Cyanophages, viruses that infect cyanobacteria, are known to be abundant throughout the world’s oceans. They are important because of the ecological significance of their hosts which are prominent primary producers. In the natural environment cyanobacteria undergo light-dark cycles, which might be expected to exert significant effects on the way in which cyanophages reproduce. The results in this study show how light plays an important role in cyanophage adsorption to the host cell using a model system consisting of cyanophage S-PM2 and Synechococcus sp. WH7803. An initial investigation of the role of light on phage adsorption revealed a striking light-dependence. In the dark, the phage S-PM2 was virtually not capable of adsorbing to WH7803, but adsorption resumed as soon as the light was switched on. This light-dependent phage adsorption was not just limited to the phage S-PM2, four out of nine other cyanophages showed the same effect. The host photosynthetic activity and light/dark cycles were demonstrated not to influence phage adsorption. The presence of the photosynthetic reaction centre gene psbA in cyanophage genomes was not associated with the light-dependent phage adsorption. No photoreceptor was detected from the phage S-PM2 particle. A phage-resistant mutant that S-PM2 can’t adsorb to WH7803 was isolated. A putative multicopper oxidase was found to be absent from the outer membrane fraction of the mutant. This outer membrane fraction in the wild type showed a moderate phage neutralisation activity (up to ~ 30%). To test whether the putative multicopper oxidase was the S-PM2 receptor, a recombinant WH7803 strain was constructed by inactivating the putative multicopper oxidase gene. As S-PM2 can still adsorb to the knockout mutant as efficiently as to the wild type, it suggests that the multicopper oxidase is not the phage receptor and that loss of the putative multicopper oxidase is probably a pleiotropic consequence of the loss of the S-PM2 receptor or other components, such as lipopolysaccharide, that is needed for a successful S-PM2 adsorption.

The objective of this study was to develop a tandem bio- chemical transformation of a feedstock material to value-added products. The chosen example is microbial fermentation of glycerol with subsequent esterification of the intermediate 1,3-propanediol with fatty acids. The use of biphasic aqueous/organic medium for the bi-catalytic system is the key feature of this study. In the first part of this work, batch and continuous fermentations of glycerol by Clostridium Butyricum bacteria were optimized to increase productivity of 1,3-propanediol. In the second part, several catalysts were screened for mono- and biphasic transformation of 1,3- propanediol. It was discovered that enzymes are the most suitable catalysts for the tandem reactions of glycerol to 1,3-propanediol derivatives compared to chemical catalysts. Biphasic enzymatic esterification of 1,3-propanediol was further optimized using Rhizomucor miehei lipase. Finally, a segmented flow tubular reactor and hollow fiber membrane contactors were designed and tested as a concept tandem reactor. The hollow fiber reactor with Rhizomucor miehei lipase immobilized onto polypropylene membrane was found to be the most effective in the biphasic linoleic acid/aqueous esterification of 1,3-propanediol. In general, the demonstrated approach and the developed system can be easily utilized in the biorefinery processes to avoid extraction and purification of the intermediate products, thus reducing time, energy and emissions.

Botulinum neurotoxin induces a potentially fatal paralytic condition in humans and various animal species collectively known as 'botulism'. It consequently poses a major problem to the food industry, due to the ability of its spores to survive in cooked foods. The incidence of wound botulism has also suffered a recent increase in the UK. The genome sequence of the C botulinum Group I strain ATCC 3502 has recently been determined. In silico analysis has revealed the presence of two distinct loci capable of encoding proteins with homology to AgrB and AgrD of the Staphylococcus aureus agr quorum sensing system. The functional characterisation of these genes has been carried out in order to determine whether they play a role in quorum sensing. To simplify laboratory procedures, C. sporogenes, the non-toxic counterpart of C. botulinum, was initially focused on. The agr regions in C. sporogenes were sequenced and their proteins compared with those of C. botulinum and other Gram-positive bacteria. Regions of conservation were observed amongst the clostridia and, to a lesser extent, between clostridia and staphylococci. Transcriptional linkage assays showed some of the genes of the C sporogenes agr regions to be co-expressed, and Real-Time RT-PCR demonstrated the maximal expression of these genes during late exponential growth. Modulation of the expression of the identified agr genes is a prerequisite to determining their function. Due to an initial lack of an effective gene knockout tool, antisense RNA expression was used for this purpose in C sporogenes, and showed that down regulation of the agrB genes affects sporulation. The development of an integrative vector system for gene inactivation in C sporogenes was also attempted. Knockout mutants in C botulinum and C sporogenes were later constructed using the newly-developed ClosTron system. These mutants were used to demonstrate that AgrDl, AgrD2 and an orphan sensor kinase protein all play a role in the control of sporulation in C. botulinum and C. sporogenes.

Pseudomonas aeruginosa is an opportunistic pathogen of animals and humans causing medical complications in burns, wounds, and cystic fibrosis. P. aeruginosa is efficient at adapting its virulence phenotype depending on the site of infection. Emerging multi-drug resistant strains and a limited number of effective anti-pseudomonal antibiotics renders P. aeruginosa infections increasingly difficult to treat. To address this need, this thesis considers targeting bacterial quorum sensing, which regulates the production of virulence factors, as an alternative prophylactic strategy. The P. aeruginosa quorum sensing system is compromised of three interlinked but independent systems, Las, Rhl and Pqs, which produce and utilise quorum sensing system molecules, 3OC12-HSL, C4-HSL and PQS respectively. Immunological targeting of the quorum sensing system molecule 3OC12-HSL, through active immunisation (vaccines), inhibits the Las system, resulting in a longer life expectancy in mice infected with P. aeruginosa in vivo. However, P. aeruginosa has the capacity to develop resistance, through compensatory mechanisms, towards quorum sensing inhibition that targets the Las system only. This emphasises the need to target all three quorum sensing systems, Las, Rhl, and the Pqs, in order to inhibit quorum sensing. The present study focuses particularly on the development of a multi-component anti-quorum sensing system vaccine that would target the three main quorum sensing system molecules, effectively compromising the quorum sensing system and minimising compensatory mechanisms. This involved the synthesis of haptens based on the quorum sensing system molecules, which were used to haptenise the immunogenic carrier keyhole limpet haemocyanin. Syntheses of the haptens, AP1 (derivative of 3OC8-HSL) and AP2 (derivative of PQS), were conducted using adapted published methods. The resulting conjugates, AP1-keyhole limpet haemocyanin and AP2-keyhole limpet haemocyanin were immunogenic in mice and rabbits. The specific anti-hapten polyclonal antibodies that were generated demonstrated cross-reactivity with the natural quorum sensing system molecules of P. aeruginosa that translated in significant and specific anti-quorum sensing system molecule activity in bioluminescence reporter assays. Anti-AP1 polyclonal antibodies were able to reduce biofilm formation at high concentrations, however, significant reduction of biofilm formation was seen when the anti-hapten antibodies were used in combination. In this study, it has been demonstrated that the inhibition of the quorum sensing system should include the three systems, Las, Rhl and PQS, and that this can be done by a multi-component anti-quorum sensing system vaccine. These data suggest that a multi-component anti-quorum sensing system vaccine takes us a step forward to a viable prophylaxis against P. aeruginosa for susceptible patients.

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.

Members of the genus Campylobacter are a major cause of food-borne disease worldwide. They can colonize the intestinal mucosa of poultry, to high levels leading to contamination of meat, at slaughter. Their numbers can be reduced in different ways including chicken treatment with bacteriophages. For such treatments to be successful, in depth understanding of the bacteriophage that infects and kills campylobacters is vital. The work in this thesis describes: isolation and comprehensive characterisation of bacteriophage candidates for future therapy applications. In order to increase the available stocks of characterized candidate bacteriophage, a number of attempts were made to isolate bacteriophages from poultry excreta. The new isolates together with some uncharacterized phages from our laboratory stocks were characterized with respect to their host range and genomic size. Some bacteriophages preparations in previous studies showed genomes of different sizes and a number of attempts were done for their separation. This raised questions about the relationship between the two different sized genomes. Prior to this work, a co isolate pair had been successfully separated and the sequence of the larger genome, CP220, was determined. Part of the work here, was performed to extend this study by obtaining the sequence of the smaller co isolate, CPX and compare it to CP220. They did not appear to have any identifiable relationship at the genetic level, but the availability of the CPX sequence will further extend our knowledge of bacteriophage genetics and this phage has clear therapeutic potential. Attempts were also made to separate and characterize a second co-isolate pair but these were unsuccessful. The availability of the DNA sequence of CP220 allowed a much closer molecular characterisation and comparison of Campylobacter phage genomes, than had previously been possible. One area that was investigated in this study was the presence of repeat regions identified in the CP220 genome, which were amplified by PCR, but could not be cloned in E. coli. Furthermore, genes encoding potential lysins were identified in the CP220 genome and they were amplified, cloned and attempts were made to express the proteins, which may have potential therapeutic value. One gene product was successfully expressed and showed evidence of lytic activity on Campylobacter and other bacterial genera. In summary, this thesis describes a much closer examination of molecular biology of Campylobacter bacteriophage than had previously been possible, including the determination of the sequence CPX phage.

It was shown that Listeria monocytogenes cells grown in a defined minimal, MCDB202, showed enhanced extracellular polymeric substances production compared to BHI. On the other hand, it was reported that in L. monocytogenes luxS mutant, AI-2 reduction and biofilm enhancement were seen. It is hypotheses that there could be a linkage between the AI-2 signaling system and the EPS formation. The expression of EPS could be induced by the reduction in AI-2. The main aim of the research is to study this EPS formation in minimal media, how is it linked to AI-2 production, the function of the EPS as well as to figure out the linkage between EPS formation with cap genes found in Listeria genome. It was shown that MCDB202 have caused an increase in surface hydrophobicity of the cells. However, cells grown in the defined media did not induced better attachment and biofilm formation towards hydrophobic surfaces. And cells grown in MCDB202 were shown less capable to infect eukaryotic cells in the cell invasion assay. On the other hand, AI-2 production was shown to be relative lower in Listeria cell grown in minimal media MCDB202) than rich media (BHI). Bioinformatics study has shown that only capA homologues, but no capBCDE homologues, were found in Listeria genome. However, the bioinformatics works have shown that the capA homologues are unlikely to be contributing the EPS seen produced in Listeria monocytogenes. This was further supported in the expression assay that the two genes were not highly expressed in MCDB media.

No description available.

A second aspect to the study involved determining the surface roughness of a variety of commercially available urinary catheters and relating their roughness to the motility rates of P. mirabilis. Motility rate was highest on the roughest catheter surfaces (hydrogel and silver/hydrogel) suggesting that manufacturers should consider topographical features as well as antimicrobial coatings when attempting to combat microbial colonisation.

Burkholderia cepacia complex (Bcc) are well known for their ability to produce antifungal agents. However, the majority of past studies have screened only a limited number of isolates, which were not taxonomically well characterized. In addition, while the activity of Bcc bacteria against plant pathogenic fungal species has been characterized, very few studies have examined the interaction between Bcc and saprotrophic wood decay basidiomycete species. The overall aim of this study was to begin to the characterization of the interactions of Bcc and other Burkholderia with the model ascomycete Candida albicans and woodland basidiomycete fungi. Methods. The study made use of a large collection (397 isolates) of taxonomically well identified Burkholderia species. A systematic screening of the antifungal activity of this collection was made against C. albicans and various wood decay basidiomycetes: Bjerkandera adusta, Trametes versicolor, Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina. A wide range of media were evaluated for Burkholderia growth, antifungal metabolite production and the ability to support the mycelial growth of basidiomycetes. An agar overlay assay was used to examine the anti-candidal activity of Burkholderia isolates, while a novel overlay assay was developed using homogenised mycelial material for the basidiomycete species. Novel and existing extraction techniques were evaluated for their ability to partially purify active Burkholderia metabolites. The purification and identification of the active Burkholderia antibiotics were performed using both preparative thin layer chromatography (TLC) and liquid chromatography combined with mass spectrometry (LC-MS). A novel TLC-bioautography assay was also developed to reveal the presence of active Burkholderia metabolites in the antimicrobial extracts. The minimum inhibitory concentration (MIC) of the semi-purified Burkholderia antifungals was also determined. The natural diversity of Burkholderia and other bacteria associated with different rhizosphere environments was evaluated by cultivating isolates from local maize crops and examining a collection of Burkholderia isolates obtained from the rainforest in Sabah, Malaysia. The genetic basis for the secretion of antifungal agents by B. ambifaria was examined using transposon mutagenesis and screening for the mutants which had lost their anti-candidal activity. Additional transposon mutants that had lost their anti-bacterial activity and had been obtained in a previous screen were also examined for alterations in antifungal activity. Finally, the Burkholderia genome database and bioinformatic techniques were used to genetically characterize these mutants and other genetic loci that had been previously implicated in antibiotic production. Results. The majority of Burkholderia isolates possessed the same antagonistic activity towards C. albicans as they did towards B. adusta. Of the 397 isolates examined, antifungal activity was the greatest in the following Burkholderia species: B. ambifaria, B. cepacia, B. cenocepacia and B. contaminans. Interestingly, no antifungal activity was observed for B. multivorans (27 isolates) and B. stabilis (18 isolates) under the experimental conditions tested. The type of medium used to study the interaction between Burkholderia and the fungi considerably effected antifungal activity, with the most suitable media for studying antagonistic activity being Sabouraud agar and an acidic minimal salts medium (BSM; pH 6) supplemented with 0.4% glycerol. Of the 397 isolates screened, 47.85% were anticandidal (190 isolates; 89.47% of these were Bcc species and 10.53% were other Burkholderia species), while 48.10% were active against B. adusta (191 isolates; 93.19% of these were Bcc species and 6.81% were other Burkholderia). Extraction using Amberlite XAD16 resin binding followed by elution with methanol was an excellent means to isolate active Burkholderia antifungals. Fractionation of these extracts using silica gel TLC and ethyl-acetate:methanol:water (20:1:0.5) as the elution solvent was optimal for the separation of the majority of the active metabolites. A TLC-bioautography assay was developed in this current study as a preliminary screening technique to detect anti-microbial components of novel Burkholderia extracts. The assay was very useful for detecting anti-candidal and anti-bacterial compounds and was adapted for use for the first time with basidiomycetes. Antifungal Burkholderia isolates produced between 1 and 10 antifungal metabolites. Several antifungal agents were identified as enacyloxin, pyrrolnitrin, bactobolin and quinolines. Novel compounds and novel bactobolin derivatives were also present. For B. ambifaria strain AMMD a number of chromosomal loci were found to be involved in the production of antifungal metabolites. Quorum sensing was essential for the expression of antifungal activity as transposon mutations in the CepI synthase-encoding gene prevented the biosynthesis of all B. ambifaria anti-candidal compounds. B. ambifaria AMMD extracts containing enacyloxin IIa also produced an inhibitory effect at low concentration against E. coli, B. multivorans and A. baumannii. Conclusions. Burkholderia are potentially a very large reservoir of known and novel antimicrobial agents, especially with nearly 50% of isolates screened exhibiting antifungal activity. Antimicrobial compounds such as enacyloxin IIa were discovered for the first time to be produced by Burkholderi and novel extraction and bioassay methods for anti-fungals were developed in this study. Overall, we now have the tools to considerably advance the isolation and characterization of antifungal Burkholderia metabolites.