Abstract
(p)ppGpp is a second messenger regulating stress response in bacteria and having a drastic effect on cell physiology by inhibiting cell growth, relocating the transcription machinery and modifying the translation. In Escherichia coli, two enzymes are responsible for the synthesis and the hydrolysis of (p)ppGpp. RelA is a monofunctional synthetase, and SpoT is a bifunctional hydrolase/synthetase. Both are activated upon different signals, like amino acid starvation for example1.Siderophores are small Fe3+-chelating molecules secreted by bacteria to scavenge free iron present in the environment2. Enterobactin is one of them, produced in the cytoplasm and sent in the extracellular medium, forming a complex called ferric enterobactin. This complex can then be delivered to the cytoplasm through different membrane transporters. There, the enterobactin hydrolase Fes can hydrolyse the complex, and Fe3+ iron will be reduced to allow free Fe2+ in the bacteria3.
In a publication from Vinella et al. in 20054, a double knock-out mutant ΔfesΔrelA in Escherichia coli was shown to accumulate (p)ppGpp upon iron starvation. They also characterized other combinations of mutants including RelA, SpoT, and the ferric enterobactin esterase Fes, all in condition of iron scarcity. They ended up proposing that iron limitation would stimulate a SpoT-dependent (p)ppGpp accumulation, and that (p)ppGpp would stimulate the synthesis of other iron uptake systems. This SpoT-dependent accumulation has not been explored further, thus it could be interesting to characterize the (p)ppGpp-dependent response to iron starvation.
To further understand this response, the first objective of this study was to confirm part of the data generated in the study of Vinella et al.4 in our Escherichia coli strain by means of a viability assay. Several regulators of SpoT have already been described, so their involvement in iron starvation was investigated. Then, two different screens were planned to find a regulator of SpoT: the transformation of the ASKA library and a CRISPRi-Seq. None of them could be achieved but experiments were set up. We although suggested the essentiality of the gene spoT in a ΔfesΔrelA genetic background.
| Date of Award | 30 Jan 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Regis Hallez (Supervisor), Anthony Perrier (Co-Supervisor) & Elie Marchand (Co-Supervisor) |