P. putida enzymatic activities implicated in the resistance and degradation of tetradecyltrimethylammonium

LIFFOURRENA, A.S.; BOERIS, P.S.; SALVANO, M.A.; LUCCHESI, G.I.

Huerta Grande, Córdoba

Jornada; I Reunión Conjunta de Sociedades de Biología de la República Argentina; 2007

Pseudomonas putida responds to tetradecyltrimethylammonium (TDTMA) through quantitative changes in membrane phospholipids (PL). The specific variations observed were increase of phosphatidic acid (PA) and phosphatidylglycerol (PG) and decrease of cardiolipin (CL). The turnover of CL might be an efficient method to replenish PA and PG pools, trough the hydrolysis of CL catalyzed by a phospholipase D activity (PLD). The first step of TDTMA degradation by P. putida is catalized by a monooxygenase activity (Mo), producing trimethylamine (TMA) and tetradecanal. PLD activity was assayed by a transphosphatidylation assay using [32P]- Pi and ethanol. When cells were exposed 5 min to 50 mg l-1 of TDTMA, a 72% increase of PLD activity relative to untreated cells, was observed. After 15 min of TDTMA-contact no significant changes were detected. The Mo activity was measured in cell-free extracts obtained of P. putida grown with TDTMA as C and N source. The enzyme was localized in periplasm and it was shown to be NAP(P)H-dependent and inhibited by TMA, effect reverted by the presence of Al3+. IEF analysis showed one band of pI 7.3 with Mo activity, which was further resolved by PAGE-SDS in 3 bands. One of these bands was identified as a 60 kDa GroEL chaperonin by MALDI-TOF and PMF. We propose that this chaperonin would be involved in the transport and folding of periplasmic Mo activity.