Clostridium difficile is the leading cause of nosocomial diarrhoea in the world, and a considerable burden to healthcare services. For colonisation of C. difficile to occur in the gut of an individual, the resident gut flora must first be quantitatively or qualitatively altered, normally through antibiotic treatment for an unrelated infection. At present, broad-spectrum antibiotics such as vancomycin and metronidazole, the frontline choices for the treatment of C. difficile infection; disturb the normal gut flora, suffer from poor recurrence rates, and have received reports of sporadic emergence of resistance. Development of novel narrow-spectrum antimicrobials would circumvent these problems but depend on the identification of novel essential genes. Molecular techniques available to identify and study essential genes in other organisms have not yet been applied to C. difficile. In this study, we identified 208 candidate essential genes via in silico analysis based upon similarity to known Bacillus subtilis essential genes. In order to provide experimental evidence of essentiality, we developed a novel method utilizing partial diploids and functionally characterised three C. difficile genes as essential; CD0274, metK, and trpS. This method not only identified CD0274, a candidate narrow-spectrum drug target and the first essential genes in C. difficile, but also provides a reliable method to identify further essential genes for novel antimicrobial targeting. In addition, we developed a lac repressor system, a rationally designed theophylline-responsive riboswitch, and most importantly, a Tet inducible promoter system able to conditionally control expression of a catP reporter through a wide dynamic range in both C. difficile and C. sporogenes, to maximum induction factors of 192.89 and 1,275.63, respectively. The combinations of these characteristics make this Tet system not only a powerful tool for identifying essential genes, but bestows a great potential for further analysing gene function far beyond the scope of this project.

Clostridium acetobutylicum is an industrially important Gram positive organism which is capable of producing economically important chemicals in the Acetone, Butanol and Ethanol (ABE) process. An orthologue of the accessory gene regulator (agr) locus of Staphylococcus aureus has been found to be present in the genome of Clostridium acetobutylicum. In S.aureus, agr encodes a quorum sensing (QS) system that controls the expression of virulence in this species. Analysis of the agr region in C.acetobutylicum was conducted using reverse transcriptase PCR which showed the agrB and agrD genes to be linked. This was also the case with the agrC and agrA genes but there was no conclusive evidence to suggest that all 4 genes resided on the same operon. The use of cat-based reporter vectors which incorporate chloramphenicol acetyl transferase were used to look at the expression profiles of the agrB and agrC putative operons. The agrC construct showed activity consistent with the expected pattern of expression but this was not repeated with the agrB construct. Antisense RNA vectors were constructed with the intention of disrupting the agr genes but had no observable effect. This work was superseded by a newly available method to knockout the agrA gene by allelic exchange and the use of the ClosTron system to obtain gene inactivation in agrB, agrC and agrA. Gas chromatography analysis of these mutants showed little or no difference in product formation and a sporulation assay was developed which revealed that these mutants were inhibited in spore production. Finally, microarray analysis has been used to look at the effect of agrB inactivation on the gene expression of C.acetobutylicum. The expression of known sporulation genes was found to be differentially regulated. This study presents some of the first evidence to support the hypothesis that agr may be a major regulator in C.acetobutylicum and may act in a cell density dependent manner via a diffusible signal molecule.

C. acetobutylicum A TCC 824 is a well characterized microorganism known for its ability to produce solvents using the Acetone-Butanol-Ethanol (AB E) fermentation process. It can utilize a variety of Cs and C6 sugars, but cannot directly access the complex lignocellulose plant cell wall material which is the most abundant source of carbon in nature. Sophisticated genetic tools are required to enhance the substrate utilisation ability of the organism by incorporating synthetic operons using a synthetic biology approach. Efficient tools were developed for making precise alterations to the C. acetobutylicum genome using either heterologous pyrE or codA genes as counterselection markers. In the case of the former, the utility of the method was also demonstrated in Clostridium difficile. The robustness and reliability of the methods were demonstrated through the creation of in-frame deletions in two genes (spoOA, amylase) using pyrE and also two genes (Cac1502 and Cac2071 (spoOA) using codA. The pyrE system is reliant on the initial creation of a pyre deletion mutant using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyre allele from C. sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a pyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE strains attractive hosts for mutagenesis studies.

