Sequence analysis of the 55.8-56.0 min region of the Escherichia coli genome has revealed a 12-gene operon designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon encodes a putative ten-subunit hydrogenlyase complex (hYdrogenase four or Hyf), a potential formate sensing 0'54_ dependent transcriptional activator, HyfR (related to FhIA), and a possible formate transporter, FocB (related to FocA). It has been proposed that Hyf in conjunction with Fdh-H forms a second formate hydrogenlyase pathway (Fhl-2) in Escherichia coli, which unlike the hyc operon encoded Fhl pathway (Fhl-l ) is a respiration-linked proton translocating Fhl complex. Initial experiments directly investigated these proposals and were conducted with hyf and hydrogenase-I, -2 and -3 mutants grown under hyf operon optimal transcriptional activation conditions. Radiolabelling experiments with 63Ni did not detect the proposed large subunit of hydrogenase-4, despite the detection of 63Ni_ associated polypeptides likely to correspond to the large subunits of hydrogenase-I, - 2 and -3. Also, Fdh-H, hydrogenase and hydrogen production assays detected no activity attributable to the hyf operon. Immunoblotting experiments with anti-HycE and anti-Hyf sera did not detect Hyf polypeptides, suggesting that expression of the hyf operon was very low under optimal transcriptional activation conditions. Transcriptional analysis of the hyf operon using a hyfA-lacZ transcriptional fusion showed that, like the hyc operon, the hyf operon is induced by formate at low pH via the formate sensing, 0'54-dependent transcriptional activator FhlA. The proposed transcriptional activator HyfR was also found to activate hyf operon transcription in a cr54-dependent manner. However the co-effector(s) used by HytR has yet to be identified. Finally bioreactors were used to analyse the growth and metabolism of hyf mutants. However, no differences in growth and metabolism attributable to the hyf operon were observed during anaerobic controlled batch cultivation and both aerobic and anaerobic glucose-limited chemostat cultivation.

The purpose of this study was to characterize blaCTX-M plasmids originating from bovine Escherichia coli and investigate their contribution to bacterial host fitness. In this study 52 bovine Escherichia coli strains collected between March and October 2007 encoding blaCTX-M, an extended spectrum β-lactamase (ESBL) gene conferring resistance to 3rd generation cephalosporins, were characterized. The majority of strains belonged to E. coli commensal phylogroups A and B1 expressing a multi-drug resistance (MDR) phenotype and harboured multiple plasmids of which 90% were transferred by conjugation. Transconjugants or transformants were made successfully from all 52 strains when selecting for resistance to cefotaxime. All plasmids were shown by PCR and sequence analysis to harbour blaCTX-M and nearly 80 % encoded multiple resistances. Plasmid sequence analysis of four plasmids encoding blaCTX-M-14b (IncI1-X1), -15 (IncFII-FIA-FIB) and -32 (IncX1 and IncB), identified genes necessary for stable plasmid maintenance and spread. Five representative plasmids encoding blaCTX-M-1, -15, -14b and -32 were assayed for their fitness impact upon the host. Efficiencies of β-lactam hydrolysis using whole cell extracts were determined in the same E. coli BL21 host strain with the most efficient encoded by blaCTX-M-14b and blaTEM-1 ESBL genes and least efficient encoded blaCTX-M-15 only. A 160 kb plasmid encoding 13 resistance genes was grown in the presence of 380 different metabolites and differences in metabolite utilisation between this and the plasmid-free BL21 strain determined. The plasmid-harbouring strain utilized less phosphor-sulphur compounds, suggesting the metabolic cost incurred by acquiring the plasmid may have implications of cellular utilization of alternative phosphate sources. There were no differences in growth was observed in nutrient rich media. The contribution of active efflux to resistance was investigated using L-phenylalanyl-L- 4 arginyl-b-naphthylamide (PAβN) in combination with ampicillin, cefotaxime or ceftazidime. Minimum inhibitory concentration (MIC) values were found to decrease ≥ 2 fold in the presence of the efflux pump inhibitor (EPI), in some cases becoming completely susceptible to ampicillin. This indicates that the possible use of EPIs in combination with previously failed antimicrobial drugs to potentially restore efficacy of treatment.

