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.

The purpose of this study was as follows: 1. Elucidate the laboratory conditions under which Anabaena circinalis produces the noxious odor component or components in maximum concentration, 2. Isolate the major noxious odorous metabolite(s), 3. Structurally define the odorous metabolite(s), 4. Quantitate the amount of metabolite(s) that a given amount of the organism produced.

Bdellovibrio bacteriovorus, is a small mono-flagellate Gram-negative delta-proteobacterium, which has a bi-phasic lifecycle, consisting of a predatory phase; in which they invade on other Gram-negative bacteria and digest the prey cell’s content to grow and septate, or host independent phase; in which they can grow and septate in media rich in amino acids as well as vitamins and cofactors. As B. bacteriovorus can kill other Gram-negative bacteria including pathogens, they have potential to be used as a ‘living antibiotic’. I have been part of this field since 2004, a time at which the first B. bacteriovorus genome (HD100) had just been sequenced and made available, and only one study into making deletion mutants had been published. During my time in this field, the research has expanded almost exponentially, with the understanding of core pathways and systems that make B. bacteriovorus so novel being highlighted and greatly understood. In addition new techniques and methodologies never before attempted in B. bacteriovorus research have been made possible and I have been lucky to be a part of this and carried out some of the work myself. In particular I have worked on the mutation and phenotype testing of genes encoding pathways for motility, prey cell lysis, B. bacteriovorus intra-cellular signalling, and bi-phasic growth switching. These advances from my work including an animal trial into the predatory nature of B. bacteriovorus have laid the foundation for its use as a novel ‘living antibiotic’ in the future.

A culture collection of clonal, axenic cultures of 35 Arthrospira strains and five strains, which were duplicate subcultures of the original isolates, was established. In addition, eight morphological mutants were isolated from cultures of these strains. All strains are different from those belonging to the genus Spirulina as concluded from TEM and PyMS studies. All Arthrospira strains, duplicates and different morphotypes were screened for variation in morphological, ultrastructural, physiological and biochemical characters for taxonomic purposes. Special emphasis was put on the analysis of factors which influence the morphology of the trichome helix, as this is the feature most characteristic forArthrospira strains. For example, the orientation of coiling was found to be influenced by genetic drift or the growth temperature, but can also be reversed by mechanical impact. Studies were also made on the ability to utilize sugars for growth. None of the strains grew on sucrose, but many grew on glucose and/or fructose. Growth in the presence of sucrose under photoheterotrophic conditions required an adaptation process, though sucrose is not being utilized for growth. Analysis of fatty acid composition of all strains revealed quantitative differences between strains, most markedly in the unsaturated fatty acid fraction. Repeat experiments showed the same results each time, indicating the value of this approach for identification purposes. Similarly, lectin-binding to cell surface structures proved to be a useful approach for differentiation between strains. Analysis of the whole cell composition by pyrolysis mass spectrometry (PyMS) did not confirm the clusters based on other phenotypic characters, but showed that there is a high similarity between duplicate strains or different morphotypes derived from the same stock. Numerical analysis of the data for 28 characters resolved the helical strains in two phenotypic clusters which show a high correlation to the two molecular clusters based on ARDRA of the ITS of the same set of strains. The helical trichome morphology of the strains showed the highest correlation to either of the clusters. Comparison of species descriptions and morphological characters of the strains, as determined in the presented work, indicate, that phenotypic cluster I is composed of Arthrospira maxima, A. fusiformis and A. indica, while A. platensis forms phenotypic cluster II

Nitrogen is an essential constituent and building unit of all living organisms, and the primary limiting nutrient on our planet such that its cycle widely depends on the diverse nitrogen-transforming microorganisms, such as denitrifiers. Oxygen minimum zones or hypoxic aquatic ecosystems account for 30-50% of all nitrogen denitrification and under dynamic transformation imbalance, of measure dependent variable modularity, little is known about discrete shifts in denitrification competition by various microorganisms of divergent metabolism; or the Fe (II) – Fe (III) redox linking process. Novel nitrate dependent Fe (II) oxidizing bacteria as rapid denitrifier and iron oxidizer can significantly oxidize various iron minerals (magnetite and ferrous mono sulfide). Evidence of nitrate dependent Fe (II) oxidation by the bacterium P. ferrooxidans sp. MAI-1 could shed light as a novel competitor at microaerophilic (<2.0mg/L DO, -100 – +100 mV) interfacial competition with cyanobacteria Microcystis aeruginosa corollary to ecosystem eutrophication and concomitant microcystin production, with the goal of abating a toxic cyanobacterial bloom. Nitrate Dependent Iron Oxidizing Bacteria (NDFe(II)OB) showed rapid nitrate reduction (>25 mg/L NO2, day 7) and consequent bright-orange iron oxides. Saturation indices (day 1 and 8 SI = log (IAP/Ksp), showed non exclusive vivianite formation i.e., 3.80 and 0.44-0.55, respectively, with near complete oxidation by day 8, significantly abating logarithmic growth over a fourteen day period (p>0.01). N-N dichotomies are not purely exclusive, as terminal PO4 competition differed by ~0.1 mg/L after a 15 day period, with approximately one five hundred times more N-nitrogen loss compared to P-phosphorus loss difference. Early logarithmic cyanobacteria cell counts under the presence of the competitor decreased by >20% by day 18 of growth. This is consistent with the classical view that under primary metabolite exhaustion, interspecific competition should lead to competitive exclusion and not niche differentiation.

