Characterization of proteins involved in lipid homeostasis of Pseudomonas aeruginosa and Agrobacterium tumefaciens

The bacterial membrane adapts to changing conditions. One possible modification is the aminoacylation of phosphatidylglycerol (PG) with alanine or lysine. This transfer RNA (tRNA)-dependent reaction is catalyzed by aminoacyl-PG synthases and increases the resistance against several antimicrobial compounds. In this study the interaction of the alanyl-PG synthase (A-PGS) from P. aeruginosa with the tRNA acceptor stem was analyzed and the key residues Lys676, Arg684 and Arg687 were identified. The protein alanyl-PG hydrolase (A-PGH) is involved in the fine-tuning of the cellular alanyl-PG content in P. aeruginosa. The molecular principles of hydrolysis should be analyzed by X-ray crystallography. The 3D structure of the N-terminal part of A-PGH (amino acids 34-215) was solved at a resolution of 1.64 Å. Structure-based alignments related the N-terminal part of unknown function to hydrolases and esterases possessing an a/ß fold. The operon encoding for A-PGS and A-PGH homologs is conserved in several Gram-negative bacteria. A. tumefaciens possesses the respective related proteins LpiA as well as AcvB and VirJ. In lpiA or acvB deletion mutants an altered membrane lipid composition which is characterized by either the complete lack of lysyl-PG (L-PG) or elevated L-PG amounts was detected. Accordingly, the L-PG hydrolyzing activity of the recombinantly produced proteins AcvB and VirJ was demonstrated in vitro. Based on site-directed AcvB mutant proteins the catalytically relevant active site residues Ser336 and His433 were identified. It was demonstrated that functional AcvB protein is crucial for the translocation of transfer-DNA during tumorigenesis and it is proposed that a fine-tuned cellular L-PG amount in the A. tumefaciens membrane might be important for this process. In a recent proteomics study PA3911 was identified as the second-most significantly upregulated protein under anaerobic biofilm conditions of the cystic fibrosis lung. A P. aeruginosa PA3911 transposon mutant exhibited a growth phenotype under acidic conditions, an increased susceptibility to specific antimicrobial compounds, enhanced twitching motility and impaired biofilm formation when related to the wild type. The PA3911 protein was recombinantly produced and subjected to in vitro studies. Under the employed conditions PA3911 was demonstrated to specifically bind to phosphatidic acid (PA). It is proposed that protein PA3911 might contribute to PA homeostasis in P. aeruginosa.