Aerobic fermentations of Escherichia coli grown with glucose accumulate organic acids in the culture medium. These by-products of glucose metabolism are synthesised and excreted when the glucose uptake rate is greater than its conversion to biomass and carbon dioxide. Acetate is the most abundant overflow metabolite produced in aerobic cultures of E. coli. Its production is strain and media specific, being greatest in dense nutrient-rich cultures. In industrial fermentations, E. coli is widely employed for production of recombinant proteins. However, acetate excretion reduces process efficiency by lowering cell growth rate, and decreasing the amount of substrate carbon converted to recombinant protein product. This thesis describes the construction of three plasmid vectors designed to express antisense RNA targeted against phosphotransacetylase and acetate kinase, which convert acetyl CoA to acetate. Antisense RNA was used as a metabolic engineering tool in this study, to enable examination of the central carbon flux distribution before, during and after enzyme downregulation. The aim was to decrease expression of these enzymes in E. coli, and thus decrease acetate production. E. coli MG1655 was transformed with a) a plasmid construct encoding an antisense RNA fragment, b) two compatible plasmids encoding different antisense RNA fragments. The resulting strains were cultivated in a 2L bioreactor, and the effect of antisense RNA expression evaluated. Assays to monitor the enzyme activities of phosphotransacetylase and acetate kinase were conducted, along with metabolite analysis to determine organic acid excretion profiles. Flux balance analysis, which is a method of modelling metabolism, was applied to the central carbon pathways in E. coli to provide insight into the partitioning of internal carbon fluxes. Information about the mechanisms used by E. coli to cope with partial shutdown of the acetate synthesis pathway was gained by comparing the flux distribution of a control strain with an antisense RNA-expressing strain.

Replication of the chromosome of E. coli starts at a single point and proceeds bidirectionally until the replication forks meet at the opposite side of the DNA molecule. All this occurs on a compacted (around 1000-fold) chromosomal DNA, which is also organized in a manner that reflects the genetic position of the loci. Any roles that DNA replication plays in helping segregate the new DNA molecules, and how replication relates to nucleoid organization, remain to be understood.

The chromosome of a slowly-growing E. coli cell has a previously unappreciated level of spatial organization. By analyzing the localization patterns of multiple segregated genetic loci and locus pairs in fixed cells, I demonstrated that the left (L) and the right (R) replichores of the circular chromosome locate to opposite halves of each daughter chromosome with 1 replication origin (oriC) in the middle. Furthermore, loci on each replichore locate in a linearmannerfromonCto one of the chromosome edge, roughly recapitulating their genetic positions on the genome map.

This thesis develops and analyses an agent based model of a bacterial Eschericia coli population in a quorum motivated by published experimental work The E. coli cells harbour a synthetic genetic regulatory network that is responsible for oscillatory behaviour across the population. The model is used to understand whether the collective oscillations seen in the experiments are an outcome of coupled oscillators synchronising or if they are an emergent property of the population. A bottom-up approach is used whereby an ordinary differential equation model is developed based on the biochemical dynamics that result from the introduced genetic network This model is studied using numerical bifurcation analysis, from the single cell level to see whether oscillatory behaviour exists and if such behaviour persists when groups of coupled cells are considered. Subsequently, the model is extended and an explicit spatial dimension is included to study any spatiotemporal effects that can affect the nature of oscillations. The spatial model is implemented in BSim, a novel software platform developed in Bristol, by students of the Bristol Centre for Complexity Sciences, to study bacterial population dynamics in silica. Finally, we study the possibility of controlling the cellular population, an area that has received a lot of attention recently in cellular biology. Specifically, we compare the performance of open and closed loop control in entraining the population to follow a non-native oscillating period. Vve also compare the ability to make the population track a non-oscillating signal. Our results indicate that the oscillatory behaviour seen in the published experiments is an emergent population level property. The onset and synchronisation of oscillations are dependent on cell density and the speed of diffusion of the coupling chemical in the environment medium. Also, the oscillating population is best controlled with closed loop methods.

Toxin-antitoxin (TA) loci, which are widely distributed in prokaryotes, are organized in small operons, consisting of genes encoding toxins and antitoxins. Transcription from the promoter is regulated by the TA protein complex. Activation of the toxin occurs when the toxin is in excess of the antitoxin, for example during nutritional stress when the labile antitoxins are rapidly degraded by cellular proteases. The biological function of TA systems is debated and several different models have been proposed. Several of the TA loci encode messenger RNA interferases (mls) that inhibit translation by cleaving mRNA. Two types are known: those that cleave mRNA codons at the ribosomal A-site and those that cleave any RNA site-specifically. In this work, it was shown that the ml, YoeB cleaved mRNA strictly dependent on translation in vivo. Furthermore, it was shown that site-specific mRNA cleavage by MazF occurs independently of translation but that translation can seriously influence MazF cleavage efficiency. Expression of the toxin Doc of phage P1 was shown to mediate mRNA cleavage, though activation of the endogenous E. coli RelE ml. In a different study, three new ml encoding TA loci of E. coli were identified and characterized. Ectopic expression of the three ml genes lead to a rapid growth arrest caused by inhibition the global rate-of-translation. Furthermore, ml induced degradation of several different model RNAs were observed in vivo. Two of the mls cleaved only translated RNAs, similar to RelE and YoeB, whereas one ml cleaved both in coding and non-coding regions of the RNAs, resembling the properties of MazF and ChpBK toxins. Transcription of the three TA mRNAs was induced by various stressful conditions and dependent on the proteases Lon and Clp. In a different section of this work, a new synthetic lethal screen was developed using antisense RNA regulated R1 plasmids. The screen was used to search for synthetic lethal of sick interactions with the molecule responsible for trans-translation, tmRNA. Two genes were found to be synthetic sick with tmRNA: the tatC gene, which encodes an essential component of the twin- ariginine translocation complex and the dksA gene, which encode a transcription factor responsible for regulation of ribosomal RNA promoters.

