RESUMO
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.
Assuntos
Compostos de Alumínio/farmacologia , Fosfatidilcolinas/metabolismo , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/metabolismo , Compostos de Trimetil Amônio/farmacologia , Proteínas de Bactérias/metabolismo , Fosfatidilcolinas/química , Pseudomonas putida/química , Pseudomonas putida/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
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.
Assuntos
Metilaminas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Espectrometria de Fluorescência/métodos , Tensoativos/análise , Tensoativos/química , Compostos de Trimetil Amônio/análise , Compostos de Trimetil Amônio/química , Alumínio/química , Carbono/química , Catálise , Flavonoides/química , Metilaminas/química , Nitrogênio/química , Tensoativos/metabolismo , Compostos de Trimetil Amônio/metabolismoRESUMO
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.