Nodularin (NODLN) is a pentapeptide produced by the filamentous cyanobacterium Nodularia spumigena that is a bloom-forming species in the Baltic Sea. NODLN is an intracellular hepatotoxin, which can have a negative effect on aquatic life including fish. Toxins are released into the water when cells are lysing, e.g. during a decaying bloom. N. spumigena filaments have previously been shown to have a negative effect on perch egg development and perch larval survival. Coastal fish such as perch (Perca fluviatilis) have suffered from recruitment problems in the Baltic Sea the last decades. However, little is known about the impact of toxic cyanobacteria on juvenile perch. In the autumn of 2007, 1+ perch were exposed, during 29 days to either whole live cells (WC) or a crude extract (CE) of broken N. spumigena cells. Chlorophyll a concentrations in the aquaria were 50 µg L -1. Perch were fed chironomidae larvae twice a day. Unexposed perch either fed (CoF) or without food (Co) served as controls. Length and weight of perch were measured at onset and termination of experiment. NODLN content was measured in N. spumigena filaments, crude extract and perch liver samples using liquid chromatography-mass spectrometry (LC-MS). Total lipids (TL) were extracted and quantified from whole-body lyophilised perch excluding livers. No significant differences for length and weight of perch were found between treatments and fed control. NODLN was detected in the crude extract samples, while no NODLN was detected in the perch livers. Moreover TL determination revealed no significant differences between treatments and fed control. Nodularia spumigena did not affect perch in this experiment, probably due to that the critical period of the first year for the perch was exceeded. Therefore, 1+ perch was not as susceptible to the cyanobacterium as eggs, larvae and younger juveniles of fish found in the literature. Perch liver did not contain NODLN, thus either the toxin was detoxicated with no recorded energetic cost or it was not ingested. The variables studied here did not show any effects of NODLN. However, other chemical methods such as enzymatic activity may disclose effects of NODLN.

Gram-negative bacteria are the most common cause of the sepsis and lipopolysaccharide (LPS) a major component of Gram-negative bacteria is known to be of major importance in the development of sepsis. Human infection with Salmonella, a Gram-negative bacterium, is associated with a number of cases of sepsis and is particularly important in childhood sepsis. During salmonellosis, monocytes and macrophages produce a number of different pro-inflammatory mediators such as TNF-α, IL-1α, IL-12, IL-18, IFN-γ, reactive nitrogen species and oxygen species. Although the production of these inflammatory mediators is required for resolution of bacterial infections, they are contraindicated in diseases such as sepsis. In the initial (acute) phase of sepsis a Systemic Inflammatory Response Syndrome (SIRS) occurs in which inflammatory mediators are produced in high concentration, which can lead to organ failure and death. The SIRS phase is then replaced by a Compensatory Anti-inflammatory Response Syndrome (CARS) phase which leads to immunosuppression. The CARS phase can lead to secondary infection and subsequent mortality within 28 days of hospital admission. To date, several studies have evaluated the role of vasoactive intestinal peptide (VIP) as an anti-inflammatory agent that may have therapeutic potential in septic patients both in vitro and in vivo. VIP has been shown to inhibit production of inflammatory mediators produced by human monocytes in response to LPS. The aim of the work described in this thesis was to investigate the therapeutic potential of VIP in sepsis using an ex vivo human monocytes model infected with viable Salmonella Typhimurium 4/74 (rather than LPS). The study shows that VIP (10-7 M) stimulates an increase in the numbers of Salmonella recovered from infected human monocytes (MOI = 10). In addition, VIP also increases the survival rate of human monocytes infected with Salmonella. These two results may suggest a detrimental effect of VIP during bacteraemia and sepsis, since monocyte death may be beneficial during sepsis and bacterial overgrowth could lead to further increased LPS (and other antigen) stimulation of the immune system. However, VIP did significantly decrease Salmonella and LPS-induced TNF-α, IL-1β and IL-6 in monocyte supernatants. VIP also had a positive effect on IL-10 production in human monocytes infected with Salmonella or stimulated with LPS. Whether this suggests a possible detrimental effect of VIP is unknown but septic patients with high serum IL-10/ low TNF-α concentration ratio have previously been shown to have a poor prognosis. Higher IL-10 concentrations in infected monocytes (due to VIP) could also increase the CARS phase of disease with increased immunosuppression. Flow cytometry and qPCR analyses showed that of all of the VIP receptors, VPAC1 was expressed most highly during Salmonella infection, or LPS stimulation, of human monocytes. Administration of VIP inhibited VPAC1 has been shown by many studies to be the most important receptor by which VIP inhibits production of inflammatory immune mediators, or increases IL-10 production from murine macrophages. Results in this thesis, therefore, suggested that Salmonella infection may promote VPAC1 expression and so provide a mechanism of inhibiting the production of inflammatory mediators in infected cells. This could then increase intracellular survival of Salmonella and provide a means of greater dissemination of the infection. To ascertain how increased VPAC1 expression on the surface of monocytes may be achieved, analysis of the expression of known intracellular endosomal and exosomal constituents was performed. Confocal laser microscopy, using specific antibodies, showed that VPAC1 on the monocyte cell membrane was internalised within early endosomes (measured by co-localisation of VPAC1 and EEA1) rather than being degraded within lysosomes (measured by immunoreactivity to LAMP1). VPAC1 is then transported via a Rab11A recycling endosome and packaged in the Trans-Golgi network (TGN), shown by co-localisation of VPAC1/Rab11A and the TGN marker (TGN46). VPAC1 was then associated with Rab3a and calmodulin. The function of these latter two proteins in the docking of exosomes to the cell membrane is well known, thus suggesting that Salmonella induced VPAC1 was also recycled to the cell membrane within exosomes. VIP inhibited the expression of both Rab3a and calmodulin but not the co-localisation of VPAC1 with these two proteins. Further studies then showed that a calmodulin agonist (CALP1) increased VPAC1 expression on the surface of monocytes, while a calmodulin antagonist (W-7) decreased expression of VPAC1 on the surface of monocytes. In conclusion, this thesis does present hitherto unknown data regarding Salmonella infection of human monocytes and the effects of VIP on infected monocytes. VIP has potential as an anti-sepsis therapy since it reduces the production of inflammatory mediators by Salmonella-infected and LPS-stimulated monocytes. However, the fact that VIP increases survival of infected human monocyte and increased growth of Salmonella in human monocytes may preclude its use in sepsis.

The carbon and energy metabolism of the human pathogen Clostridium difficile is poorly understood. Amino acid metabolism by the Stickland reactions has previously been described as a primary source of energy in a number of Clostridium species, especially when grown in a medium containing only amino acids. Deeper insights may be gained by metabolic analyses using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platforms, but such experiments are best performed in well-defined growth media. A number of C. difficile strains have been successfully cultivated on defined media but these media provide an excess of nutrients, particularly in terms of amino acid provision, resulting in undesirable background growth in the absence of glucose. To overcome these challenges, three variants of a defined medium were developed that contain the essential nutrients that support the growth of this bacterium, in the presence of a carbon and energy source such as glucose together with an LC-MS/MS method that will simultaneously measure all twenty amino acids. Since the focus in this study was amino acid utilisation, a comprehensive and most effective technique that will provide as much information as possible for understanding the metabolic requirements of eight Clostridium difficile strains (CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F) and two transposon mutants (CRG-2979, CRG-3887) was required. Due to high water solubility and the range of ionic characteristics of amino acids, an aqueous normal phase chromatographic method was considered to simultaneously separate a mixture of amino acids without derivatisation. Aqueous normal phase chromatographic method represents an important new technology with a capability of the silica-hydride-base stationary phases, offering a distinct advantage for practical application with a high degree of reproducibility and long-term stability of polar and non-polar compounds. The developed methods were subsequently adapted to study amino acid utilisation in the presence or absence of a fermentable carbohydrate and/or selenium. OD determinations were performed by measuring absorbance at 600 nm (or OD600) using a Biomate 3 spectrophotometer. The concentration of individual amino acids remaining in the spent C. difficile culture media was measured after 24 h and/or 48 h using the developed LC-MS/MS method in order to determine which amino acids were being utilised by these organisms and in which order. This analysis should give further insight on the importance of amino acids for the survival of this bacterium in the gut, which may possibly lead to discovery of novel fermentable products and metabolic pathways and/or eventually aid in the successful control of the disease. Data obtained for CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F strains showed that they all grew on the fully defined medium, with different growth profiles in terms of lag phase, growth rate, and maximum OD reached. The LC-MS/MS data generated suggest that cysteine, glutamine, isoleucine, leucine, serine, threonine