Phosphatidylcholine synthase is involved in the synthesis of phosphatidylcholine in

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

Rosario, Argentina

Congreso; Sociedad Argentina de Microbiologia General (SAMIGE); 2009

Pseudomonas putida A ATCC 12633 responds to tetradecyltrimethylammonium (TTAB) and Al3+ through quantitative changes in membrane phospholipids. 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, indicating that the negative charges of the headgroups of phospholipids are the primary membrane-associated factors for the response to TTAB. In the presence of Al3+, phosphatidylcholine (PC) increased three fold. In prokaryotes, PC can be synthesized by two pathways, the methylation pathway mediated by phospholipid N-methyl-transferase (Pmt) or the CDP-choline pathway that involves phosphatidylcholine synthase (Pcs) that condenses choline directly with CDP-diacylglyceride. Only Pcs activity was detected in cell-free extracts obtained of P. putida grown in choline-free medium with TTAB as carbon and nitrogen source. The methylated intermediates of a Pmt pathway were not detected in such extracts. Cell-free extracts obtained from P. putida grown with TTAB and exposed to Al3+ during 15 or 180 min contained approximately 3 fold more Pcs activity than cell-free extracts from TTAB. When these cell-free extracts were extensively dialyzed, the concentration of Al3+ decreased from about 7 to 1 nmol mg protein-1 and in these extracts the Pcs activity was similar to the activity detected in extracts from cultures without AlCl3, indicating that Al3+ is an activator of the enzyme. Using the Sinorhizobium meliloti Pcs sequence as query for BLAST we identify a candidate gene, pp0731, that might code for the enzyme Pcs in P. putida KT2440. Using specific oligonucleotides, pp0731 was amplified from genomic DNA and cloned into the expression plasmid pET9a. Expression of pp0731 from P. putida KT2440 in Escherichia coli BL21 (DE3) (pLysS) leads to the formation of PC. Based on these results, we suggest that the Pcs pathway might be the only pathway for PC biosynthesis when P. putida grow with TTAB. Thus, we expect that a P. putida mutant defective in Pcs can be an useful model to elucidate the synthesis of PC and to clarify the role of PC in Al3+ stress responses.