Pseudomonas aeruginosa is an opportunistic bacterium that can infect immunocompromised people, and is especially prevalent in patients with cystic fibrosis. Treatment of P. aeruginosa is complicated due to resistance to many classes of antibiotics. This is partly due to biofilm formation, which is not simply a diffusion barrier, but also has a distinct mechanism for resisting the action of antibiotics. P. aeruginosa quinolone signal (PQS) has an important role in quorum sensing, which is involved in biofilm formation. PqsBC is a condensing enzyme in the biosynthesis of the PQS. The crystal structures of PqsBCC129A and PqsBCC129A-Fe3+ were collected to 2.04 Å and to 2.3 Å, respectively. The crystal structure showed that PqsB and PqsC have a pseudo 2-fold symmetry that mimics the FabH homodimer as well as the presence of a catalytic diad instead of the typical catalytic triad in PqsBC, seen in other FabH family enzymes. The PqsCC129A active site volume is twice the volume as those of FabH enzymes or PqsD, with a calculated volume of 761Å3, compared to 389Å3 for PqsD and 367Å3 for FabH.The PqsBCC129A-Fe3+ crystal structure shows that Fe3+ binds to nitrogen atoms from PqsB His282 and PqsC His2 along with oxygen atoms from PqsB Glu48 and Glu280. Therefore, there are four bonds involved in the interaction between Fe3+ and PqsBCC129A. These bond lengths are very similar to those observed in the structures of Azurin and FutA1 complexed with iron. These crystal structures of PqsBC provide a unique insight into substrate recognition and establish a scaffold for structure guided drug design of novel antimicrobial agents.

This thesis details research in three areas, in attempts to produce more effective inhibitors of the staphylococcal agr quorum sensing system. The non-ribosomal peptide synthase biosynthetic cluster responsible for the production of the aureusimines represents roughly 0.5% of the S. aureus genome. However, their function is yet to be elucidated. Research detailed herein develops a new reliable method for synthesis for these natural products. Efforts to discover the biological target or function of these compounds using affinity chromatography is reported. Further biological investigations revealed that the aureusimines are mild antagonists of the agr system. Weak inhibition of CCL-2 mediated chemotaxis of monocytes and staphylococcal biofilm formation is also observed. However, the main biological function of these natural pyrazinones is yet to be discovered. The staphylococcal bioreporter assays were used to eludicdate the structure-activity relationship of a series of truncated AIP-based antagonists against the AgrC1 receptor. Promising inhibitors are then evaluated against AgrC2, AgrC3 and AgrC4. Several compounds were found to be potent low nanomolar inhibitors across all four agr groups. A bioreporter assay based on the mutant receptor A101T T104V AgrC1 in which (Ala5)AIP1 is an agonist was also used to evaluate the panel of compounds. This revealed that most of these truncated AIP-based compounds are agonists of the mutant receptor, similar to (Ala5)AIP1. However, (Ala2, Leu4, Tfh5)trAIP1 (3.23) effectively inhibited activation of this bioreporter by AIP1. Compound 3.23 was also a sub-nanomolar inhibitor of AgrC1 and a low nanomolar inhibitor in other agr groups. Compound 3.23 is the most potent AgrC1 inhibitor discovered to date and, furthermore, its effects are likely to be less susceptible to mutations within the AgrC receptor. The depsipeptide natural products, solonamides, were synthesised using two uniquely different strategies. Development of a new synthetic strategy produced analogues with a high yield and diastereomeric excess in contrast with previous low yielding or non-stereoselective strategies. Their previously reported inhibition of the agr system was confirmed and fully quantified. Solonamide A and B inhibit activity in all four agr groups. Expression of toxic shock syndrome toxin-1 (TSST-1) and α-hemolysin were reduced in Staphylococcus aureus KH1187A. Schild analysis of data from agr bioreporters revealed that the inhibition is not competitive (as previously reported), but the solonamides act as negative allosteric modulators of both the AgrC1 and AgrC2 receptors, interacting with a new putative conserved allosteric binding site. Weak agonism at high concentrations was also discovered, which has not been previously observed. A panel of analogues was produced to assess the SAR with AgrC1. Modifications of the solonamide scaffold achieved mild improvements of physical characteristics and potency.

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.

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.