Functional and structural annotation of pseudomonas chemoreceptors

Resumen

Bacteria possess different systems to sense and respond to environmental signals (Galperin, 2018). Most abundant are one-component systems (Ulrich et al., 2005), two-component systems (Stock & Zhulin, 2017) and chemosensory pathways (Porter et al., 2011). While one- and two-component systems mainly control gene expression (Stock & Zhulin, 2017, Ulrich et al., 2005), chemosensory pathways mediate chemotaxis or are associated with alternative functions (Porter et al., 2011, Wadhams & Armitage, 2004). The analysis of the increasing number of bacterial genomes shows that about half of bacteria have genes encoding chemosensory pathways (Ortega et al., 2017). The central element of a chemosensory pathway is the ternary complex formed by chemoreceptors, the CheA autokinase and the CheW coupling proteins (Wadhams & Armitage, 2004). Ligand mediated stimulation of the chemoreceptor causes an alteration of CheA activity that in turn modulates transphosphorylation to CheY that in its phosphorylated form binds to the flagellar motor causing ultimately chemotaxis (Wadhams & Armitage, 2004). The chemoreceptor typically is composed by a periplasmic ligand binding domain, a transmembrane module and a cytoplasmic signaling domain (Parkinson et al., 2015). The function and signals recognized of most chemoreceptors are unknown. Casting light into this issue is essential to understand the forces that have led to the evolution of chemoreceptors in bacteria that inhabit different ecological niches. In this thesis I report the functional annotation of a number of chemoreceptors of Pseudomonas putida KT2440 and Pseudomonas aeruginosa PAO1, which are important models to study chemotaxis and chemoreceptors. Approaches are multidisciplinary and include experimentation in the fields of biochemistry, biophysics, microbiology, bioinformatics and structural biology. Chemoreceptors annotated were found to recognize organic acids (McpP, McpQ, McpK and CtpM), polyamines (McpU and TlpQ) or nitrate (McpN). McpP is the major chemoreceptor that mediates chemotaxis toward the C2- and C3-carboxylic acids acetate, pyruvate, propionate, and L-lactate. Its sCACHE-type ligand binding domain recognizes theses ligands as monomer (Garcia et al., 2015). McpQ has a HBM ligand binding domain which recognizes citrate and citrate in complex with metal ions and complements the broad ligand range homologue McpS that is little sensitive to citrate (Martin-Mora et al., 2016b). These organic acids serve as carbon sources for P. putida KT2440 growth and are abundantly present in root exudates. Since P. putida KT2440 efficiently colonizes plant roots, chemotaxis to organic acids is likely to drive root colonization. McpK of P. aeruginosa PAO1 was identified as a specific chemoreceptor for α-ketoglutaric acid. This receptor has an HBM-type ligand binding domain that recognizes α-ketoglutaric acid with positive cooperativity (Martin-Mora et al., 2016a). α-ketoglutaric acid is a key metabolite and is a signal molecule that controls the carbon and nitrogen balance. Another organic acid chemoreceptor studied is CtpM, which mediates chemotaxis toward the C4-dicarboxylic acids L-malic, D,L-bromosuccinic and D,L-citramalic acids (Martin-Mora et al., 2018). L-malic acid is a central metabolic molecule, and D,L-bromosuccinic and D,L-critramalic acids are present naturally in the environment. The ligand binding domain of CtpM is a sCACHE domain. It recognizes not only L-malic, D,L-bromosuccinic and D,L-critramalic acids, but also D.L-methylsuccinic and D,L-citraconic acids. However, methylsuccinic and citraconic acids do not generate a chemotactic response (Martin-Mora et al., 2018). Interestingly, methylsuccinic and citraconic acids act as antagonists, competing with malic, bromosuccinic and citramalic acids for binding at CtpM, decreasing the chemotactic response toward malate (Martin-Mora et al., 2018). McpU and TlpQ are two homologous chemoreceptors from P. putida KT2440 and P. aeruginosa PAO1, respectively (Corral-Lugo et al., 2018, Gavira et al., 2018). Their ligand binding domains recognize and mediate chemotaxis toward histamine and polyamines (spermidine, putrescine, cadaverine, agmatine, ethylenediamine) (Corral-Lugo et al., 2018, Gavira et al., 2018). The ligand affinities of TlpQ are in the nanomolar range and correspond to the highest affinities ever measured for chemoreceptors (Corral-Lugo et al., 2018). The three-dimensional structures of the ligand binding domains of both chemoreceptors has been resolved during these thesis and site-directed mutagenesis was employed to study the role of individual amino acids in ligand recognition (Corral-Lugo et al., 2018, Gavira et al., 2018). Polyamines were found to support bacterial growth as carbon and nitrogen sources. Lastly, the McpN chemoreceptor from P. aeruginosa PAO1 was found to possess a PilJ-type ligand binding domain that recognizes and mediates chemotaxis specifically towards nitrate (Martin-Mora et al., 2019). The three-dimensional structure of McpN revealed that nitrate is recognized by a single site on the symmetry axis of the McpN-LBD dimer (Martin-Mora et al., 2019). Sequence alignments with McpN homologues identified a conserved sequence motif around the nitrate binding site, termed N-box, that may help to identify nitrate binding PilJ domains (Martin-Mora et al., 2019). Nitrate is of central physiological relevance in bacteria. In P. aeruginosa, it was found to serve as nitrogen source for growth and final electron acceptor for anaerobic respiration. The results presented in this thesis increase the knowledge about the chemotactic chemosensory pathways, reveal the signals sensed and cast light into the sensing mechanisms, which is important information to understand important physiological processes such as bacterial migration, colonization, infection and virulence. Future work will determine the potential application of this knowledge, for instance the determination of the role of these chemoreceptors in the promotion of plant root colonization or infection. The demonstration that chemoreceptors respond to chemoeffectors and structurally related antagonists may be a potential strategy to interfere specifically with chemotaxis and virulence. This abstract has the approval of the thesis supervisor, Dr. Tino Krell.