published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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

A facultative anaerobe such as Escherichia coli is able to switch between the aerobic and anaerobic modes of metabolism in response to O2 availability. This adaptation is primarily controlled by a global transcription regulator called FNR (fumarate nitrate reduction). The key property that allows FNR to act as an O2 responsive transcription factor is its capability to dimerize and being activated upon binding of an O2 labile [4Fe-4S]2+ cluster. Previous functional studies have largely focused on the regions of FNR analogous to CRP (cAMP receptor protein), a prototype CRP/FNR family protein which X-ray crystal structure has been resolved. However, E. coli FNR contains extra N- and C-terminal regions that are conserved among various FNR orthologs but are absent in CRP. The functions of these two regions have not been resolved. In this study, their functions and physiological significance to the O2 sensing capacity of FNR were systematically investigated. A three-alanine (3-Ala) scanning library on amino acid 2-19 and 236-250 of FNR was constructed and selective 3-Ala substitution mutants exhibited variable defects. These defects were found to be due to their impairment of intracellular FNR protein levels which was unique only among FNR mutations in these two regions. Introduction of 3-Ala substitution at the residues 239-244, resulting in LAQ239-241A3 and LAG242-244A3 respectively, caused an especially accelerated degradation and decrease of intracellular FNR proteins. These variants were found to be degraded by the ClpXP protease. Sequence alignment of FNR orthologs revealed a highly conserved “L239XXL242XG244” motif, and my experimental data further revealed that L239 and L242 were important residues and were responsible for the defects of LAQ239-241A3 and LAG242-244A3, respectively. Circular dichroism analysis revealed that introduction of LAQ239-241A3 caused conformational changes with a significant loss of secondary structures in FNR. These studies taken together suggest that the N- and C-terminal regions of FNR play an important role in mediating the intracellular protein level of FNR. My studies also specified the ClpXP signals as the N-terminal RR9-10 and C-terminal VA249-250, and indicated that VA249-250 is a more important site than RR9-10 in targeting FNR to proteolysis. The second topic of the thesis involves exploration of the regulatory mechanism of an anaerobically activated multidrug efflux pump MdtEF in E. coli. MdtEF is an important multidrug efflux pump that causes antibiotic resistance upon overexpression. Previous studies revealed that expression of MdtEF was significantly upregulated under anaerobic conditions, but its regulatory mechanism was unknown. In the current study, systematic analyses on the unusually long promoter region of the gadE-mdtEF operon which drives the expression of MdtEF were performed. It was found that unlike FNR, mdtEF was not regulated at post-translational level by proteolysis, but at transcriptional level through the promoter region of gadE. My study showed that anaerobic activation of mdtEF was mediated by the anaerobic regulator ArcA and nitrate responsive regulators NarL and NarP. Important promoter regions P3 and P1 were also identified. This study provides essential molecular basis for the upregulation of MdtEF in a host and clinically relevant conditions. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy