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| Author(s): |
Henrique Iglesias Neves
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2020-04-16 |
| Examining board members: |
Beny Spira;
Regina Lúcia Baldini;
Rodrigo da Silva Galhardo;
Teca Calcagno Galvao
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| Advisor: | Beny Spira |
| Abstract | |
The PHO-regulon of Escherichia coli comprises genes which transcription is regulated by the concentration of ortophosphate (Pi) in the periplasm. The transcription of PHO-genes is controlled by the two component system PhoB-PhoR and by the Pi transporter PstSCAB-PhoU. PHOconstitutive mutants, which express PHO genes regardless of Pi concentration, can be selected in media where the only carbon source is glycerol-2-phosphate (G2P). These mutants express large amounts of alkaline phosphatase, that hydrolyses G2P in the periplasm. The emergence of these mutants follows a pattern similar to that observed for adaptive mutations, i. e., they emerge after contact with the selective medium and keep accumulating thereafter. Unlike other mutational systems, pre-existing PHO-constitutive mutants are not observed and all mutants appear after the third day of incubation. However, there is no theoretical impediment to the occurrence of these mutations in the non-selective culture. The first part of this thesis describes the apparent absence of spontaneous PHO-constitutive mutations. We found out that this phenomenon is due to growth inhibition exerted by the wild-bacteria on the PHO-constitutive mutants, preventing their growth in the early days. This growth inhibition depends on physical proximity between the two populations, but physical contact is not required. Screening of a library of E mutants. coli indicated that for inhibition to occur, the inhibiting bacteria must be able to use glycerol as a carbon source. The results point to a \"tragedy of commons\" situation where the glycerol produced by G2P hydrolysis is consumed by wild-type bacteria before it could be taken up by the PHO-constitutive mutant. The actual rate of PHO-cinstitutive mutations is approximately 2, 5×10-6 mutations/generation, close to the general rate expected in E. coli. It was calculated in plates seeded with low bacteria density, a condition in which the growth of the PHO-constitutive mutant is not inhibited. In the second part of this work, the transport of Pi via the phosphonate transpor system encoded by PhnCDE was analysed. A spontaneous 8 bp deletion in the phnE ORF in the triple mutant ΔpitA ΔpstSCAB-phoU ΔugpBAEC was obtained. This insertion is common in derivatives of E. coli K-12 and inactvates transport through PhnCDE. Surprisingly, this strain could grow with Pi as the sole source of phosphorus. Growth and Pi uptake experiments showed that the PhnCDE system is indeed able to take up Pi with an efficiency similar to that of PitA. (AU) | |
| FAPESP's process: | 15/13730-2 - Study of the arising of PHO-constitutive mutants in Escherichia coli |
| Grantee: | Henrique Iglesias Neves |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |