Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 145-174).

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 102-112). Also available in electronic version. Access restricted to campus users.

The effect of expression of foreign proteins on the physiology of the bacterium <I>Escherichia coli </I>has been investigated. To quantitate accurately protein production a model system was developed based upon the expression of an α-2 Interferon fragment, (amino acids 4-155). A polyclonal antibody-based enzyme linked immunosorbant assay (ELISA) for α-2 IFN was developed for rapid determination of accumulated protein. As with many foreign proteins expressed in <I>E. coli,</I> α-2 IFN (4-155) was accumulated in an entirely insoluble form as an inclusion body. For accurate determination of α-2 IFN (4-155) by ELISA, protocols for the solubilisation of the foreign protein were developed. Foreign proteins could be accumulated at up to 30% of total cell protein with no significant difference in the specific growth rate of the recombinant cell compared to its parent. Determination of RNA: protein ratios indicated that the protein synthetic capacity of parental and recombinant cells were not significantly different. A model is proposed in which the expression of certain proteins was reduced to accommodate the extra translational load of recombinant protein production. Since RNA pools were unaffected by recombinant protein production it is inferred that the ribosomes and other proteins involved in translation are not significantly affected. The data predict that many <I>E. coli </I> proteins are synthesised at rates faster than those needed to sustain specific growth rate. The susceptibility of recombinant cells to environmental challenge is, however, increased indicating proteins, that are accumulated to lower levels during foreign protein expression, have a role in the ability of <I>E. coli</I> to adapt to a changing environment.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

ompW encodes a widespread outer-membrane porin protein in Gram-negative bacteria. It has been implicated in bacterial responses to various antibiotics and environmental substances such as antibiotics, drugs and mouse mucus etc. Little is known, however, about its regulation and physiological roles during bacterial stress responses. Recently, comparative genomics studies revealed that the ompW gene is a core regulon of the global transcription factor FNR (Fumarate Nitrate Reduction) which mediates the transition from aerobic to anaerobic lifestyle of facultative bacteria. Anaerobiosis represents a predominant challenge encountered by many bacteria in their natural ecological niches and human hosts. This thesis thus aims to elucidate the molecular mechanism of FNR-dependent regulation of ompW expression and its relevance to the anaerobic adaption of the model facultative bacterium E. coli. Regulation of ompW expression by several other key physiological signals related to the anaerobiosis of E. coli, as well as the physiological significance, is also explored systematically. In the first half of the thesis, FNR-dependent regulation of ompW is confirmed by in vivo transcriptional activity assay, and then further confirmed at mRNA and protein level by RT-qPCR and western blotting. EMSA combined with transcriptional activity assay reveals that FNR directly binds with two sites centered at -81.5 and -126.5 bp respectively on ompW promoter (PompW). While binding to the -81.5 site by FNR activates the transcription of ompW, interaction with the -126.5 site represses it, and repression through the -126.5 site is dependent on primary occupancy of the -81.5 site by FNR. Based on these molecular mechanisms, a novel regulatory model of ompW expression during anaerobic adaptation of E. coli is proposed. Growth competition assay further confirmed the physiological significance of this fine-tuned regulation of ompW by FNR in facilitating the fitness and adaptation of E. coli during the transition from aerobic to micro-aerobic and anaerobic lifestyles. In the second half of the thesis, it is demonstrated that two other physiological signals related to the anaerobiosis of E. coli participate in the regulation of ompW, i.e. carbon and electron sources. The molecular mechanisms of how the relevant transcription factors, namely CRP and NarXL, mediate ompW transcription were elucidated: CRP activates the transcription of ompW by binding with the -42.5 site on PompW when glucose is absent; NarL represses the expression of ompW via its binding with the -18.5 site on PompW in the presence of nitrate (the most preferred electron source of E. coli during anaerobic growth). Fumarate is estimated to enter the central channel of OmpW and rescues OmpW-mediated colicin S4 killing of E. coli, suggesting OmpW is a receptor for fumarate and revealing its role in facilitating C4-dicarboxylates utilization. In summary, my study reveals a previously unrecognized, highly co-ordinated and dynamic regulation network for the expression of the widely distributed Gram-negative bacterial minor porin protein OmpW. Given the high conservancy of both the ompW gene and its promoter regions in several pathogenic bacterial species, my study contributes to the understanding of the pathogenicity of these species in the host relevant environment of anaerobiosis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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