PDRP is an unusual bifunctional ADP-dependent phosphotransferase involved in the regulation of C4 plant photosynthesis. Despite thirty years of research since its discovery, biophysical, kinetic, and structural characterisations have remained elusive. The discovery of a homologous protein (YdiA) in E. coli provides an unparalleled opportunity to obtain such characterisations. For the first time, YdiA has been cloned, overexpressed, and the biophysical characteristics have been determined. The protein was found to be dimeric by gel filtration, native-PAGE, and AUC. ITC was used to characterise binding affinity for the putative substrates. The protein binds in a largely temperature and pH-independent manner to ADP, AMP, phosphate, and pyrophosphate, in the uM range, with KdS of \.24 ± 0. 76, 5.23 ± l.l0, 15.54 ± 4.30, and 24.18 ± 2.70 respectively. The putative macromolecular substrate, PPS, was cloned, overexpressed, and characterised. This substrate was found to be dimeric at high concentration by gel filtration, and monomeric at low concentration by native-PAGE. YdiA was shown to interact with PPS in the presence of ATP, by gel filtration. The biophysical data for this interaction could not be obtained. Initial data using peptidcs howcver suggest that YdiA could bind to PtsI. To determine the structure of YdiA, mutagenesis, bioinformatics, and X-ray crystallography were employed; however, it was not possible to determine the structure. Finally, two new coupled assays were developed to obtain a kinetic characterisation of YdiA. This involved the cloning, overexpression, and characterisation of five genes and four proteins.

The mammalian host responds to E. coli by producing an arsenal of oxidative and nitrosative stresses, including peroxynitrite, which is formed by the near diffusion- limited reaction of nitric oxide with superoxide. Peroxynitrite, the main subject of this thesis, is regarded as a highly toxic species due to its multiple targets that include lipids, DNA and proteins; however information regarding specific bacterial targets is lacking. Toxicity of peroxynitrite was found to be dependent upon both O2 availability and numbers of target cells. A sublethal concentration of peroxynitrite was selected to stress E. coli and uncover the transcriptional response under tightly controlled chemostat conditions. Up-regulation of cysteine and arginine biosynthetic pathways was observed in addition to the oxidative stress response and systems involved in iron-sul fur cluster assembly/repair, glutathione import and phosphate transport. Expression of karG, encoding a catalase-peroxidase, was particularly elevated. Consequently. KatG was characterised to determine whether it exhibited the capacity to break down peroxynitrite, an activity attributed to the same enzyme from Mycobacterium tuberculosis. However. KatG was an inefficient peroxynitritase. The S-nitrosoproteome of E. coli generated in response to GSNO was examined. Reductive chemiluminescence was used to quantify S-nitrosothiol formation within cells and to establish the contribution of selected systems in protecting the cell from this post-translational modification. An I7srR mutant was more susceptible to GSNO- mediated S-nitrosation than wild type cells, whereas separate mutations in hmp and ytfl: had negligible effects. A mutation in I7IjA however caused a reduction in 5- nitrosothiol production relative to wild type cells. suggesting that cytochrome c nitrite reductase promotes S-nitrosation. SNO capture (SNOCAP). a proteomic method applied to identify S-nitrosated proteins, was also applied to E. coli cells stressed with GSNO. Targets were generally categorised into those involved in the oxidative stress response, protein biosynthesis and intermediary metabolism. The global impact of NO on metalloproteins in E. coli was investigated by electron paramagnetic resonance. Spectral features corresponding to haern-nitrosyls and iron- sulfur nitrosyls were uncovered. Additionally. this technique was applied to cells stressed with peroxynitrite and revealed a diminished signal corresponding to iron- sulfur centres with a concurrent increase in signal attributable to free iron. This thesis represents the first detailed integrated study of ONOO' toxicity in E. coli.

Many bacteria can express polysaccharide capsules on their cell surface, which may be important virulence factors. Escherichia coli is able to express more than 70 distinct capsular polysaccharides, or K antigens, which are organised into four groups on the basis of biochemical, genetic and regulatory properties. The K5 antigen belongs to group 2 and is expressed by extra-intestinal isolates of E. coli associated with urinary tract infections and septicaemia.