Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are diarrhoeagenic human pathogens that colonize intestinal epithelial cells via an attaching and effacing (A/E) lesion. Intimate adherence is mediated through binding of the intimin adhesin to the bacterial translocated intimin receptor, Tir. EPEC adheres to all regions of the human intestine in the in vitro organ culture (IVOC) system. In contrast, although EHEC is associated with colonic pathology in human infections, a prototypical strain of EHEC has shown a restricted tropism towards follicle-associated epithelium (FAE) of the terminal ileum without evidence of colonic adhesion. This thesis used the human IVOC system to further examine the intestinal interaction of EHEC 0157:H7 and related EPEC serotypes. To address the apparent paradox of non-adherence of EHEC to colonic tissue in the IVOC experimental system, the role of environmental, host and bacterial factors in modulating EHEC colonisation and tissue tropism were studied. No environmental factor (modulation of IVOC system, bicarbonate, serum, and mannose) was found to induce colonic adhesion. The investigation of the hypothesis that prior host-bacterial interactions might enhance subsequent EHEC colonic adhesion found that exposure to FAE promoted subsequent colonic adhesion, but in a non-intimate manner, demonstrating a novel form of interaction with human intestine. Great diversity was found in EPEC and EHEC in relation to the presence and sequence of tir, tccP and espJ fccP-negative strains expressing TireHEc were identified indicating that novel Nck-like molecules may be awaiting discovery. Tir was essential for EPEC and EHEC adhesion in IVOC but its phosphorylation (EPEC) and its interaction with TccP (EHEC) were not necessary for colonisation and A/E lesion formation on IVOC, questioning the role of pedestal formation in enterocyte infection. This is in direct contrast to cell culture findings and demonstrates the importance of IVOC in establishing 0157:H7 human pathogenesis.

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Most bacteria have an inherent need for iron, as it is a component of various enzymes involved in vital metabolic processes. Therefore, bacteria have evolved several mechanisms to acquire iron from the environment. Bacterial iron transporters allow the active uptake of both ferrous and ferric iron. Escherichia coli is believed to possess two high-affinity ferrous iron transport systems: EfeUOB (which is induced under acidic conditions) and Feo (which is anaerobically induced). Feo consists of three proteins, FeoA, FeoB and FeoC, encoded by a three-gene operon,jeoABC. FeoB is an integral membrane protein, which is believed to act as a ferrous permease. It is a unique protein, possessing a G-protein domain - combination of a G-domain to a membrane protein has only so far been associated with higher organisms. Its role in FecB function is not clear, however it has been demonstrated that FeoB function is dependent on this domain. The role of FeoA and FeoC within the Feo system is uncertain. However, it has been suggested that FeoA may function in assisting the G-protein function of FeoB and that FeoC is a gene regulator. This study was undertaken to elucidate the function of FeoA, FeoB and FeoC and what kind of role they play in ferrous iron uptake. Firstly, it was shown that FeoA and FeoB are necessary for Feo function in E. coli, as l!.jeoA and 6.feoB mutants exhibited a poor anaerobic growth phenotype both under high and low iron availability. Raising the dosage of FeoB enhanced growth, indicating a predominant role for FeoB in Feo function. Regulatory studies with the aid of lacZ- fusions indicated that FeoC negatively regulates feoABC expression where a ~ 19-fold induction [high iron] and a ~ 10- fold induction [Iow iron] in feo-lacZ expression was observed compared to wild-type. However, expression of feo-lacZ remained iron- dependent in the feoC mutant. feoA promoter truncations were generated and studied. Results indicated binding sites for at least 3 eis-regulatory elements located between: (1) the -35 and +1 site, (2) downstream from the +1 site and (3) ~ 30 bp upstream from the -35 site. Studies using a GTPase assay confirmed that G-protein domain of FeoB has GTPase activity, which was enhanced by crude preparations of FeoA, indicating that FeoA may act as a GTPase-Activating Protein. This is the first experimental data indicating a specific role for FeoA in Feo function and a regulatory role for FeoC. ii

Microorganisms growing as collective communities called 'biofilms' are widely recognised today. Biofilms impact almost all aspects of human life: personal, medical and industrial. Escherichia coli are model organisms for the study of biofi lms. In this work, eight enteropathogenic E. coli (EPEe) and three Shigella sonnei strains (a bacterium that is very similar to E. coli and is often implicated in diseases that are similar to those caused by EPEe) were investigated for their biofilm fonning abilities in relation to a number of factors. These included nutrient availabi lity, pH, temperature, time, sodium chloride concentration and iron avai lability. E. coli were able to form biofilms under all conditions tested; however, S. sonne; was not able to form any biofi lms under any of the tested settings and therefore studies on this microorganism were discontinued. The results of the investigations into biofilm-fonning abilities directed the subsequent research. These included est imation of antibiotic resistance, examination of structural development of biofilrns and the inspection of biofi lm behaviour at genetic and proteomic levels. A modification of Calgary Biofilrn Device was employed to estimate resistance to nine antibiotics in biofi lm form. A Stovall Flow Cell was employed to study the structure of biofilms formed by one (selected) strain E. coli NCiMB 555 which was transformed to express gfp in order to visualize the biotilm morphology. E. coli NelMB 555 (un-transformed) biofilms were also grown to study proteins expression and gene regulation of six targeted genes. Of these four genes were up-regulated, one was down-regulated and onc was expressed at a similar level to planktonic forms. This study contributes to the existing knowledge of the biofilm formation by E. co/; under a number of conditions, their antibiotic resistance and gene- and protein-expression associated. The findings of the work have highlighted the need for more work especially in the field of antibiotic resistance. 4

The Zur (zinc uptake regulator) regulon of a range of pathogenic and non-pathogenic E. coli strains was explored by combining in silico Zur binding site predictions with experimental approaches. Initially, novel Zur-binding sites were predicted in the genomes of K-12 MG1655, 0157:H7 str. Sakai, H10407 and CFT073 using patser. The effect of both zinc depletion and zur deletion was probed in each strain by transcriptomics analysis, in order to identify direct Zur targets. Known Zur targets, including zinT, an auxiliary component of the znuABC zinc uptake system, and ykgM, an alternative non-zinc-binding ribosomal protein were up-regulated in all strains under both zinc depletion and zur deletion conditions. In contrast, up-regulation of the high affinity zinc uptake system znuABC, was more modest and variable. Transcription of pUG (ycgK), encoding a periplasmic G-type lysozyme inhibitor, was up-regulated under zinc depletion conditions and upon zur deletion in all strains, and also possessed a high-scoring predicted Zur binding site within its promoter region, leading us to believe that it is a novel member of the conserved Zur regulon. Additionally, a novel Zurregulated operon, c1265-7 was predicted in the CFT073 genome, and validated by microarray data and the use of a promoter-lux fusion. C1265 appears to be a TonBdependent heme/hemoglobin receptor, whilst C1266 and C1267 showed significant homology to components of metal uptake systems. Deletion of c1265J 7, even in the presence of a znuABC deletion, showed no clear phenotypic outcome under a range of conditions tested. C1265-7 was found to be almost exclusively distributed in ExPEC strains suggesting it is of importance to extra-intestinal survival. Additionally, the severe growth defect of CFT073 D.zur, even in zinc-deplete media suggests the CFT073 zur regulon may include extra genes involved in zinc uptake. iii ... \ ...

DNA ligases are essential repair enzymes required for maintaining genomic integrity in cells. The first ligase to be discovered was Escherichia coli DNA ligase; a 670 amino acid, 74 kDa, NAD+ dependent ligase. This work reports a series of studies into the behaviour of His-tagged E.coli ligase. Order-of-addition studies on singly-nicked oligoduplexes under steady state conditions revealed that ligase undergoes an obligatory off-step from the DNA after sealing a break in a phosphodiester strand before readenylation in solution. These results corroborate the findings of Lehman that a sequential model is the normal mode of Ligase operation. Ligase affinity for its substrates NAD+ and DNA were 3.5 μM and 3.5 nM respectively. Length dependency studies on singly-nicked PCR substrates revealed that when two different DNA lengths were in the same solution, the initial association rate was always faster for the longer DNA substrate. For example, 40 bp versus 902 bp gave initial rate values 0.06 nM/min (40 bp) and 0.28 nM/min (902 bp); increasing the length 22 fold increased the initial rate 4 fold. This hints that Ligase uses DNA flanking a nick to locate its specific site. Processivity studies were achieved to determine the one- or three-dimensional pathway of Ligase using doubly-nicked DNA. Nicks were either directly repeated (on the same DNA strand) or inverted (opposite strands). Results revealed Ligase is weakly processive; 32% processive. However, when beta-clamp and gamma-loader were added to the reaction processivity significantly increased.

Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences)