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Diego Carriel

Structure-function relationships of the lysine decarboxylase LdcA from Pseudomonas aeruginosa

Published on 15 May 2017


Thesis presented on May 15, 2017

Abstract:
The lysine decarboxylase (LDC) belongs to a family of decameric PLP-dependent enzymes that catalyse the reaction transforming L-Lysine into cadaverine while consuming a proton. They have been extensively characterised in enterobacteria, where they have been shown to play a crucial role during acid, oxidative stress and antibiotic resistance.
Since mechanisms allowing bacteria to counter stress challenges are important for displaying full virulence, we wondered if the opportunistic bacterium Pseudomonas aeruginosa could be using LdcA to counter stress conditions that have already been described for enterobacteria. During my PhD, we addressed this question by using different but complementary.
First of all, we used promoter-gene fusions and western-blot analysis to determine the conditions in which ldcA was expressed and its product synthesized.
In parallel, we constructed an ldcA mutant and its complemented strain to understand whether LdcA was involved in acid and oxidative stress response. We used manual screenings and high-throughput technologies (Biolog) and we discovered that the cadaverine produced by LdcA is needed for full growth fitness when growing in minimal medium using L-glutamate as carbon source. Since slow growing phenotypes are linked to heightened bacterial persistence and because cadaverine has been shown to reduce the persisters population, we also examined if the presence of LdcA is modifying the amount of persisters during carbenicillin treatment.
Finally, by combining phylogenetic and structural analysis, we discovered that LdcA belongs to a different subgroup of bacterial LDCs. Sequence alignments show that key residues needed for binding ppGpp are not present in the predicted binding site which also suggests that the enzymatic activity is not inhibited by this molecule. This observations are coherent with the fact that LdcA seems to be close to Arginine Decarboxylase (ADC) from E. coli which does not bind ppGpp.
Our work shows that, in spite of the fact that LdcA catalyses the same enzymatic reaction and shares the same structural fold than enterobacterial lysine decarboxylases, it is not implicated in acid stress or oxidative stress responses. Its role is linked to physiological effects of cadaverine and to the relationship between L-lysine and L-Arginine catabolism.