The ability to sense and rapidly respond to oxygen availability is crucial to the survival and physiology of facultative anaerobes. In many gram negative bacteria such as Escherichia coli, this process is primarily controlled by the dimeric, [4Fe〖-4S]〗^(2+) containing global transcription factor FNR, which regulates transcription of genes necessary for the anaerobic metabolism. Activity of FNR is directly regulated by the presence of oxygen, which inactivates FNR by oxidizing the [4Fe〖-4S]〗^(2+) cluster and causing the dissociation of the FNR dimer. Although the biological function of FNR has been well established, structural and biochemical characterization of the FNR dimer has been limited due to its extreme lability to oxygen.
In the current study, I conduct molecular engineering on FNR protein and obtain oxygen stable variants that are suitable for in vitro biochemical studies. By combining several approaches including covalently linking two FNR monomers using a flexible peptide linker, amino acid substitutions to promote dimerization, and removal of protease recognition sites to prevent proteolysis, a series of FNR variants which are potentially active in the presence of oxygen are constructed. Various in vivo and in vitro assays led to the identification of the construct (FNRD154A)2 which covalently links two copies of FNRD154A, an FNR variant that has greater dimerization capability, in tandem displays significantly improved transcription regulation and DNA binding to various FNR regulated promoters in the presence of O2. Circular Dichroism analysis showed that this variant maintains a similar secondary structure as that of native FNRD154A and in vivo transcription assay demonstrated that this protein retains other properties of the native FNR dimer including [4Fe〖-4S]〗^(2+) cluster binding, oxygen sensing, and capability to support the anaerobic growth of E. coli. All these together led the conclusion that an FNR variant that retains structural and functional properties of native FNR has been constructed, but with significantly improved O2 stability. Thus, it has the potential to be widely used in various biochemical and structural studies of FNR in the presence of oxygen.
In addition to the major project of molecular engineering of FNR protein, in this thesis, I also initiated the study of using metabolomics approaches to identify the cellular substrates of the multidrug efflux pump MdtEF. MdtEF is an important efflux pump in E. coli and its expression has been shown to be induced under a number of stressed conditions. It is thus proposed to have a general detoxification function in E. coli, but the cellular substrates it expels have not been identified. In this study we established and applied metabolite profiling on the wild type and ΔmdtEF E. coli strains and confirmed that indole red, a metabolic by-product formed during anaerobic respiration of nitrate, is one of the cellular substrates of MdtEF under anaerobic conditions. This study provides a general methodology to identify endogenous substrates of efflux pumps and contributes to the understanding of the physiological roles of multidrug efflux pumps in bacteria. / published_or_final_version / Biological Sciences / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/193014 |
| Date | January 2012 |
| Creators | 单越, Shan, Yue |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | Creative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| Relation | HKU Theses Online (HKUTO) |
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