and tyrosine are preferentially utilised, both in the presence and absence of glucose. Several other amino acids, including asparagine, glycine, phenylalanine, proline and valine were also utilised but to a lesser extent. Notable in this study was the lack of glutamate utilisation, except by strain L26, and the excretion of alanine after its initial uptake by most of the tested stains. The excretion of alanine may be due to the use of pyruvate as an amino acceptor during the degradation of preferentially fermented amino acids, whereas, glutamate is not a substrate for most C. difficile strains. Thus, LC-MS/MS profiling confirmed that these organisms derive most of their carbon and energy from the fermentation of a selected range of amino acids. Given the importance of selenium-dependent Stickland reactions to the growth of this bacterium, further studies were undertaken to evaluate the metabolism of amino acids by two different C. difficile strains (630∆erm, R20291) and two transposon mutants CRG-2979 (defective in hadB, encoding one of the two subunits of hydroxyisocaproyl-CoA dehydratase required for reductive degradation of leucine) and CRG-3887 (defective in selA encoding selenocysteine synthase) in the presence or absence of glucose/selenium. LC-MS/MS data reveal that amino acid utilisation was affected by the presence of selenite, notably proline utilisation, which could be explained by the presence of the enzyme proline reductase and the lack of glycine consumption, known to be selenium-dependent. The non-utilisation of glycine could be explained by the presence of proline which represses the formation of the necessary enzyme systems required for glycine degradation. Data generated for CRG-2979 reveals that this mutant could only thrive in the presence of selenium when glucose is present, possibly due to the presence of proline replacing leucine as the major Stickland acceptor. The results of the transposon mutant CRG-3887, were much of a surprise too, because this mutant was predicted to be deficient in proline and glycine breakdown which is also reliant on selenoenzymes. This suggests the presence of selenium independent proline fermentation pathway although this hypothesis is not supported by the existing literature. Experimental data provided further evidence about the ability of this bacterium to obtain its carbon and energy in the absence of a fermentable carbohydrate; by Stickland reactions and that the presence or absence of certain amino acids could repress the utilisation or biosynthesis of other amino acids.

Unbranched heterocytous cyanobacteria exhibit complex filament and colony architectures and variable life strategies from symbionts to free living planktic and non-planktic species. They are counted among microbial groups showing an extensive production of secondary metabolites, resulting in both pharmaceutically important and toxic compounds. The main focus of this thesis is to broaden our knowledge on bioactive secondary metabolite potential in this widespread group of cyanobacteria. An effective combination of methods including whole genome sequencing, bioinformatic analysis, and analytical chemistry techniques are applied to accomplish this task. The discrepancies in distribution of various classes of compounds among ecological groups defined by different life strategies are discussed. Additionally, the thesis endeavours to test multidisciplinary approaches to tackle taxonomic assignments of unresolved unbranched heterocytous cyanobacteria using morphological, phylogenetic and ecophysiological methods, including a meta-analysis of morphological traits.

Clostridium difficile is the leading cause of health care associated diarrhoea and remains a burden for the NHS. Disease symptoms can range from mild diarrhoea through to fulminant pseudomembranous colitis, resulting in mortality for some patients. Recurrence is a major problem and estimates are that 20% of all patients with disease will either relapse (with the same strain) or have a re-infection (with a different strain). Arguably, the main virulence factors are toxins A (TcdA) and toxin B (TcdB) which cause disease symptoms. The genes encoding TcdA and TcdB are located within the pathogenicity locus (PaLoc) along with three accessory genes; tcdR, tcdE and tcdC. The regulatory network has been studied but we aimed to add to this knowledge by using two under investigated strains R20291 a so-called hypervirulent strain and VPI 10463 a strain known to produce higher levels of toxin. Two different methods of investigation were employed during this study to improve our understanding of both the regulation of TcdA / TcdB but also the genetic mechanisms behind clinical relapse. These methods were; using forward and reverse genetic analysis to assess phenotypic differences and using bioinformatics to identify genes and / or single nucleotide variants (SNP) that may play a role. Using a combination these methods we have identified potential regulators of toxin production in both strains. We have also identified unique genes and SNPs that might provide a fitness benefit to strains of C. difficile that were isolated from patients who had suffered relapse episodes.

