Accessing transcriptional regulation of Escherichia coli biofilm formation using promoter and genomic libraries

• Ano de defesa: 2023
• Autor(a) principal: Medeiros, Ananda Sanches
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Biofilm; Biofilme; Eschericha coli; Gene expression regulation; Promoters; Promotores; Regulação da expressão gênica; SortSeq
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/17/17136/tde-08082023-144205/

Biofilms are complex structures formed by bacterial communities of the same or different species, embedded in an extracellular matrix composed of polymeric extracellular substances (EPS), such as polysaccharides, proteins (as curli structures), and nucleic acids. The regulatory networks of biofilm formation are composed of the integration of environmental and intracellular stimuli. One of its layers is transcriptional regulation, which is capable of drastically altering bacterial gene expression, converting it from a free-living style to a sessile behavior. To better understand how biofilm-related genes are regulated by different transcription factors, we aimed to apply the SortSeq technique to deeply investigate the architecture of central promoters of biofilm formation. We started this work with 21 promoters of interest, associated directly with biofilm formation and motility. A library containing these 21 promoters mutated randomly at a 10% rate was constructed and tested. The preliminary tests showed that this library had too many mutations and truncations per promoter sequence, and few promoter variants, making unfeasible the acquisition of results with it. This way, we decided to focus on two main promoters of genes for biofilm formation, related to curli synthesis: csgBAp, and csgDp. Their promoters are restricted to the same intergenic region between their genes and have a strict relation, once csgD is a master regulator of transcription of the csgBA operon. We divided this intergenic region into four parts and built libraries with region-focused random mutations. These four libraries were individually inserted into the original promoters, which were assembled in a low-copy number plasmid and modulate the expression of sfgfp and mCherry reporter genes (csgBD-reporter plasmid). To access how the promoter mutations could affect the expression of each gene, we transformed the four libraries into the E. coli W3110 RpoS+ and cultivated it to the stationary phase. Reproducing the SortSeq technique, the cultures were analyzed by flow cytometry and sorted into four tubes according to their fluorescence for sfGFP, mCherry, sfGFP, and mCherry, and Negative (no expression for both reporters). Triplicates of this experiment were mini-prepped and deep-sequenced using Illumina platform. This new library design and SortSeq approach allowed us to better understand how each promoter region can affect the expression of both genes individually and at the same time, helping us to confirm or deny binding sites already predicted. We also found new possible binding sites for transcription factors not described yet, which were used for the prediction of new regulators to these promoters. This library gave us some insights into the logic involved in csgB bistability regulation. To expand the investigation of csgBA and csgD promoters we constructed a genomic library of the E. coli W3110 RpoS+ strain, using a barcoded-Tn5 plasmid. The Tn5 transposase present in the plasmid inserts randomly a unique barcode in a unique region of the genomic DNA. Then, we map the barcode to its corresponding genomic region, allowing the mutated site identification only by sequencing the barcode. We transformed our csgBD-reporter plasmid (without any library insertion) into it and used the SortSeq approach to sort the bacteria in the same four phenotypes and sequence the inserted barcode through the NGS technique. This second approach led to the identification of new players in the gene regulation of these important genes associated with biofilm formation. Combining these two types of libraries, we had new insights about the csgBA and csgD promoters\' regulation that will be tested and confirmed in the future.