The acetogenic bacterium Clostridium autoethanogenum possesses the inherent ability to produce acetate and ethanol during growth on industrial waste gases such as carbon monoxide and carbon dioxide using the Wood-Ljungdahl Pathway (WLP) for carbon fixation. With the urgent need to reduce greenhouse gas emissions and produce chemicals and fuels in a more sustainable manner, autotrophic organisms such as C. autoethanogenum have received considerable industrial interest over recent years. However, the metabolic pathways present in C. autoethanogenum and therefore its capability to produce industrial relevant carbon building-blocks and biofuels have not yet been sufficiently examined. Therefore, the investigation of pathways leading to the production of industrially relevant carbon building blocks is seen as a worthwhile undertaking at the interface of fundamental and applied research. In this project, the metabolism of C4 dicarboxylic acids in C. autoethanogenum in conjunction with the production of succinate was investigated. This analysis revealed a previously unrecognised carbon and energy source, fumarate, and unveiled the combination of the autotrophic WLP with reactions of the branched tricarboxylic acid (TCA), Krebs “cycle” using in vivo NMR techniques. Under the conditions employed, the reducing equivalents gained from the oxidative breakdown of fumarate to acetate were used to partially re-assimilate the CO2 that was liberated during that process. Accordingly, inactivation of the fumarate hydratase led to a disruption of fumarate metabolism. Additionally, through the introduction of a fumarate reductase and its overexpression in the organism, the resulting strain was able to produce succinate in amounts of up to 3.54 g l-1 and yields of up to 0.78 g g-1 fumarate. This study therefore presents an essential basis for the possible establishment of succinate production with C. autoethanogenum and a better understanding of its C4 dicarboxylic acid metabolism.

Clostridium acetobutylicum is renowned for the ability to convert sugars into acids and solvents, including the potential biofuel butanol. However, regulation of its fermentation metabolism remains poorly understood, especially regarding the shift from acid to solvent metabolism. Several RNPP-type cell-cell communication systems have recently been discovered within C. acetobutylicum ATCC 824. The aim of this study was to investigate one such system, Quorum Sensing System B (QssB), consisting of a regulator, QsrB, and a signalling peptide of unknown structure derived from a precursor protein, QspB. The primary objectives were to characterise this system in greater detail, its effects on metabolism and endospore formation, and to gain insights into the nature of the signalling process. Overexpression of qsrB confirmed a dramatic reduction of solvent production and sporulation, and revealed increased production of acetate and butyrate, with considerably decreased consumption of glucose. These phenotypes were overcome by the addition of synthetically produced peptides matching the C-terminal region of the conjugate signalling peptide QspB. In doing so, the minimum peptide sequence required to relieve the effects of qsrB overexpression was shown to be the heptamer AEPTWGW. The system was shown to be highly selective for this sequence, with an alanine scan showing the sequential reduction in biological activity. QssB did not engage in cross talk with the other RNPP-type systems present within the organism, or with a homologous system discovered in Clostridium roseum, further evidence of selectivity. Attempts were made to identify the Specific Binding Protein (SBP) required for QspB uptake via ABC transport. ClosTron mutation of two SBP encoding genes, CA_0176 and CA_C3632, conferred resistance to the effects of QsrB overproduction on sporulation. An iTRAQ-based proteomics approach was used to study the wider effects of qsrB overexpression, as well as their alleviation via synthetic peptide. This analysis confirmed that the qsrB overexpressing strain remained in an acidogenic state which could be overcome though peptide addition. Although glucose consumption was decreased upon QsrB overproduction, key glycolysis enzymes were found to be upregulated. The stationary phase regulator CA_C0957 was found to be downregulated under these conditions, whereas proteins associated with motility and chemotaxis increased in abundance. GusA reporter assays supported the former, whilst swimming and swarming assays remained inconclusive. Enzyme assays revealed increased acetate and butyrate kinase activity, agreeing with the acidogenic state suggested from the iTRAQ analysis. QsrB was confirmed to be a repressor of solvent production and the production of endospores, with new evidence indicating further complexity.

