiento & purificación , Colina/farmacología , Medios de Cultivo , Fosfatos/farmacología , Reproducibilidad de los Resultados , Fosfolipasas de Tipo C/efectos de los fármacosRESUMEN
Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg2+, the Km values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The Ksi values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.
Asunto(s)
Fosfatasa Ácida/metabolismo , Pseudomonas aeruginosa/enzimología , Colinesterasas/metabolismo , Etanolaminas/metabolismo , Cinética , Nitrofenoles/metabolismo , Compuestos Organofosforados/metabolismo , Fosforilcolina/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.