The flagellar motor of Escherichia coli is a bidirectional rotary nano-motor, powered by a transmembrane influx of protons. The maximum speed of rotation is about 300 Hz. The motor rotates either counterclockwise (CCW) or clockwise (CW) and the rotation direction is controlled by a chemotactic protein, CheY-P. Despite extensive structural and functional studies, precise mechanisms regarding the torque generation and the directional switching processes remain unclear. In this work, a bottom-up strategy is proposed and followed to investigate this motor. This strategy, named as a multiscale modeling approach, integrates various publicly available experimental data and ‘state-of-the-art’ molecular modeling methods to build structural models for the two most important parts of the motor, the C ring and the stator. Starting from the primary sequences of the composition proteins of these two substructures, tertiary structures are predicted by means of comparative modeling or de novo prediction when the comparative modeling is not available. Quaternary structures of these proteins’s complexes are then predicted by data-driven protein-protein docking or multiscale molecular dynamics (MD) simulations. Finally, structural models of the C ring at CCW and CW rotational states are constructed by cryo-EM aided assembling methods (constraint search that is based on the multiscale modeling tools and under the constraint of the EM images). In the case of the stator, its composition proteins, MotA and MotB, are assembled by coarse-grained MD simulations. This is the first molecular model based atomistic details for the stator. A new molecular mechanism for rotational switching is proposed based on the structural models of the C ring and the stator. The two states of the C ring display significant differences in the interfaces among the self-assembled FliMs and the orientations of FliG C-terminal domain. Based on protein docking results, a binding site of CheY-P is identified on FliM which is close to the interfaces of FliMs for self-assembling. Thereby, I propose that the CheY-P binding interferes with the interactions between neighboring FliM proteins, and thus, induces ~65° rotation of the FliGc domain with respect to FliM. Subsequently, the interactions between the stator and FliGc domains are altered and the rotation direction is changed. Furthermore, a mechanism accounting for the directed rotation of the flagellar motor is proposed based on systematic MD studies on the dynamics of FliGc. It is found that the C-terminal subdomain of FliGc is capable of rotating by ~180° with respect to the N-terminal subdomain of FliGc. If this dynamics is considered in the framework of the whole C ring, the rotation of the C-terminal subdomain of FliGc exhibits an asymmetric feature. As a result, the C ring is decorated with asymmetric teeth on the outer periphery and hence similar with Feynman’s ratchet. The preference of the motor in CCW rotation or in CW rotation is then explained based on the Feynman’s ratchet model. / published_or_final_version / Chemistry / Doctoral / Doctor 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