Clostridium difficile is an anaerobic, Gram-positive, endospore forming bacillus and is the leading cause of nosocomial infection. Symptoms range from mild diarrhoea to the potentially fatal intestinal complications pseudomembranous colitis and toxic megacolon. The prerequisite for C. difficile infection (CDI) is the perturbation of the healthy microbiota of the gut by broad spectrum antibiotics. It is therefore important to develop therapies which take this in to account, either by minimal disruption of the resident gut microbiota, or by reinstating the protective nature of the gut microbiota. The novel antimicrobial fidaxomicin (FDX) is the first in a new class of macrocyclic antibiotics, with a narrow spectrum of activity for C. difficile. FDX exerts its bactericidal activity by binding to RNA polymerase (RNAP) and inhibiting transcription. The minimum inhibitory concentrations were determined for six clinically relevant isolates of C. difficile and the effect of the drug on spore germination and outgrowth was assessed. Inhibition of C. difficile occurs at low concentrations (0.03 – 0.05 μg/mL) and it was found that FDX does not inhibit the initiation of spore germination, but effectively halts outgrowth at an early stage. The effect of mutations in the β subunit of RNAP were also investigated in terms of susceptibility to FDX and any potential fitness costs incurred to the bacterium. Three separate single nucleotide polymorphisms (SNPs), T3428A, T3428G, G3427T, in the rpoB gene were found to confer reduced susceptibilities to FDX. However, the clinical relevance of these mutations is unclear, as mutants appeared to be attenuated in terms of growth, toxin production and virulence in the hamster model of infection. Clostridium scindens is a member of the healthy gut microbiota and is thought to be a key organism in providing colonisation resistance against C. difficile. C. scindens is the most active transformer of primary bile acids to secondary bile acids, known to inhibit the growth of C. difficile. This occurs due to the gene products of the bile acid inducible operon and a presently unknown reductive arm of the pathway. An RNA extraction protocol for high quality total RNA from C. scindens was developed to aid in the study of the transcriptome of C. scindens cultured with the primary bile acid, cholic acid (CA). This has enabled the identification of potential gene candidates for the reductive arm of the bile transformative pathway of C. scindens. To further study these potential genes, the ability to transfer DNA in to C. scindens is desirable in order to create gene knock outs. The genome of C. scindens ATCC 35704 was assembled and annotated and used to identify potential barriers to DNA transfer. The methylation pattern of this strain identified two type I, twelve type II and one type IV restriction methylation (RM) systems. RM systems were further characterised in an effort to circumvent the RM system barrier to DNA transfer.

El objeto de este trabajo es la vida y la obra de la artista americana Mary Cassatt, adscrita al movimiento impresionista y poco conocida en nuestro país, centrándonos en dos aspectos principalmente: su condición de mujer moderna y su larga época como pintora de retratos de mujeres con niños. Así, repasaremos algunos de los mitos de la era moderna, pasearemos por el París de finales del siglo XIX y debatiremos sobre la condición femenina en las postrimerías de dicho siglo, lo que nos llevará a cuestionarnos las ideas de libertad defendidas y difundidas por la revolución francesa, cuyas asimetrías y falacias continúan afectando aún hoy a nuestra sociedad occidental. Mary Cassatt fue una mujer luchadora que combatió con singular inteligencia y habilidad las convenciones de su siglo. Sus retratos muestran acertadamente la psicología de la dama burguesa, pero también van un paso más allá; en este trabajo hablaremos de cómo Cassatt pasó de plasmar las escenas cotidianas de los rituales burgueses que tanto atraían a los impresionistas a convertirse en pintora por excelencia de la maternidad, y cómo este cambio fue y sigue siendo malinterpretado, no solo por la crítica masculina, sino, muy especialmente, por la feminista. Trataremos de explicar cómo una mujer fuerte y trabajadora, y, tal vez paradójicamente, soltera y sin descendencia, pudo defender los derechos y la dignidad de su género retratando un tema tan manido y edulcorado, y sin embargo tan hermoso y preñado de posibilidades como las relaciones materno-filiales. Este trabajo tratará pues de unir los aspectos humanos y artísticos de una gran pintora, y, a la vez, relacionarlos con la época que le tocó vivir, como un todo inextricable. Creemos que solo así podemos entender lo comprometido de su postura, lo valiente de sus decisiones y el objeto de su obra; Mary Cassatt trató de cambiar su mundo mediante la elegancia de su arte: sus armas fueron su inteligencia y su talento, y con ellas logró hacerse respetar en un mundo de hombres, y, quizás, contribuir a un tímido cambio hacia una sociedad más justa, cambio que aún no ha terminado de hacerse efectivo en nuestros días. Intentaremos explicar cómo lo logró, en qué triunfó y en qué fracasó y el porqué de que su obra no goce hoy en día de la popularidad que merece. / This thesis is about the life and work of the American artist Mary Cassatt, who was a member of the Impressionist movement, but whose paintings are poorly known in our country; we focus in two main aspects: her condition as a modern woman and her long period as a portrait painter of women with children. With these themes in mind, we will examine some of the modern-age myths, walking through the streets of Paris as they were at the end of the XIXth century, and we will debate about the turn-of-the-century concept of femininity, which will lead us to question the idea of freedom defended and disseminated by the French Revolution: its asymmetries and fallacies are still affecting nowadays our western civilization. Mary Cassatt was a combative woman who fought against her century’s conventions with unusual ingenuity and skill. Her portraits reflect cleverly the bourgueois woman’s psychology, but they also go beyond that; in this thesis we will talk about how Cassatt made the transition from capturing the everyday scenes that fascinated her Impressionist colleagues so much, to become the quintessential painter of motherhood, and how this change was, and still is, much maligned and misunderstood, not only by regular critics, but also, and specially, by feminist ones. We will try to explain how this strong, hard-working woman, and one who was, perhaps paradoxically, single and without children, could become the staunch defender of her genre’s rights and dignity devoting herself to such a trite and watered-down – but also so beautiful and full of possibilities in the proper hands – topic. So in this thesis we will try to combine the humane and artistic aspects of a great painter, and, at the same time, relate them to her day and age, as an inextricable blend. We believe that this is the only way to understand her commitment and braveness, her decisions and the purpose of her work; Mary Cassatt tried to change her world using the elegance of her art: her weapons were intellect and talent, and with them she achieved respect in a world ruled by men, and, perhaps, she contributed to a timid advance towards a more unbiased society, a change which is still in progress during our days. We will try, in essence, to explain how she managed to do that, her triumphs and defeats, and the reasons why her work doesn’t have today the popularity it deserves.