The work of this dissertation examined the secondary metabolites of several blooms of the marine cyanobacterium Lyngbya collected in Guam and Florida with an emphasis on the isolation and structure elucidation of novel biologically active compounds. The introduction in Chapter One provides a brief history of marine natural products, a description of cyanobacteria and a summary of peptides isolated from Lyngbya collected in the Caribbean. In Chapter Two, a bioassay-guided fractionation of a Floridian collection of Lyngbya polychroa led to the isolation and structural determination of the cytotoxin desacetylmicrocolin B and the known compounds microcolins A and B. The structures were established by nuclear magnetic resonance (NMR) spectroscopic analysis. All three compounds inhibited the growth of cancer cell lines HT-29 and IMR-32 at nanomolar concentrations. Microcolins A and B were found to have little activity in the ecological assay against the marine fungus Dendryphiella salina. Chapter Three describes the isolation and structure elucidation of the glycosidic, acyl proline derivative tumonoic acid J from a sample Lyngbya sp. collected in Guam. The planar structure was determined by 1D and 2D NMR spectroscopy in conjunction with high resolution-mass spectrometry (HR-MS) data. Tumonoic acid J showed moderate activity in the ecological assay against the marine fungus D. salina. In Chapter Four, NMR-guided fractionation of a Floridian sample of Lyngbya majuscula led to the isolation of two novel cyclic peptides porpoisamides A and B. The planar structures were determined by 1D and 2D NMR spectroscopy with HR-MS data. The absolute configurations of these two compounds were defined through chiral chromatographic methods and derivatization techniques. / The porpoisamides showed only moderate activity in cytotoxicity assays against cancer cell lines HCT-116 and U2OS. Finally, Chapter Five examines a potential ecological role of compounds isolated from marine cyanobacte ria. These secondary metabolites may function as chemical defenses against competing microorganisms within marine environments. Compounds isolated from cyanobacteria were tested for anti-fungal activity against the saprophytic marine fungus D. salina. Three of the six compounds tested produced inhibitory activity at or below their natural concentration. / by Theresa Meickle. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Currently, fossil fuels contribute a large number of high value chemicals that are used on a daily basis. Crude oil is cracked to give a number of high value chemicals, including vehicle fuels as well as chemicals and solvents that are used daily both commercially and industrially. However, fossil fuel reserves are in decline, with research going into alternatives to obtain these useful chemicals, one of which is biofuels. Biofuels can be generated in a number of ways, one of which is the fermentation of acetogenic bacteria, microorganisms that generate acetate as a product of anaerobic metabolism. Clostridium autoethanogenum is an acetogen that can grow on one carbon gases as its feedstock, and can be used to generate valuable chemicals, with scope to develop the range of metabolic products further. One aim of this project was to investigate the metabolic flux through pathways of the bacterium using isotopically labelled compounds, which would be assessed by mass spectrometry and NMR. Following on from this, design of inhibitors for the enzymes of the pathways with a view to drive the metabolic processes towards higher value chemical compounds by ‘switching off’ other branches of the pathway. Putative small molecule mimics of acetyl-CoA, SNAC thioesters, were synthesised and tested for uptake and activity in whole cell growth experiments with C. autoethanogenum, and determined to be unsuccessful. Further to this, compounds were designed and synthesised to replace pantothenic acid in the growth media, which were not tested in growth experiments. A library of inhibitor compounds was synthesised and tested against recombinantly purified acetate kinase. A number of compounds were shown to inhibit the enzyme, and the mode of inhibition was determined, as well as IC50 and Ki values for each. This project operated as part of a larger GASCHEM project in the Synthetic Biology Research Centre at the University of Nottingham.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

