tRNA-abhängige Biosynthesis of Phospholipids in Pseudomonas aeruginosa
Final Report Abstract
Lipid homeostasis is a fundamental process for understanding antimicrobial susceptibility. Modification of the polar head group of phosphatidylglycerol into the respective aminoacylester of phosphatidylglycerol is a widely used strategy to mediate bacterial resistance. We resolved the structures of the catalytic domains of aminoacyl-phosphatidylglycerol synthases from Pseudomonas aeruginosa and Bacillus licheniformis. These prototypical enzymes specifically catalyze the tRNA-dependent synthesis of alanyl-phosphatidylglycerol (A-PG) and lysyl-phosphatidylglycerol (L-PG), respectively. A central tunnel architecture facilitates binding of the polar aminoacyl-tRNA molecule opposite the hydrophobic lipid substrate as a fundamental principle for the catalysis at the water–lipid interface. Structure based biochemical experiments allowed us for the molecular understanding of the unusual tRNA-dependent catalysis. For P. aeruginosa we could specify that ORF PA0919 encodes an A-PG hydrolase which facilitates the tuning of the cellular A-PG content. On the cellular level it was demonstrated that an imbalanced A-PG physiology drastically increases the antimicrobial susceptibility. Agrobacterium tumefaciens transfers oncogenic T‐DNA via the type IV secretion system into plants causing tumor formation. It has been described that the acvB gene encodes a virulence factor of unknown function required for plant transformation. We could specify AcvB as a periplasmic L-PG hydrolase. An L-PG synthase / L-PG hydrolase regulatory circuit for the adaptation of the cellular L-PG content was elucidated. Absence of the hydrolase resulted in ~10‐fold increase in L‐PG in Agrobacterium membranes and abolished T‐DNA transfer and tumor formation. This results suggested that elevated L‐PG amounts are responsible for the observed virulence phenotype. Our work identifies AcvB as a novel virulence factor which is essential for membrane lipid homeostasis and T‐DNA transfer in A. tumefaciens. In P. aeruginosa the so-far uncharacterized protein PA3911 revealed drastically increased abundance under anaerobic growth conditions. The physiological relevance of ORF PA3911 was demonstrated since the related mutant strain showed increased susceptibility in the presence of antimicrobials, enhanced twitching motility and impaired biofilm formation. PA3911 was identified as a soluble, cytoplasmic protein which facilitates the specific binding of phosphatidic acid. Key residues as part of the proposed lipid binding cavity were identified by site-directed mutagenesis. A comparative shotgun lipidomics approach revealed an imbalanced lipid homeostasis for the PA3911 mutant strain. From the overall project we conclude that lipid homeostasis is a promising target to render pathogenic bacteria more susceptible to antimicrobial treatment.
Publications
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(2009). Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanylphosphatidylglycerol. Mol Microbiol 71, 551-565
Klein, S., Lorenzo, C., Hoffmann, S., Walther, J. M., Storbeck, S., Piekarski, T., Tindall, B. J., Wray, V., Nimtz, M. and Moser, J.
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(2011). Alanyl-Phosphatidylglycerol Synthase: Mechanism of substrate recognition during tRNA-dependent lipid modification in Pseudomonas aeruginosa. Mol Microbiol 80, 935-950
Hebecker, S., Arendt, W., Heinemann, I.U., Tiefenau, J.H.J., Nimtz, M., Rohde, M., Söll, D. and Moser, J.
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(2012). Resistance phenotypes mediated by aminoacyl-phosphatidylglycerol synthases. J. Bacteriol 194, 1401-1416
Arendt, W., Hebecker, S., Jäger, S., Nimtz, M. and Moser, J.
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(2013). Identification and characterization of a periplasmic Aminoacyl-Phosphatidylglycerol Hydrolase responsible for Pseudomonas aeruginosa lipid homeostasis J. Biol. Chem. 288, 24717-24730
Arendt, W., Groenewold, M., Hebecker, S., Dickschat, J. and Moser, J.
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(2015). Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine. Proc Natl Acad Sci USA. 112, 10691-10696
Hebecker, S., Krausze, J., Hasenkampf, T., Schneider, J., Groenewold, M., Reichelt, J., Jahn, D., Heinz, D. W. and Moser, J.
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(2018). A phosphatidic acid-binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in Pseudomonas aeruginosa. Biochem J 475, 1885-1907
Groenewold, M. K., Massmig, M., Hebecker, S., Danne, L., Magnowska, Z., Nimtz, M., Narberhaus, F., Jahn, D., Heinz, D. W., Jänsch, L. and Moser, J.
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(2018). Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysylphosphatidylglycerol hydrolase AcvB. Mol Microbiol 111, 269-286
Groenewold, M. K., Hebecker, S., Fritz, C., Czolkoss, S., Wiesselmann, M., Heinz, D. W., Jahn, D., Narberhaus, F., Aktas, M. and Moser, J.
