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postgraduate thesis: Functions and physiological significance of the N- and C- terminal regions of the Escherichia coli global transcription factor FNR

TitleFunctions and physiological significance of the N- and C- terminal regions of the Escherichia coli global transcription factor FNR
Authors
Issue Date2013
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Pan, Q. [潘庆]. (2013). Functions and physiological significance of the N- and C- terminal regions of the Escherichia coli global transcription factor FNR. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4985852
AbstractA 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.
DegreeDoctor of Philosophy
SubjectTranscription factors
Escherichia coli - Molecular aspects
Dept/ProgramBiological Sciences
Persistent Identifierhttp://hdl.handle.net/10722/196081

 

DC FieldValueLanguage
dc.contributor.authorPan, Qing-
dc.contributor.author潘庆-
dc.date.accessioned2014-03-28T07:05:43Z-
dc.date.available2014-03-28T07:05:43Z-
dc.date.issued2013-
dc.identifier.citationPan, Q. [潘庆]. (2013). Functions and physiological significance of the N- and C- terminal regions of the Escherichia coli global transcription factor FNR. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4985852-
dc.identifier.urihttp://hdl.handle.net/10722/196081-
dc.description.abstractA 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.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshTranscription factors-
dc.subject.lcshEscherichia coli - Molecular aspects-
dc.titleFunctions and physiological significance of the N- and C- terminal regions of the Escherichia coli global transcription factor FNR-
dc.typePG_Thesis-
dc.identifier.hkulb4985852-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4985852-
dc.date.hkucongregation2013-

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