Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 180-192.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 106-119).

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.

The neurotoxic amino acid β-­‐N-­‐methylamino-L­alanine (BMAA)is present in environmentally ubiquitous cyanobacteria and bioaccumulates and biomagnifies within the environment. The implication of BMAA in the development of neurodegenerative disease has raised concerns over the potential risk of human exposure to this neurotoxin, and has focussed attention on identifying possible routes of exposure that include direct contact with cyanobacteria and the ingestion of BMAA-­‐containing plant and animal products.

Cyanobacteria are known to produce a variety of toxic compounds. β-N-methylamino-L-alanine (BMAA) is one of the neurotoxins produced by most cyanobacteria. BMAA has been implicated in amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS / PDC) and was suggested to contribute to this pathology after biomagnification and slow release of BMAA from a protein associated form. The uptake and accumulation of BMAA by the aquatic macrophyte Ceratophyllum demersum has recently been shown, but the consumption of aquatic macrophytes by humans is not typical. The uptake by, and accumulation in, crop plants (Nasturtium officinale and Daucus carota) was therefore investigated so as to establish the existence of any risk to humans from the consumption of plants irrigated with water from dams with high cyanobacterial biomass and therefore high BMAA levels. After the exposure to the BMAA through the growth medium, BMAA had no effect on growth and development of N. officinale and D. carota. The uptake and bioaccumulation of BMAA was observed in N. officinale and D. carota, and was found to be concentration-dependent. Both free and bound cellular BMAA was detected following BMAA exposure through the growth medium. The photosynthetic apparatus of N. officinale was not significantly damaged. The uptake and accumulation of BMAA in edible terrestrial plants may constitute another route of human exposure to BMAA; it may now be prudent to avoid spray irrigation of edible plants with waters from dams with high cyanobacterial biomass and therefore high BMAA levels. After uptake by plants, the cyanotoxins may induce oxidative stress. A recent study showed that BMAA has a significant inhibitory effect on the oxidative stress enzymes in C. demersum. Therefore, the toxicological effects on selected plants were investigated by a range of biochemical enzyme assays in order to establish the plant stress response to exogenous BMAA. The inhibition of antioxidant enzymes upon exposure of N. officinale to BMAA through the growth medium was observed. The inhibition of antioxidant defence enzymes by BMAA correlated with the BMAA bioaccumulation in N. officinale. Further investigations are needed to analyze the uptake, accumulation, and ecotoxicology of BMAA in other crop plants, and to examine the fate of BMAA in these plants particularly its distribution and metabolism.

Cyanobacterial and algal blooms can negatively affect water quality particularly when producing toxins that affect human health and wildlife. While reports of blooms are on the rise globally, their underlying causes remain unclear. The goal of this thesis was to determine temporal changes in cyanobacterial abundance and composition through sediment cores in relation to (1) altered land-use leading to cultural eutrophication and (2) warmer air temperatures that have been recorded in the past few decades. This involved evaluating the use of DNA extracted from lake sediments to quantify cyanobacterial abundance and composition. Lake sediments preserved under appropriate storage conditions showed the potential to yield high quality DNA for downstream molecular applications. Cyanobacteria, quantified using the 16S rRNA gene, were found to have increased over the last three decades in comparison to historical averages (since the 1850s) both inside and outside a protected area in western Quebec, Canada, in concert with recent regional warming. Copy numbers of 16S rRNA genes specific to cyanobacteria largely correlated to temporal trends in cyanobacterial pigments. Larger percent increases were seen in cyanobacterial genes in recent times compared to changes in the eubacterial glutamine synthetase (glnA) gene. The mcyD gene was quantified as a proxy for microcystin production through sediment cores from two lakes of western Canada. Copy numbers of both mcyD and Microcystis 16S rRNA correlated with chemical analyses of microcystin through time in cores. Cyanobacteria in the more eutrophic of these lakes shifted toward less diverse assemblages and more toxigenic taxa in recent years. Lastly, temporal and spatial changes in cyanobacterial diversity were analyzed through pyrosequencing of cyanobacterial 16S rRNA along a latitudinal transect representative of northern Canada. Significant shifts towards less diverse assemblages were found, composed of potentially toxigenic strains, suggestive of climate warming in northern latitudes. These results support recent reports of increased abundance and geographic expansion of cyanobacteria and point to increases in cyanotoxins in some cases. Using DNA archived in sediments to determine the historical state of cyanobacterial abundance and diversity could help inform lake management policies.

No description available.