Characterization of PHO-constitutive mutations in Escherichia coli

• Grant number: 17/04130-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: May 01, 2017
• End date: April 30, 2018
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Beny Spira
• Grantee: Cláudia Ramos de Carvalho Pinto
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

E. coli PHO regulon consists of more than 30 genes, which together respond to the availability of orthophosphate (Pi) in the medium. The excess of Pi in the periplasm represses a transcription of the PHO genes, whereas, at low concentrations of Pi in the medium, genes belonging to the PHO regulation have their expression activated. Transcriptional control of PHO regulon is performed by the two-component PhoB-PhoR system and the PstSCAB-PhoU transport system. Other two systems related to Pi uptake are important for this project: the Pi transporter, PitA, the glycerol-3-phosphate transporter, Ugp and an alkaline phosphatase enzyme, which is the reporter used to identify the level of activation of the PHO regulator. PHO-constitutive mutants are those that lose the ability to repress a transcription of the PHO genes in the presence of excess Pi in the medium. Such mutants can be isolated in the presence of glycerol-2-phosphate (G2P) as the sole carbon source. All of the already isolated PHO-constitutive mutants contain mutations in one of five pstSCAB-phoU operon genes or no phoR gene. The objective of this project is to locate and identify molecularly isolated PHO-constitutive mutants, in laboratory, in G2P plates. The gene affected by the mutation will be defined through in-trans complementation assays and then sequenced. In addition, the level of constitutivity of each mutant (activity of alkaline phosphatase) will be measured and correlated with the affected gene and the type of mutation (indel or substitution; and the polarity of the mutation). The locus and type of mutation will be correlated with the day of arising of the colony at the selection plate. (AU)