Chemotaxis in Pectobacterium atrosepticum Scri1043: Functional and structural studies on chemotaxis adaptation proteins and chemoreceptors

• Autores: Félix Velando Soriano
• Directores de la Tesis: Tino Krell (codir. tes.), Miguel Ángel Matilla Vázquez (codir. tes.)
• Lectura: En la Universidad de Granada ( España ) en 2023
• Idioma: inglés
• ISBN: 9788411950671
• Número de páginas: 225
• Enlaces
  • Tesis en acceso abierto en: DIGIBUG
• Resumen

  • Bacteria possess different systems to sense and respond to environmental signals. Onecomponent systems, two-component systems and chemosensory pathways are the most commonly found. While one- and two-component systems mainly control gene expression and are present in all bacteria we currently know, chemosensory pathways are approximately found in half of the bacterial genomes described to date and primarily mediate chemotaxis. The core of a chemosensory pathway is the ternary complex formed by chemoreceptors, the CheA autokinase and the CheW coupling proteins. Chemoreceptors sense ligands and modulate CheA activity, leading to transphosphorylation of the response regulator CheY. CheY-P interacts with the flagellar motor, and this results in chemotaxis, or movement towards or away of compounds. In this process, adaptation proteins are important to enable bacteria to react appropriately to physicochemical gradients. These adaptation proteins, CheR and CheB, are tasked with methylation and demethylation of chemoreceptors, respectively; a mechanism that causes changes in their sensitivity to their cognate ligands. A canonical chemoreceptor typically consists of: (i) a periplasmic ligand binding domain; (ii) a transmembrane module; and (iii) a cytoplasmic signaling domain. Chemoreceptor genes are easy to predict bioinformatically, but the function and signals recognized for most of them remain unknown. Plant-associated bacteria have a higher number of chemoreceptors than bacteria inhabiting other niches, and exploring the function of these receptors can help to understand the evolutionary pressures that have driven chemoreceptor evolution in this particular ecological niche. In this thesis, the enterobacterium Pectobacterium atrosepticum, a phytopathogen of global relevance, was used as a model to explore chemotaxis. Strain SCRI1043 has a single chemosensory pathway and 36 chemoreceptors. Interestingly, 19 of them present carboxyterminal pentapeptides, which, in Escherichia coli and other species, were found to function as docking sites for the adaptation enzymes, CheB and CheR. I demonstrated that the chemosensory pathway of P. atrosepticum SCRI1043 regulates chemotaxis and characterised the interaction between CheR/CheB and the chemoreceptors and identified the function of four chemoreceptors that were termed PacA, PacB, PacC and PacP. I found that CheR of SCRI1043 (CheR_Pec) binds, with different affinities, all 9 different C-terminal pentapeptides present in the chemoreceptors of this strain. In addition, I showed that the cellular concentration of CheR_Pec is subject to changes during growth and is in the range of the Kd values for the interaction of CheR with the different pentapeptides, suggesting a new mechanism of regulation of the chemotactic output. Contrary to these results, CheB of SCRI1943 (CheB_Pec) is unable to bind pentapeptides, and solving the three-dimensional structure of CheB_Pec by X-ray crystallography revealed that the region corresponding to the pentapeptide binding site in the E. coli CheB is disordered in CheB_Pec, which most likely accouts for the lack of pentapeptide binding. These results supported that CheB methylesterases can be divided into pentapeptide dependent and independent enzymes. PacA was functionally described as a chemoreceptor with a dCache ligand binding domain that binds and mediates chemotaxis towards quaternary amines (e.g. choline, betaine, L-carnitine) and the amino acid L-proline. During this thesis, the three-dimensional structure of the sensor domain of PacA was solved. Through comparison with that of the sensor domain of the PctD chemoreceptor of Pseudomonas aeruginosa, a receptor that has a similar profile of ligands, but which also recognizes the neurotransmitter acetylcholine, progress was made towards understanding quaternary amines binding by dCache domains. Along with PacA, I described two additional chemoreceptors of SCRI1043 that recognize amino acids, indicating that amino acids are important signals for bacteria. PacB has a dCache ligand binding domain that recognizes a wide range of proteinogenic and non-proteinogenic amino acids. Instead, PacC, the sole chemoreceptor encoded in the chemotaxis gene cluster, was shown to be homologous to the Tsr chemoreceptor of E. coli, but was shown to bind and mediate taxis to D- and L- aspartate and L-asparagine. Its evolutionary history was described in this thesis. Finally, PacP, the only chemoreceptor with a sCache domain in SCRI1043, was described as the first identified chemoreceptor for phosphorylated compounds. PacP binds compounds related to the glycolysis as well as glycerol 3-phosphate and glycerol 2-phosphate. It is shown here that it is part of a family of chemoreceptors that primarily recognize glycerol 3-phosphate - a potential stress signal in plants. Evolutionary analyses revealed that this subfamily of chemoreceptors originated from sCache domains that recognize organic acids. It was concluded that the recognition of families of homologous proteins with similar ligand binding capabilities from in silico docking analyses is a promising tool to rapidly increase knowledge about sensing capabilities of all kinds of bacterial receptors. Overall, the findings in this thesis have led to significant advances in the knowledge of the chemotactic capacities of the important phytopathogen P. atrosepticum, including the function of several chemoreceptors and how the adaptation enzymes CheR and CheB recognize chemoreceptors. The role of chemotaxis in plant colonization and disease development by P. atrosepticum is now open for scientific experimentation, and insights gleaned on this thesis can be applied to other soft rot Pectobacteriaceae species. Collectively, soft rot Pectobacteriaceae phytopathogens have a great impact on a wide range of crops and plants worldwide. Future work will allow the characterization of new chemoreceptor proteins of P. atrosepticum, as well as describe their homologue families in other species, thereby facilitating progress in the study of bacterial sensing and signaling mechanisms.