Due to the recent persistence of Listeria monocytogenes in food factory environments and an increase in outbreaks of Listeriosis, in particular some associated with duck meat products, an investigation was carried out to establish the potential effects of different growth conditions on the hydrophobicity of L. monocytogenes cells and to determine the behaviour of the cells in a minimal nutrient environment. It was found that duck meat extracts increased growth rate but did not alter the surface charge of the cells and when grown in minimal 010 and MCDB202 media the cells flocculated and showed more hydrophobicity than when grown in the rich media BHI. The modified surface of the organism behaved like an emulsifier and this led to the discovery for the first time, that there was formation of capsular exopolymeric substances (EPS) on the surface of planktonic cells of L. monocytogenes. After confirmation of the capsular EPS by two capsule stains, namely Nigrosin and Giemsa, the EPS was purified and proved to play a role in holding the cells together. It was also found to absorb water rapidly and can retain water for long periods suggesting that the EPS can contribute to the desiccation tolerance of L. monocytogenes cells embedded in a biofilm matrix. Chemical characterization of the EPS showed high levels of glycerol and phosphate indicating that the EPS is amphiphatic and may contain mainly glycerolphosphates.

Listeria monocytogenes is a common food borne pathogen which is an important contaminant found in various food factory environments, because of its ability to survive in a wide range of environmental conditions, and to grow at refrigeration temperatures. L. monocytogenes has caused both occasional outbreaks and sporadic cases of food-borne illness characterised by high mortality rates. In the UK and other European countries, there has been a conspicuous rise in the number of reported cases of “Listeriosis” recently. Hence, development of efficient and rapid methods for detection of this microorganism in various foods is of great significance for the food industry; and is needed to ensure the safety of foods that are considered at high risk of contamination. Conventional bacteriological methods (e.g. ISO 11290-1/A1) for the detection and quantification of L. monocytogenes are laborious and time consuming. Therefore, development of a rapid and reliable test capable of detecting very low numbers of the organism in ready-to-eat products is required. To address this, a phage amplification assay has been developed as a rapid method for the detection of L. monocytogenes using the broad host range phage A511. Successful development of the assay required identification of a virucide that could achieve inactivation of the phage without affecting the viability of the target cell to be detected. Several different substances were evaluated as potential virucides, and among the tested materials, tea infusions were found to be the most effective virucidal agent for this experiment. The efficacy of the new assay was tested using Stilton cheese, as a representative high risk dairy product, and a method was developed to use centrifugation to concentrate bacterial cells present in samples of half-Fraser broth enrichments. The cells were detected by using the new phage amplification assay and this combination of techniques was shown to be able to detect low numbers of cells in shorter times than can be achieved using conventional culture methods. An additional molecular identification step was also developed so that the identity of the cells detected could be confirmed using a multiplex PCR which targeted conserved regions of the Listeria 16S rDNA genes. In this assay, two amplified DNA fragments were generated confirming the presence of Listeria genus (400 bp band) and also L. monocytogenes species (287 bp band). An advantage of this combined phage-PCR method its ability to detect only viable cells in food samples. The combined assay was then tested on a wide range of spiked food samples, including Camembert cheese, pasteurised milk, minced meat, turkey meat and smoked salmon. The obtained results showed that the limit of detection was as low as 20 (± 5) cfu per 25 g, and duration needed for the detection and molecular conformation of speciation was 2 days (44 h), compared to 5 days using conventional culture methods. The combined phage-PCR assay was able to achieve a sensitive and specific identification of viable L. monocytogenes present in foods within 48 h, and therefore would allow for rapid screening of food products prior to release from the factory.