In addition to nitrogen and phosphorus, iron is an essential nutrient for oceanic primary productivity. Unlike nitrogen and phosphorus however, negligible amounts of iron are supplied to surface waters through recycling or mixing but instead from the limited and sporadic input of aeolian particulate. The low concentration of iron that becomes biologically available from the dust places a serious constraint on the heavily iron-dependent processes of photosynthesis and nitrate reduction which affect primary productivity. As much as 47% of the total oceanic primary productivity can be attributed to cyanobacteria making them critical organisms in the biogeochemical cycles. This thesis addresses the effect of iron on primary productivity using a combined approach of physiological and molecular biology. The physiological response of three marine strains of Synechococcus to growth on different concentrations of FeEDTA was investigated. Cells grown with higher concentrations of iron had greater cell density, more Chl- and phycobiliproteins and higher carbon fixation rates than cells grown at limiting iron concentrations (l0-8 M Fe). Iron enrichment of iron limited cultures stimulated carbon fixation, growth rate, and pigment and protein synthesis. Iron limited cells spiked with SJ.l.M Nlf4Cl prior to short term incubations had higher dark carbon fixation than cells gro·wn at higher iron and also spiked to 5J.1M Nlf4Cl. The addition of ammonium relieves a restricted nitrogen assimilatory pathway in the low iron cells that is evidenced by increased dark carbon fixation. We propose that this measurement of enhanced dark carbon fixation could be a useful assay in supporting the contention that populations of Synechococcus in nitrate rich waters are iron limited. Molecular genetic techniques were used to look for the presence of an iron uptake gene in cyanobacteria. Preliminary results indicate that there is a gene that is homologous to the ferric uptake regulation (fur) gene in E. coli. This hybridization occurred in siderophore-producing cyanobacteria, but not in marine cyanobacteria that do not produce siderophores. The fact that marine Synechococcus do not produce siderophores and did not hybridize to the fur gene suggest that fundamentally different mechanisms for iron uptake operate in high biomass freshwater cyanobacteria and cyanobacteria from dilute oligotrophic waters.

The prevalence of the cyanobacterial metabolites: MIB, geosmin and microcystin in drinking water is a major concern to the water industry as these metabolites can compromise the quality of drinking water. Consequently, effective removal of these metabolites from drinking water is paramount. The combination of ozone (O3) and granular activated carbon (GAC) has been shown to be effective for the removal of these metabolites from drinking water. In this study, the ozonation of MIB and geosmin was affected by the character of natural organic material (NOM). In particular, NOM containing compounds of high UV absorbing properties and high molecular weight (MW) resulted in greater destruction of MIB and geosmin due to the formation of hydroxyl (OH) radicals. In addition, alkalinity also affected the ozonation process, with waters containing higher alkalinity resulting in decreased destruction of MIB and geosmin. Laboratory scale minicolumn experiments, coupled with the homogenous surface diffusion model (HSDM), were found to be ineffective in predicting the GAC breakthrough behaviour of MIB and microcystin at two different pilot plants. This can be attributed to the biological degradation of the metabolites at the pilot plants which cannot be modelled by the HSDM. In addition, the volume of GAC used in the minicolumn experiments may not have been appropriate for the predictions, rather, larger laboratory scale columns were found to be more applicable in mimicking pilot plant results. Microcystins were shown to be readily degraded by the bacteria attached to the GAC. Furthermore, the lag period prior to the onset of degradation, which is indicative of most biological degradation studies, was effectively eliminated and in one instance abated. This finding suggests that biological filtration of microcystin is practically feasible especially since the occurrence of microcystins in water supplies is seasonal. This study expands on previous research in the area of O3 and GAC for the treatment of MIB, geosmin and microcystin. With the imminent increase of the use of O3 and GAC in Australian water treatment plants (WTPs), this study provides valuable information for the use of these processes both alone and in combination, particularly since minimal research in this area has been conducted in Australia. / thesis (PhDAppliedScience)--University of South Australia, 2004.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 120-137).