The diminishing choice of effective antibiotics against resistant pathogens has forced clinicians to revive the use of old antibiotics. Hence, fosfomycin has been frequently suggested for alternative therapies given its track record of low resistance rates despite extensive use. However, there have been recent reports of plasmid-mediated fosfomycin resistance among animals and healthy humans in Asia. Accordingly, comparison of shared fosfomycin resistance mechanisms between animals and humans will shed light on the spread of resistance and guide future use of antimicrobials. This study aimed to investigate plasmid-mediated fosfomycin resistance in E. coli isolates collected from patients and animals in Hong Kong. Non-duplicate E. coli isolates were cultured from 1711 urinary isolates and 167 blood clinical samples collected from multiple centres during 1996-2008 and from fecal samples of 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents during 2008-2010. A total of 2106 animal samples yielded 1693 E. coli isolates of which 831 were ESBL-producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be ESBL-producers and multidrug resistant (≥3 antimicrobial classes). Of the 101 fosfomycin-resistant isolates, 97 (96.0%) were fosA3 positive and 94 (93.1%) were blaCTX-M positive. Of the 1878 clinical isolates, 18 were fosfomycin-resistant of which six were fosA3-positive and two were positive for another fosA variant (designated fosKP96). All but one fos-carrying clinical isolate was ESBL-producing. The majority of the fos-carrying E. coli strains belonged to diverse clones under two main clonal complexes CC58 accounting for 38 (36.2%) strains and CC10 for 32 (30.5%) strains. PCR mapping showed that all fosA3-containing regions were followed by a downstream IS26. In all clinical isolates and 81 (83.5%) of animal isolates, an IS26 was also found upstream. In 14 (14.4%) of animal isolates, the fosA3 gene was preceded by an upstream blaCTX-M-14-containing transposon-like structure (ΔISEcp1-blaCTX-M-14-ΔIS903 or ISEcp1-IS10-blaCTX-M-14-ΔIS903). For the remaining two animal isolates, the upstream region could not be defined. In a random subset of 18 animal isolates, fosA3 was carried on transferable plasmids with sizes of 50–200 kb and the following replicons: F2:A-:B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), IncHI2 (n = 3), IncN (n = 2), IncI1 (n = 1), B/O (n = 1) and untypeable (n = 5). Among six fosA3-carrying clinical isolates, the distributions were: F2:A-:B- (n = 2), IncN (n = 1), multi-replicon F-:A-:B1/IncN (n = 1) and untypeable (n = 2). Both fosKP96-carrying plasmids belonged to IncN. Restriction fragment length polymorphism analysis showed that the four F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes from a pig, dog and two patients shared an almost identical pattern. The complete plasmid sequences further demonstrated their homology. This study demonstrated the emergence of fosA3-mediated fosfomycin resistance among E. coli isolates from multiple sources. Highly similar IncFII plasmids and IS26 transposon-like structures appear to be the main vehicles for dissemination. This study also highlighted occurrence of plasmids carrying fosKP96 that may have been overlooked by others as this variant could not be detected by established PCR markers. / published_or_final_version / Microbiology / Master / Master of Philosophy

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein), which binds the intron RNA to promote RNA splicing and intron mobility. Mobility occurs by intron RNA reverse splicing directly into a DNA strand and reverse transcription by LtrA. I used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to the cellular poles in both Escherichia coli and L. lactis. This polar localization occurs with or without co-expression of the intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three non-overlapping LtrA subsegments, reflecting dependence on common redundant signals and/or protein physiochemical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. In E. coli, the Ll.LtrB intron inserts preferentially into the chromosomal ori and ter regions, which are pole localized during much of the cell cycle. Thus, the polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in these regions. I established a high throughput method using cellular array and automated fluorescence microscopy for screening transposon-induced mutants, and identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) in which disruptions result in increased proportion of cells having diffuse LtrA distribution. This altered localization is correlated with a more uniform distribution of Ll.LtrB insertion sites throughout the E. coli genome. Finally, I find that altered LtrA localization in all five disruptants is correlated with accumulation and more diffuse intracellular distribution of polyphosphate, and that a ppx disruptant, which also results in polyphosphate accumulation, shows similar LtrA mislocalization. These findings may reflect interaction between LtrA and intracellular polyphosphate. My findings support the hypothesis that the intracellular localization of LtrA is a major determinant of Ll.LtrB insertion site preference in the E. coli genome. Further, they show that alterations in polyphosphate metabolism can lead to protein mislocalization, and suggest that polyphosphate is an important factor affecting intracellular protein localization.

No description available.

Campylobacter is the most commonly reported bacterial cause of gastrointestinal disease in developed countries. Campylobacteriosis is an infectious disease that causes severe diarrhoea, abdominal cramps, vomiting, blood in stools and fever, along with the inability to carry out normal activities for an estimated 3-5 days. Long term sequelae associated with Campylobacter infection includes Guillain Barré syndrome, irritable bowel syndrome and reactive arthritis. The incidence of human campylobacteriosis in the Grampian region was 138.8 per 100,000 people in 2011 which was one of the highest incidence rates within Scotland. Identified areas of limited knowledge in Campylobacter research include: population exposure to risk factors, financial burden and public acceptability of interventions to reduce Campylobacter in the poultry process. This thesis utilises questionnaire methods to gather data from the Grampian population to expand our knowledge in these research areas to assist in the reduction of human campylobacteriosis.

published_or_final_version / abstract / toc / Ecology and Biodiversity / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy