Pseudomonas aeruginosa synthesises heterogeneous mixtures of mono and di-rhamnolipid (RL) biosurfactants. The complex genetic and metabolic regulation surrounding RL biosynthesis is one of the major hurdles for the large scale production of these metabolites. To date there is still a lack of quantitative understanding of the interaction between the metabolic and regulatory networks governing RL biosynthesis in P. aeruginosa. The initial part of the thesis details the first systematic validation of reference gene stability under nutrient limiting conditions for the development of a reliable and accurate RT-qPCR method specific for RL biosynthetic gene expression. A specific UPLC-MSMS method was simultaneously developed and validated for the quantitative determination of RL yield and congener distribution in P. aerllginosa. This is the first report of individual RL quantification using UPLC-MSMS. The second part of the thesis investigated the effect of hydrophilic and hydrophobic carbon substrates on RL production. Consistent with such a high level of regulatory complexity for P. aerllginosa, transcriptional analysis of the RL biosynthetic genes revealed a highly conserved gene expression profile. RL biosynthetic genes rhlAB (responsible for mono-rhamnolipid synthesis) were not induced until early stationary phase when growth had significantly slowed down. Expression of rhlC (responsible for di-rhamnolipid synthesis) was induced during later-stationary phase after maximum up-regulation of rhlAB expression. Choice of carbon substrate for RL production significantly influenced the expression level and timing of RL biosynthetic genes, with hydrophobic substrates inducing the highest overall up-regulation of RL genes. UPLC-MSMS revealed a conserved RL congener distribution , mono-RLs were more abundant during early-stationary phase followed by a predominance of di-rhamnolipids accumulating during mid to late stationary phases. RL composition was regulated by the selective incorporation of CIO ~-hydroxy fatty acids into RLs with Rha-Rha-C1o-C IO and RhaC10- C IO the most abundant congeners produced irrespective of carbon substrate metabolised. UPLC-MSMS also showed that inactivation of rhlC led to the production of mono-RL only congeners in yields and composition comparable to wild-type P. aeruginosa. The data suggests that expression of rhlA B is not only cell density regulated but also stringently regulated by nutritional cues (i.e. carbon source and macronutrient limitation). Thus de novo RL biosynthesis is prudently regulated at the transcriptional level, the regulation of rhlAB is the rate limiting step which dictates the extracellular RL profile. From a biotechnological point of view, this prudent metabolic regulation may explain why RLs are synthesised in such relative low yields and is perhaps a mechanism by which Pseudomonas aerllginosa has evolved to adapt to nutrient limiting environments. This thesis presents a systems metabolic engineering approach for quantitatively assessing RL production under nutrient limiting conditions

Abstract. Recent studies have indicated that macrolide antibiotics, in doses well below their minimum inhibitory concentration (MIC), may have a specific effect on quorum regulated systems in Gram negative bacteria. Vibrio fischeri was used in this study as a paradigm to observe the effects of sub-MIC erythromycin on quorum sensing. Concentrations of 1/100th of the MIC inhibited bioluminescence but did not affect growth in V. fischeri. Reduction of bioluminescence was reversible with the addition of exogenous acyl-HSL. To elucidate the effects of macrolides on this quorum regulated system, reverse-transcriptase PCR was used to compare transcription of luxD, luxR and a 'housekeeping gene', 16s rRNA, when grown with sub-MIC macrolides with/without acyl-HSL.The luxR and 16s rRNA genes were unaffected but transcription of luxD was reduced by sub-MIC erythromycin, suggesting that sub-MIC erythromycin affected genes under quorum sensing control. As both acyl-HSL and erythromycin contain a lactone ring structure, competitive inhibition could explain these results. An Escherichia coli strain that overproduced LuxR protein was used to investigate whether acyl-HSL and macrolide antibiotic competed for a site on or near to LuxR. Radiolabelled acyl-HSL was produced by adding tritiated methionine to growing cultures of V. fischeri. Radiolabelled acyl-HSL bound to the LuxR protein when compared to the control, supporting the hypothesis that macrolides may block binding of acyl-HSL to LuxR transcriptional activator. Clinical strains of P. aeruginosa were transformed with luxCDABE genes under a constitutive promoter and used to investigate the effect of sub-MIC macrolides on growth and light output. In contrast to results obtained from V. fischeri, where the lux genes are quorum regulated, sub-MIC macrolides did not have a differential effect, indicating that the low dose macrolide results observed in V. fischeri are due to an effect on regulation of the lux genes. Sub-MIC macrolides have been reported to decrease virulence production in P. aeruginosa; to investigate their in-vivo effects a Caenorhabditis elegans P. aeruginosa model was developed. Worms fed on a clinical isolate of P. aeruginosa were killed in 24 hours and this lethal effect was reversed in the presence of 0.4 mg/L erythromycin. Further experiments with a selfbioluminescent construct of the clinical P. aeruginosa isolate demonstrated that 0.4 mgll erythromycin did not alter bacterial viability withilJ worms, whilst clearly inhibiting virulence factor production. The results of this study demonstrate that sub-MIC erythromycin affects quorum regulated systems in V. fischeri and P. aeruginosa and indicate a possible competitive inhibition effect between sub-MIC erythromycin and acyl-HSL.

The bacterium Campylobacter jejuni is the most common cause of food borne disease in the UK, causing a 5-7 day enteritis including profuse watery diarrhoea, abdominal pain, fever, headache and occasionally vomiting. In rare cases leading to the paralysing autoimmune disease, Guillain-Barré syndrome. C. jejuni are highly motile cells, propelled through the environment by flagella, their motility is directed through a behaviour called chemotaxis. Cells are able to detect attractants or repellents and reposition the cell accordingly. Chemotaxis is central to C. jejuni colonisation as non-motile and non-chemotactic mutant strains poorly colonise their usual hosts. In Escherichia coli chemotaxis is regulated by the Che proteins which form a two component phospho relay system. In previous studies In silico comparison of E. coli Che proteins identified homologues in C. jejuni, which display altered chemotactic phenotypes in Δche mutant strains. Studies of interactions between the Che proteins using bacterial and yeast two hybrid systems, suggested ways in which the homologues may interact, but to further discern these mechanisms required in vitro study. For the purpose of this study the C. jejuni Che homologues were cloned, expressed and purified, for use in in vitro experiments. Radiolabelled Phosphotransfer assays confirmed CheA as a histidine kinase, and demonstrated Pi transfer to the response regulators of CheY, CheV and CheA, in that order of preference. Affinity tag pull-down assays found the predicted decrease in affinity between phosphorylated CheY and CheA, but also an increase in the affinity of phosphorylated CheV for the receptor, TLP[subscript 1]. The results of this study confirm the two component backbone of the C. jejuni Che model, and suggest how CheV may regulate methylation adaption in a system devoid of a CheB response regulator.

The major food borne pathogen Campylobacter jejuni utilizes chemotaxis to colonise the chicken gastrointestinal tract. Similar to the Escherichia coli chemotaxis system, C. jejuni produces a CheA-CheW-CheY backbone that transduces signals from surface chemoreceptors to direct flagellar rotational direction. In contrast to E. coli, C. jejuni also expresses CheV and CheA has a response regulator domain. The phosphorylation level of CheY in E. coli is modulated by the phosphatase CheZ, however until recently C. jejuni was thought to lack a CheZ homologue. The Hp0170 protein of Helicobacter pylori was shown to be a CheZ remote homologue. As C. jejuni Cj0700 has homology with HP0170, it was postulated that Cj0700 acts as a CheZ orthologue in campylobacters. The aim of this project was to characterise the role of Cj0700 in C. jejuni chemotaxis and demonstrate the phosphatase activity of the protein on C. jejuni CheY, CheV and the response regulator domain of CheA (CheA-RR). A mutant (Δcj0700) and the cognate complement (Δcj0700, Cj0046::cj0700) were constructed in C. jejuni NCTC 11168. No growth differences were seen between Δcj0700, ΔcheY and wild-type. On semi-solid agar the Δcj0700 mutant strain showed reduced motility relative to the wild-type and this phenotype was reversed in the complemented strain. The effect of a mutation in cj0700 was also demonstrated in C. jejuni strains 81-176 and 81116. Cj0700 was expressed as a His-tagged protein and was found to dephosphorylate C. jejuni CheY protein. Expressed Cj0700 also dephosphorylated CheA-RR-P and CheV-P, but less efficiently than CheY-P. The ability of Cj0700 to interact with CheY, CheA-RR and CheV was investigated using an in vitro pull down assay and also in a two hybrid system in E. coli. Using these approaches Cj0700 was shown to interact with CheY, CheA-RR and CheV, with the strength of interaction correlating with the observed cognate phosphatase activity. Although attempts to crystallise Cj0700 were not successful, a combination of bioinformatics, CD and NMR approaches indicated that expressed Cj0700 contains significant levels of disorder. These findings indicate that cj0700 has a role in C. jejuni chemotaxis and shows that Cj0700 can promote dephosphorylation of CheY and hence is likely to be a CheZ orthologue. Furthermore, Cj0700 also modulates the phosphorylation level of the response regulator domain on CheA and may also affect the related domain in CheV.

Salmonella are a leading cause of food borne illnesses in humans. Even though symptoms are often mild, these enterobacteria are responsible for over 200,000 deaths worldwide every year. There is considerable concern over the emergence of drug-resistant strains of Salmonella. Lactate dehydrogenase (LDH) from Salmonella typhi is believed to be an essential enzyme for the bacteria and hence a potential drug target. This enzyme is a D-speCific LDH and hence belongs to a class of enzymes much less studied than the more common L-specific LDHs. In this study the expression, structural and functional characterisation of the Salmonella D-LDH enzyme is reported. Crystal structures of the enzyme indicate a number ' of unusual features within its active site that may facilitate specific targeting by novel inhibitors . . Preliminary enzymatic activity data confirm the expected Dspecificity of this enzyme, and that oxamic acid is a weak inhibitor with a Ki of 10.5 mM. The crystal 'structures and enzymatic analyses provide a basis for structure-based design of novel inhibitors. This thesis also describes preliminary studies aimed at determining the structure of a cytochrome P450 enzyme from Jeotga/icoccussp. (oleTJE). This enzyme, a member of the CYP152 family, has peroxygenase activity and can be used to produce alkenes (olefins). It is therefore of interest for the development novel biofuels. Its crystallization is reported, although initial attempts to determine its crystal structure have so far been unsuccessful.

Salmonella enterica remains a major source of human and animal infection worldwide. Despite the identification of over 2,500 Salmonella serovars, the majority of our knowledge on Salmonella pathogenicity and host response mechanisms has developed through studies on a limited number of strains from a few serovars. Strain variation within and between serovars has not been fully investigated. Here, 18 Salmonella strains representing six serovars frequently recovered from human cases of infection have been characterised. All strains exhibited similar growth and motility characteristics but significant variation was observed in their abilities to form biofilms and to invade epithelial cells. Strain heterogeneity was evident both within and between serovars. Furthermore, the overall invasion profiles observed were conserved between epithelial cell lines and did not alter with the application of mild centrifugation. GFP reporter constructs confirmed that the Salmonella Pathogenicity Island (SPI) 1 structural gene, prgH, was only expressed in a subpopulation of Salmonella and this bistable expression returned after populations were sorted based on GFP expression and then allowed to recover. GFP and LacZ reporter constructs revealed a close correlation between SPI-l prgH promoter transcription at")d invasion in MDCK I cells at late log phase in the majority of strains examined, with low invasion associated with low levels of prgH promoter activity. Strains which lacked the effector protein, Salmonella outer protein E1 (SopEl) induced slower and smaller membrane ruffles in comparison to SopEl positive strain (SopEl +) S. Tm SL1344. However some SopE1+ strains were observed inducing membrane ruffles with comparable induction kinetics and ruffle diameters as strains which lacked SopEl. The presence of SopEl, large membrane ruffles or the fast induction of membrane ruffles did not ,correlate with invasion. These data demonstrate that significant strain variation exists between strains and serovars which may have important implications for Salmonella and other microbial infection studies

Mycobacteria are present in a wide range of environments. They contain characteristic complex mixtures of mycolic acids in the cell wall together with other lipids. The high resistance of mycobacteria to the majority of antibiotics, therapeutic agents and disinfectants is thought to be related to the unique structure of the mycobacterial cell wall. Mycolic acids are high molecular weight a-alkyl-branched B-hydroxy long chain fatty acids (60-90 carbon atoms). Different species of Mycobacteria may produce different classes of mycolic acids including a-, methoxy, keto, and wax ester mycolic acids, each present as mixtures of homologues. These may contain different functional groups such as ester, keto, methoxy, hydroxyl, and alkene. The most reported lipids present in the cell wall are sugar esters, e.g mycolyl trehalose esters. These mycolic acids and mycolyl trehalose esters show very interesting toxic and immunological properties; these offer considerable potential for application in the detection, control, and treatment of mycobacterial infection, and also in developing sensors for detection of disease. This study will seek to explore these potentials. This project involved the synthesis of several mycolic acids, wax esters and trehalose esters. The biological activities of these synthetic compounds would be studied, particularly their suitability as specific antigens to detect mycobacterial infections in serodiagnostic assays. The objectives of the project are discussed in four parts. The first part of this project involved the first synthesis of wax ester (A) and its corresponding ω-carboxymycolic acid (B), one component of the complex mixture isolated from Mycobacterium avium. Once the synthesis had been optimised, synthesis of the wax ester (C) and its corresponding ω-carboxymycolic acid (D) were also achieved, the latter compounds being isolated from Mycobacterium gordonae. In addition the wax ester (E) was also prepared introducing longer chain lengths than the natural wax ester. This was to study whether or not the chain length has any effect on the biological activities.

No description available.

Clostridium botulinum is an anaerobic, endospore-forming, Gram-positive species comprising four distinct groups (I, Il, Ill, and IV) based on physiological and metabolic characteristics, that all share the ability to secrete an exceptionally potent neurotoxin, the causative agent of the potentially fatal paralytic condition known as "botulism". Food-borne botulism arises when C. botulinum. spores are able to contaminate food to be sealed inside anaerobic packaging, where the spores are then able to germinate, grow and elaborate toxin. Due to the potential severity of the condition, C. botulinum has remained the principal target for the food processing industry for over a century, prompting a heavy emphasis on research into increasing the efficacy and efficiency of intervention and preservation systems. Due principally to the extreme potency of the neurotoxin and its potential for use in bioterrorism, the government has labelled the Botulinum Neurotoxin as a category A Select Agent. This fact, alongside the necessary safety measures required to safely manipulate the organism, restricts the use of the C. botulinum to only a handful of specialised research facilities, resulting in the need for a surrogate organism for routine development of intervention and preservation systems in food. This study outlines the development of novel genetic tools, and subsequent generation and characterisation of a neurotoxin negative reporter variant of a representative Group I strain of Clostridium botulinum, suitable for use in the assessment of the efficacy of current and novel commercial food preservation techniques on the ability of the strain to survive, grow and elaborate toxin.

Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. This thesis reports first on the Sco-like protein from Streptomyces lividans (Sco sl) and presents a series of experiments that firmly establish a role for Sco sl as a copper metallochaperone as opposed to a role as a thiol-disulfide oxidoreductase that has been assigned to other bacterial Sco proteins.