The synthetic polymers reported in this thesis are able to bind the small molecule autoinducer-2 (AI-2) in the Quorum Sense (QS) pathways of the marine organism with high affinity, and some of the polymers are also able to sequester rapidly the same bacteria from suspension. Specifically, the Alizarin Red S (AR-S) assay was used to compare binding interactions of boric and boronic acid with diol species, and interactions were further probed by 11B-NMR spectroscopy and Mass spectrometry. Dopamine was considered as a potential AI-2 scavenger for polymeric QS control owing to the high binding affinities for boron. Therefore, poly{N-(3,4-dihydroxyphenethyl) methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide} [p(DMAm-c-DMAPMAm)] and poly(3,4-dihydroxy-L-phenylalanine methacrylamide) [p(L-DMAm)] were prepared via Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization and characterized by 1H-NMR spectroscopy. The activities of these catechol polymers and carbohydrate-based poly(β-D-glucosyloxyethyl methacrylate) (p(GlcEMA)) in QS interference was demonstrated by bioluminescence assays with the Vibrio harveyi MM32 strain and by bacterial aggregation experiments. Polymersomes were then investigated as artificial protocells, with a view to establishing polymer vesicle containers as both reservoirs of QS mediated molecules, and of binding QS agents and bacteria. Hydrophobic monomers N-(2-Ethylhexyl) acrylamide [p(2-EHAm)] and N-phenylacrylamide [p(PAm)] were therefore polymerized into block copolymers from p(L-DMAm)-RAFT agents. The membrane permeability of polymersomes was measured via encapsulation and release of dyes, while the morphologies were examined with Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Polymersomes were also investigated for potency in QS quenching via the bioluminescence assay and bacterial aggregation experiments. Initial studies of a communication feedback loop between bacteria and polymersome-encapsulated QS agents were performed again via bioluminescence assays. The results reveal that the investigated polymersomes exhibit potent activities in QS quenching, and further development might act as components of a synthetic biology approach to combating microbial pathogenicity.

Clostridium difficile is the leading cause of hospital acquired diarrhoea and a major burden to healthcare services worldwide. Endospore production plays a pivotal role in infection and disease transmission, but in order to cause disease these spores must germinate and return to vegetative cell growth. Therefore, knowledge of spore germination is important and may have direct applications in future disease prevention. Germination has been well studied in Bacillus and in some clostridia, but the mechanisms of C. difficile spore germination remain unclear. Apparent homologues of genes important for germination in other spore formers have been identified in the C. difficile genome and ClosTron technology was used to inactivate homologues of sleC, cspA, cspB and cspC (Clostridium perfringens) and cwlJ, sleB and cwlD (Bacillus subtilis) in both C. difficile 630Δerm and a BI/NAP1/027 isolate (a ‘hypervirulent’ type associated with outbreaks of increased disease severity). Using a combination of several different assays to study these mutants in detail, a number of the identified target genes appear to be essential for germination and outgrowth of C. difficile spores. This is the first report of using reverse genetics to study the germination of C. difficile spores and the first gene characterisation by mutagenesis in a BI/NAP1/027 isolate of C. difficile. Furthermore, this study uncovered evidence of significant variation in the sporulation and germination characteristics of different C. difficile strains, but this variation did not appear to be type-associated.

