Refactoring the interplay of Pseudomonas putida with solid surfaces for programming lifestyle decisions

• Autores: Angeles Hueso Gil
• Directores de la Tesis: Víctor de Lorenzo Prieto (dir. tes.), Belén Calles Arenales (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2019
• Idioma: inglés
• Número de páginas: 144
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Genética
      • Ingeniería genética
    • Microbiología
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • The control of bacterial biofilm formation is a clear objective for health and Industrial Microbiology because of the problems it usually causes, but it is also a goal with biotechnological relevance. In this field, biofilm manipulation could allow some advantages, such as direction of bacterial consortia, precise localization of metabolic reactions and production of compounds of interest with low diffusion within media. Pseudomonas putida KT2440 is an environmental bacterium whose particular tolerance to stresses and high reducing power makes it a perfect chassis for multiple functions, biofilm formation amongst them. In this Thesis we have studied some mechanisms that regulate biofilm in this microorganism and explored the possibility of controlling its formation. To do so, we addressed the issue from three points of view. First, we performed a transcriptomic comparison between P. putida incubated in solid media vs. incubated in liquid media at 6, 12 and 24 hours in order to know what genes and promoters were implicated in the changes associated with both lifestyles and conditions. Results showed that P. putida incubated in solid media was more metabolically active, differentially regulated translation activities and it was more exposed to oxidative stress at 6 hours. However, at 12 and 24 hours, most flagellar and motile genes notably reduced their transcription levels, in addition to similar changes in translation, metabolism and oxidative stress as compared to activity levels noted at 6 hours. Second, we characterized the P. putida Wsp complex. It is well known in Pseudomonas aeruginosa , where it is composed of 7 proteins that form an intermembrane complex and regulate c-di-GMP, which is the main secondary messenger controlling motility and biofilm formation. We have analysed the homologous operon in P. putida through deletion mutants of wsp genes. Examining mutants behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas sp. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa complemented P. putida mutants. It was also noticed that c-di-GMP produced by WspR defined biofilm using the regulator YgcR under a citrate regime at 24 hours. Finally, our third approach consisted of the development of a light-driven switch that could control c-di-GMP amounts and biofilm formation in P. putida. For this purpose, we used CcaSR two component system from Synechocystis sps. In those cyanobacteria, CcaSR activates transcription of pcpcG2 promoter when green light is present. The system had been previously adapted to Escherichia coli, but its performance in P. putida required an optimization as its direct transfer produced leakiness and low dynamic range. With that goal in mind, we carried out a mutagenesis process with randomized oligos that were targeted to the regulatory regions of CcaSR components, as we assumed that the expression balance between them was a key point for proper light sensing. Using this technique, we generated comprehensive libraries that were sorted to find an optimal clone. Selected mutants produced higher amounts of the reporter GFP under the presence of light and had a very low basal level when cultures were kept in darkness. When the optimized CcaSR system was used to express a constitutively active diguanylate cyclase PleD, a 30-fold increase in the production of biofilm could be reached after green light induction. Additionally, biofilm basal activity was similar to wild type P. putida’s, demonstrating the efficiency of the construct.