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Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-109). Also available in print.

Zugl.: Berlin, Freie Univ., Diss., 2008

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 163-176). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 174-188).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 89-102).

Thesis (M. Sc.)--University of Hong Kong, 1970. / Mimeographed.

The translocation of the T7 genome into the cell is a multistep process. Following adsorption, approximately 850bp of the 40kb linear genome is internalized to expose host-specific promoters in the leading end to transcription components. There are three strong early promoters, PA1, PA2, and PA3 on this leading 850 bp. Further T7 genome internalization is coupled to transcription and I have measured internalization rates to characterize the rate of transcription by E. coli RNA polymerase in vivo. E.coli RNAP internalizes the entire 40kb and distal parts of the genome are internalized nearly as efficiently and at the same rate as the leading end. I have shown that processivity is dependent on the antitermination element boxA, located 63 bp downstream from PA3, and on only one of the three early promoters. However, when any one of boxA, PA3, or the host antitermination factor nusB is mutated the efficiency, rate, and apparent processivity of transcription -- and thus the efficiency of genome internalization are all significantly reduced. The PA3 promoter, boxA, and E. coli nusB are all non-essential for T7 growth, but they confer a fitness benefit to wild-type phage by increasing the rate of genome internalization. In T7, the minimal requirement for antitermination is promoter PA3 and the boxA sequence. I have found that transcripts initiating at PA1 and PA2 are not effectively antiterminated by boxA, however those from PA3 alone do. Upon further investigation it was shown that there is a requirement for sequences upstream of the -35 hexamer of PA3 to confer full antitermination. After T7 expresses its own single-subunit RNA polymerase, bacteriophage T7 must shutoff host transcription via the phage proteins gp0.7 and gp2. In the absence of host RNAP shutoff, T7 DNA is degraded and the infection fails. I have found that the absence of either promoter PA3 or boxA,gene 2 is unnecessary for growth. These results argue the target for shutoff is actually antiterminating transcription. / text

published_or_final_version / Dentistry / 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