The important human pathogen Pseudomonas aeruginosa causes a broad-spectrum of diseases including life threatening infections. A cell density-dependent regulatory network termed quorum sensing (QS) is pivotal in the control of P. aeruginosa pathogenicity, and the signal molecules employed are N-acyl-L-homoserine lactones (AHLs) and the Pseudomonas quinolone signal (PQS). Production of these QS signal molecules (QSSMs) requires precursors including fatty acids, S-adenosyl-L-methionine (SAM) and aromatic amino acids. SAM is derived from the activated methyl cycle (AMC) which is an important pathway dedicated to the degradation of the toxic metabolite S-adenosyl-L-homocysteine (SAH). Through removing the genes encoding the AHL synthases, RhlI and LasI from the complex hierarchical system of P. aeruginosa by expressing them in the heterologous host, Escherichia coli, this study has measured the influence of AHL production upon bacterial metabolism. AHL profiles were broader than previously reported, correlated with a reduction in the intracellular concentrations of several metabolites, and were more pronounced in the E. coli strain producing the LasI synthase than the RhlI enzyme. Production of foreign QSSM synthases had a knock-on effect on the native E. coli QSSM, autoinducer-2 (AI-2). We hypothesize that AI-2 production was significantly reduced since it also requires AMC metabolites for its synthesis. The influence that these metabolic perturbations had on cell fitness was manifest through reduced growth in minimal media. Complementation of growth by exogenously added metabolites confirmed our hypothesis that QSSM synthesis creates a drain on metabolite levels with consequences for cell fitness. Site-directed mutagenesis of key catalytic residues in the QSSM synthases was performed to directly prove that the effects observed were due to the function of the synthases, and not the production of a heterologous protein. Moreover, complete profiling of P. aeruginosa PA01 AHL synthase mutants is unravelling the interrelationship between metabolism and cell-to-cell communication in P. aeruginosa.

This project has focused on the relationship between the green seaweed Ulva, commonly found in the intertidal zone of the UK coastline and its cognate bacterial community. It has previously been reported that motile Ulva zoospores are attracted to N-Acylhomoserine lactones (AHLs), signalling molecules utilised by Gram-negative bacteria in a density dependent form of cellular communication termed quorum sensing (QS) and produced by several biofilm dwelling species of marine bacteria. The species represented in the bacterial community associated with Ulva spp. were identified by generating a 16S rDNA phylogenetic clone library from bacterial DNA isolated from the surface of the seaweed. These data revealed that the majority of the population belonged to the Proteobacteria or Bacteroidetes phyla. In order to investigate whether QS signalling affected the rate of zoospore germination in addition to zoospore attraction, Ulva zoospores were settled and allowed to grow on synthetic AHLs, biofilms derived from AHL-producing model organisms and strains relevant to the Ulva epiphytic population which were shown to produce AHLs. Results from these experiments revealed that AHLs affected zoospore germination and the early growth of the Ulva germling as zoospores germinated and grown in the absence of AHLs were significantly longer than those germinated in the presence of AHLs. We therefore hypothesise that reduced germling growth in the presence of AHLs allows Ulva to obtain a healthy epiphytic bacterial community that is vital for the seaweed’s later development. Further understanding of Ulva growth biology could have potential applications in preventing marine biofouling by this genus of seaweed. This study progressed to characterise AHL production in a number of strains of Shewanella and Bacteroidetes bacteria, which, for differing reasons were deemed relevant to Ulva biology. Although data presented by this thesis showed AHL production in these bacterial groups, AHL synthase and response regulator sequences could not be identified in the published genome sequences from either Shewanella or the Bacteroidetes. This study also identified an AHL inactivating acylase enzyme in an environmental Shewanella isolate. This acylase, AacS, was shown to degrade a variety of synthetic AHLs and the AHLs produced by Yersinia pseudotuberculosis. This study has therefore increased the range of marine bacteria known to be producing AHLs, however the lack of AHL synthase and response regulator genes in the genomes of these bacteria leads to the conclusion that many marine bacteria possess novel, yet to be characterised AHL-mediated QS systems. Finally, this study screened a number of extracts from marine microalgae for compounds that act as agonists or antagonists to AHL-mediated QS. Although no AHL mimics were identified data presented by this thesis showed extracts to affect the luminescence produced in lux-based AHL bio-reporters in the presence of exogenously added signal, affect a number of QS regulated phenotypes in marine pathogens and effect QS regulated genes in the human pathogen Pseudomonas aeruginosa. As such, we hypothesise that these microalgae have the ability to produce quorum-quenching compound(s). Further characterisation of quorum-quenching compound(s) produced by microalgae may be beneficial in the bio-control of pathogenic bacteria in aquaculture and may act as candidates for novel antibiotics.