<p>Nodularin (NODLN) is a pentapeptide produced by the filamentous cyanobacterium <em>Nodularia spumigena</em> that is a bloom-forming species in the Baltic Sea. NODLN is an intracellular hepatotoxin, which can have a negative effect on aquatic life including fish. Toxins are released into the water when cells are lysing, e.g. during a decaying bloom. <em>N. spumigena </em>filaments have previously been shown to have a negative effect on perch egg development and perch larval survival. Coastal fish such as perch (<em>Perca fluviatilis</em>) have suffered from recruitment problems in the Baltic Sea the last decades. However, little is known about the impact of toxic cyanobacteria on juvenile perch. In the autumn of 2007, 1+ perch were exposed, during 29 days to either whole live cells (WC) or a crude extract (CE) of broken <em>N. spumigena</em> cells. Chlorophyll <em>a </em>concentrations in the aquaria were 50 µg L ^-1. Perch were fed chironomidae larvae twice a day. Unexposed perch either fed (CoF) or without food (Co) served as controls. Length and weight of perch were measured at onset and termination of experiment. NODLN content was measured in <em>N. spumigena </em>filaments,<em> </em>crude extract and perch liver samples using liquid chromatography-mass spectrometry (LC-MS). Total lipids (TL) were extracted and quantified from whole-body lyophilised perch excluding livers. No significant differences for length and weight of perch were found between treatments and fed control. NODLN was detected in the crude extract samples, while no NODLN was detected in the perch livers. Moreover TL determination revealed no significant differences between treatments and fed control. <em>Nodularia spumigena</em> did not affect perch in this experiment, probably due to that the critical period of the first year for the perch was exceeded. Therefore, 1+ perch was not as susceptible to the cyanobacterium as eggs, larvae and younger juveniles of fish found in the literature. Perch liver did not contain NODLN, thus either the toxin was detoxicated with no recorded energetic cost or it was not ingested. The variables studied here did not show any effects of NODLN. However, other chemical methods such as enzymatic activity may disclose effects of NODLN.</p><p> </p>

This thesis details investigations of marine cyanobacterial secondary metabolism, with emphasis on a strain of Phormidium sp. collected in Indonesia. These studies assessed the effects of nineteen putative elicitor compounds on the growth and metabolite production of five species of marine cyanobacteria, biosynthetic investigation of an intriguing secondary metabolite, phormidolide, and the discovery of one novel halogenated peptide, phormidamide. The growth, biomass production and the ratio of the components of the extract were affected by some of the elicitors in most of the cyanobacterial species tested. However, production of a novel secondary metabolite or a significant change in the bioactivity of the extracts was not observed. Biosynthetic investigations of a brominated brine shrimp toxic polyketide, phormidolide, were conducted on a cultured Phormidium sp. strain originally isolated from Indonesia. Stable isotope feeding experiments confirmed its polyketide nature and established a new example of a general trend in cyanobacterial metabolism where both S-adenosyl methionine and C2 of acetate contribute to the biogenesis of pendant methyl groups. At the same time, feedings with deuterated acetate provided insight into the HMG-CoA synthase-like mechanism by which addition of pendant methyl groups from C2 of acetate takes place. Studies of phormidolide production in bromine-depleted medium showed that two analogs are produced, debromophormidolide with a terminal olefin in place of the vinyl bromide, and iodophormidolide, introducing iodine in place of bromine from the trace amounts present in the medium. Supplementation of the bromine-depleted culture medium with iodine resulted in a 10-fold increase of iodophormidolide production, while bromine supplementation resulted in a more moderate (2.5 fold) enhancement in phormidolide yield. A novel halogenated cytotoxic peptide, phormidamide, was isolated and a planar structure is proposed, pending confirmation by X-ray crystallographic analysis. Phormidamide contains a unique bromophenylalanine functionality, three chlorine atoms, and a very high number of quaternary carbon atoms which have hindered structural elucidation efforts through spectroscopic methods. / Graduation date: 2004