Acquisition of Pi-uptake genes through mutation and horizontal gene transfer in Escherichia coli

• Grant number: 25/17481-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2025
• End date: November 30, 2028
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Beny Spira
• Grantee: Marina Caldas Leite
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Horizontal gene transfer mediated by bacteriophages is a major mechanism by which bacteria acquire genetic variability, potentially increasing their fitness. Several environmental studies have reported phages carrying genes related to inorganic phosphate (Pi) acquisition. This project aims to investigate the presence of such phages in wastewater from a stream contaminated with domestic sewage and to evaluate the transfer of Pi uptake genes via transduction or lysogenic conversion between Escherichia coli strains. To facilitate this, we constructed strain 4¿ (¿pitA ¿pitB ¿pst ¿phn), which lacks all canonical and alternative Pi transport systems and is thus unable to grow in minimal medium with Pi as the sole phosphorus source. This strain will serve as a recipient in transduction and lysogenic conversion experiments using wastewater samples as the source of transducing or lysogenic phages. In parallel, DNA sequencing analyses will be conducted to detect Pi-related genes within phage genomes. The results will provide direct experimental evidence of evolution via horizontal gene transfer in an ecosystem affected by anthropogenic activity, contributing to our understanding of bacterial evolution in urban environments and the ecological interactions between phages and their bacterial hosts. Additionally, to identify new phage-mediated transduction systems in E. coli, phages will be isolated from wastewater and tested for their ability to transduce genetic markers using both laboratory strains (K-12 derivatives) and natural isolates. Isolated phages will be sequenced, annotated, and characterized morphologically via electron microscopy. Lastly, we will explore bacterial adaptation throughmutation. Strain 4¿ will be grown in a chemostat under conditions of excess Pi and limiting glycerol-3-phosphate (G3P). We hypothesize that selective pressure will drive the emergence of mutations restoring Pi uptake. Another chemostat system will also be employed to monitor gene transfer events involving Piacquisition via phages.