Regulation of lifestyle transition by the transcription factors AmrZ and FleQ in the bacterial model for rhizosphere colonization Pseudomonas fluorescens F113

• Autores: Esther Blanco Romero
• Directores de la Tesis: Marta Martín Basanta (dir. tes.), Miguel Redondo Nieto (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2021
• Idioma: inglés
• Número de páginas: 267
• Tribunal Calificador de la Tesis: Juan Imperial Ródenas (presid.), Patricia Bernal Guzmán (secret.), Miguel María Cámara García (voc.), Francisco Manuel Cazorla López (voc.), María Trinidad Gallegos Fernández (voc.)
• Programa de doctorado: Programa de Doctorado en Microbiología por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Genética
    • Microbiología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Motility and biofilm formation are two crucial traits in the process of rhizosphere colonization by Pseudomonas fluorescens F113. The regulation of both traits requires a complex signaling network. Recently, the study of the direct regulon of AmrZ and the phenotypic analyses of an amrZ mutant in P. fluorescens F113 have shown that this protein plays a crucial role in the regulation of several cellular functions, including motility, biofilm formation, iron homeostasis, and bis-(3′-5′)- cyclic dimeric guanosine monophosphate (c-di-GMP) turnover. On the other hand, FleQ is the master regulator of flagellar synthesis, and, in other Pseudomonas species, its functions are being recently expanded.

    In this work, we have analyzed the indirect and direct regulons of AmrZ and FleQ transcription factors in diverse growth conditions to deepen the regulation of P. fluorescens F113 environmental adaption using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) techniques. These analyses have allowed the description of the AmrZ/FleQ regulatory hub in the motile to sessile lifestyle transition and iron homeostasis in this bacterium. Furthermore, both transcription factors have been described as global regulators, controlling many cellular functions crucial for rhizosphere colonization, including nitrogen and amino acid metabolism, secretion systems, and transport.

    On the other hand, we have studied the presence of several genes and gene clusters involved in the synthesis of extracellular matrix components in P. fluorescens F113 and their distribution in the Pseudomonas genus. This analysis has allowed the in silico description of two new components, the Pseudomonas acidic polysaccharide (Pap) and a new type of fimbrial low-molecular-weight protein/tight adherence (Flp/Tad) pilus. Moreover, this work has demonstrated that AmrZ indirectly regulates these genes' expression through FleQ and the second messenger c-di-GMP. The c-di-GMP-mediated regulation by AmrZ not only controls the expression of polysaccharide biosynthetic and extracellular proteins in this bacterium but is also responsible for the reduced biofilm formation and hypermotile phenotypes of an amrZ mutant. On the other hand, despite the amrZ mutant is hypermotile and motility has been linked with better performance in the rhizosphere, it is severely impaired in this process. In this work, we have also shown that, unlike biofilm formation and motility, the complementation of the c-di-GMP levels in the amrZ mutant is insufficient to restore the competitive rhizosphere colonization phenotype.

    Finally, to deepen the biological role of extracellular matrix components in P. fluorescens F113, several mutants have been constructed in this work: papA- , pgaA-, alg8-, fapB-, fapC-, fapF-, mapA-, psmE-, and flp-1. As a result, this work has shown that the polysaccharides poly-β-1, 6-N-acetylglucosamine (PNAG) and alginate play a key role in biofilm formation in abiotic surfaces, being alginate also required for proper motility and rhizosphere competitive colonization. On the other hand, the extracellular protein PsmE and the Flp/Tad pilus are also important for biofilm formation in abiotic surfaces. Furthermore, PsmE, Flp/Tad, and MapA are also required for a correct rhizosphere colonization in this bacterium. The alteration in these components could partially explain the observed phenotypes in an amrZ mutant.

    This work reveals the complexity underlying the rhizosphere colonization process in P. fluorescens F113, which involves several regulators and determinants.