In Pseudomonas putida A ATCC 12633 phosphatidylcholine act as a temporary reservoir of Al3+

BOERIS, P.S.; LUCCHESI, G.I.

Jornada; II Reunión Conjunta de Sociedades de Biología de la República Argentina; 2011

In this work we have cloned and sequenced a gene in P. putida A ATCC 12633 that encodes a phosphatidylcholine synthase (PCS) and characterized a pcs-deficient mutant. Sequence analysis of P. putida KT 2440 revealed the ORF PP0731, which has 26 % sequence identity to a PCS protein in Sinorhizobim meliloti. The orf pp0731 was cloned and expressed in Escherichia coli BL21, a phosphatidylcholine- deficient organism. The P. putida A ATCC 12633 pcs homologue was disrupted to generate a corresponding mutant. In the pcs-deficient mutant, detectable levels of phosphatidylcholine were not found, and the mutant was much more sensitive than the wild-type strain when challenged with Al3+. Only 50% of the mutant cells managed to grow in the presence of the ion (the amount of viable cells decreased from 1012 ufc ml-1 to 106 ufc ml-1 15 min after Al3+ addition). Also, Al3+ in the membrane of wild-type and mutant strains was visualized using the fluorochrome 2´,3,4´,5,7- pentahydroxyflavone (morin reagent). In the mutant strain, the distribution of green fluorescence was quite similar to that of wildtype cells, but they had a lower level of fluorescent intensity in the cell membrane, consistent with a reduced presence of Al3+. These results support that phosphatidylcholine is involved in the response of P. putida to Al3+ and acts as a temporary reservoir of available ions.