Antibiotic-associated diarrhoea (AAD) and colitis, with the causative agent being the Gram-positive anaerobe, Clostridium difficile, are some of the most important hospital-acquired infections and significant burdens to healthcare services worldwide. Treatment of the infection is often ineffective and currently no vaccine is available against C. difficile infection (CDI). Research to identify novel virulence factors potentially leads to the development of new therapeutic and prophylactic drugs. As lipoproteins have been shown to play key roles in the virulence of several pathogens, the aim of this project was to investigate whether lipoproteins are involved in the virulence of C. difficile. Lipoproteins are anchored to the extracellular side of the cytoplasmic membrane in Gram-positive bacteria. Two enzymes are involved in the biosynthesis of lipoproteins: lipoprotein diacylglycerol transferase (Lgt) attaches lipoproteins to the membrane, and lipoprotein signal peptidase (Lsp) cleaves the signal peptide from the amino-terminus of lipoproteins. In order to study lipoprotein processing in C. difficile, lgt and lsp mutants of the C. difficie 630Δerm strain were generated using the ClosTron system. Antibody reactivity of 14 C. difficile lipoproteins was also investigated. It was shown in this study that lgt mutation caused changes in the lipoproteome of C. difficile. Therefore, inactivation of the lgt gene allowed investigation of the global contribution of lipoproteins to bacterial processes. The physiology and virulence of the lgt mutant was studied in vitro and in vivo. Surprisingly, many of the assayed phenotypes were not significantly affected by disruption of the lgt gene. Nevertheless, the ability of the lgt mutant to adhere to Caco-2 cells was markedly reduced. In addition, the phenotype of the lgt mutant observed in mice suggests that the faecal shedding of C. difficile is affected by Lgt inactivation. In further studies, the CD0873 lipoprotein as a potential adhesin of C. difficile was identified by in silico approach. Contribution of the CD0873 lipoprotein to the adherence of C. difficle was investigated by several different assays and the results strongly suggest that the CD0873 lipoprotein is directly involved in adhesion

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

Au cours des dernières décennies, la fermentation IBE (isopropanol, butanol and éthanol) a connu un regain d'intérêt en vue de la production de carburants ou de composés chimiques à partir de matériaux renouvelables. Dans cette étude, nous avons étudié la production d'IBE avec le producteur naturel Clostridium beijerinckii NRRL B593 et avec des souches modifiées de Clostridium. acetobutylicum ATCC 824. En culture discontinue, la souche C. beijerinckii NRRL B593 excrétait 13.2 g/L d'IBE (dont 4,5 g/L d'isopropanol). Afin d'améliorer la production d'IBE, le gène codant pour l'alcool déshydrogénase secondaire (s-Adh) de NRRL B593, ainsi que différentes combinaisons des gènes des enzymes actives de la conversion de l'acétoacétyl-CoA en acétone, c.-à-d. l'acétoacétyl-CoA acétate/butyrate transférase (ctfA et ctfB) et l'acétoacétate décarboxylase (adc), ont été exprimées dans la souche productrice d'ABE (acétone, butanol éthanol), C. acetobutylicum ATCC 824. Les résultats montrent que la sur-expression des gènes ctfA et ctfB augmentait significativement la productivité et les concentrations finales en IBE tandis que la surexpression du gène adc n'avait qu'un effet limité. Cultivée en discontinu, la meilleure souche, exprimant les gènes adh, ctfA, ctfB et adc a produit 24.4 g/L d'IBE dont 8.8 g/L d'isopropanol avec une productivité de 0.7 g/L h. Cultivée en mode continu à un taux de dilution de 0.1 h-1, la productivité en IBE a été portée à 1.7 g/L h. Puisque le mélange IBE est considéré comme un additif carburant de qualité, les transformants obtenus constituent une avancée réelle vers le développement d'un procédé IBE industriel de production de biocarburants. / Over the past decades, the IBE fermentation (isopropanol, butanol and ethanol) has received a renew interest for the production of fuels or biochemicals from renewable materials. In the present study, we have investigated the IBE fermentation using the natural producer C. beijerinckii NRRL B593 and genetically-modified strains of Clostridium acetobutylicum ATCC 824. In batch culture, C. beijerinckii NRRL B593 was found to excrete 13.2 g/L IBE of which 4.5 g/L was isopropanol. To increase IBE production, the gene coding the secondary alcohol dehydrogenase (s-Adh) of C beijerinckii NRRL B593 and different combinations of genes coding for enzymes active in acetoacetyl-CoA to acetone conversion i.e. acetoacetate decarboxylase (adc) and acetoacetyl-CoA: acetate/butyrate: CoA transferase subunits A and B (ctfA and ctfB) were expressed in the ABE (acetone, butanol ethanol) producer C. acetobutylicum ATCC 824. Results showed that the overexpression of the ctfA and ctfB genes significantly increased both speed and extent of the IBE production while the overexpression of the adc gene had only a little effect. In batch culture, the best mutant (expressing adh, ctfA, ctfB and adc) produced 24.4 g/L IBE (of which 8.8 g/L was isopropanol) and displayed an IBE productivity of 0.7 g/L h. Cultivated in continuous mode at the dilution rate of 0.1 h-1, IBE productivity was increased to 1.7 g/L h IBE. As the IBE mix has been considered as a valuable fuel additive, the transformants obtained are a real step forward towards the development of an industrial IBE process for biofuel production.