Unveiling the effect of global regulators in the regulatory network for biofilm formation in Escherichia coli

• Autor(a) principal: Amores, Gerardo Ruiz
• Data de Publicação: 2017
• Tipo de documento: Tese
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: http://www.teses.usp.br/teses/disponiveis/17/17136/tde-17042018-140449/
• topic: Biofilm; Biofilme; Redes regulatórias; Regulação da transcrição; Regulatory network; Transcriptional regulation

In nature, biofilm is a complex structure resulted of multicellular bacterial communities that provide important nutritional functions and the acquisition of protective traits such as antibiotics resistance and horizontal gene transfer. The development from the planktonic, lonely bacteria, to the mature multilayered biofilm structure consists of three main phases: motility, attachment and biofilm maturation. At cellular level, the process is controlled by several genes such as flhD, fliA, rpoS, csgD, adrA, cpxR all acting as master regulators. Additionally, the global regulators CRP, IHF, Fis, and others in less frequency, have been related to biofilm formation, although blurry information has been provided. In this thesis we used synthetic, molecular and cellular biology approaches to understand the effect of CRP, IHF and Fis in the transcriptional regulatory network in the bacterium Escherichia coli. In the first chapter, we employed network analysis to reconstruct and analyze part of the entire regulatory network described to modulate the flagella-biofilm program. With this analysis we identified some critical interactions responsible for the planktonic-biofilm transition. Next, we selected the top ten effectors nodes of the network and cloned the promoter region of those genes in a reporter system. As extensively explained in chapter II, this system allowed us to validate as well as suggest new interactions in the network. Additionally, the measurement of the promoter activity during bacterial development show that CRP, IHF and Fis differentially modulate most of the surveyed genes suggesting that those Global Regulators participate to modulate gene expression in different phases of the planktonic-biofilm development. At chapter three, to get a better overview of the entire process, we performed motility, adherence/early biofilm and mature biofilm assays. We describe the intrinsic ability of E. coli to perform motility, adherence and mature biofilm at 37?C. In contrast, the absence of ihf, fis as well as Carbon Catabolite Repression (CCR), lead to altered phenotypes at both motility and biofilm development. At the end, we discussed how the changes of promoter activity of target genes, together with our network analysis, could explain part of the altered phenotypes observed. For instance, we observed changes at the main stress responders rpoS and rpoE that, in combination with alterations at specific genes such as fliA, can explain the enhanced motility in the E. coli ?ihf strain. Altogether, in this thesis, we provided evidence that CRP, IHF and Fis control the activity of the promoter regions of genes involved in the planktonic-biofilm development.