The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation.

Access to renewable energy and reduction of carbon emissions represent two major issues facing humankind in the twenty first century and beyond. The underlying driving forces behind both are multi-faceted and often intrinsically connected, ranging from environmental concerns over climate change to improving economic security through self-sustaining energy production. Possible solutions to reliance on non-renewable, carbon-emitting fossil fuels have been explored over recent decades, with significant interest placed on biofuels. Due to ease of integration into liquid-based petrochemical fuel infrastructure, these renewable alternatives have been a consistent topic of both industrial and academic interest. Despite offering renewable energy, conventional crop-based biofuel production has faced criticism due to consumption of land, water and other resources associated with agriculture. Acetogens provide a solution to conventional biofuel production due to their utilisation of carbon monoxide and carbon dioxide gas as carbon and energy sources, rather than plant matter. This allows generation of a range of chemical products from a broad range of sources, including industrial waste gases and gasified solid waste. Acetogens offer the double benefit of both renewable energy production, and carbon emission sequestation. This study outlines the development of genetic tools to provide a foundation for using synthetic biology approaches to improve performance of acetogens as industrial chassis. Specifically, development of tools and techniques for the acetogen Clostridium autoethanogenum are described, with further applications of such technology to other Clostridia.

Phosphotransferase systems (PTS) represent an important method of sugar uptake in bacteria and have been well described in the past. However, research into PTS within the genus Clostridium has been mainly restricted to the non-pathogens. Analysis of the genome of Clostridium difficile 630 revealed over 40 intact PTS; this is over three times as many as in other pathogenic Clostridia, such as Clostridium perfringens and Clostridium botulinum. Previously, carbon catabolite repression has been shown to affect toxin production in C. difficile. Being capable of utilising different carbohydrates efficiently could be important for C. difficile to adapt to, grow, and survive in the human gut. So far, little work has been done to corroborate the role of individual PTS in carbohydrate uptake, sensing of environmental stimuli and regulation of virulence, i.e. toxin expression. A deeper understanding of the PTS in C. difficile, and their importance in virulence, could lead to the development of new drug targets. The aim of this study is to characterise the main PTS of C. difficile, determine their role in carbohydrate uptake, and their effect on regulation of virulence. To date, we have chosen the main candidates thought to be involved in mannitol and sorbitol uptake, and have inactivated these PTS using the ClosTron and in-frame deletion methods. Phenotypic characterisation of these mutants was undertaken to prove their role in uptake of the relevant sugar and to determine their role in virulence regulation. This study has demonstrated, by growth assays and HPLC, that the operons at CD630_0762-8 and CD630_2331-4 respectively encode PTS specific for sorbitol and mannitol uptake. In the case of the mannitol operon, it has been proved (through the use of cytotoxicity assays, which showed reduced bacterial toxicity in the presence of the sugar) that the suppression of toxin synthesis in the presence of mannitol is dependent upon uptake of the substrate via this operon. With sorbitol, toxin levels are, seemingly, not directly reliant upon uptake of the sugar, resulting in, mainly, an increase of toxin in sorbitol. Presently, it is not possible to say whether these systems have a distinct role or not in the motility of the organism.

abstract: The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Made available in DSpace on 2014-06-11T19:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-09Bitstream added on 2014-06-13T20:16:35Z : No. of bitstreams: 1 silva_cm_me_sjrp.pdf: 624368 bytes, checksum: 527ab11904f1d5551c5d3ed9ee3c5385 (MD5) / Embora com hábito primariamente aquático, a flora de algas terrestres, em especial de cianobactérias, é relativamente luxuriante e tais organismos desempenham papel ecológico importante. Dentre estes organismos, Scytonema é um dos gêneros mais frequentes, abundantes e diversificados em ambientes terrestres e a taxonomia infragenérica tem se revelado problemática. Considerando-se a importância e representatividade do gênero, tornam-se necessárias investigações para melhor definição e distinção de espécies de Scytonema, bem como um melhor estabelecimento dos limites com gêneros próximos (Petalonema e Brasilonema, por exemplo). Tal estudo tem como objetivos aprofundar os estudos taxônomicos dos gêneros Brasilonema, Petalone e Scytonema; comparar espécies de Scytonema de ambientes aerofíticos brasileiros com registros das espécies encontradas em diferentes ambientes mundiais inicialmente quanto a características morfológicas e, em alguns casos, segundo características moleculares; reavaliar critérios taxonômicos utilizados na classificação; avaliar relações biogeográficas de espécies selecionadas. Os trabalhos de campo resultaram na coleta de 44 amostras com a presença de 22 táxons pertencentes às Nostocales com ramificações falsas. Foram identificadas 14 populações como Scytonema, três como Brasilonema e cinco como Petalonema, sendo descritas morfologicamente e apresentadas as condições ambientais de ocorrência. Na tentativa de se obter culturas desses organismos, foram utilizados diferentes meios de cultivo, diferentes temperaturas e irradiância, entretanto não foi uma etapa bem sucedida. As análises moleculares foram positivas para sete populações, com sequencias de aproximadamente 600 pb. As análises revelaram que há grande similaridade molecular entre espécies do gênero Brasilonema e aqueles inicialmente identificados como Scytonema... / Not available

16S rDNA sequences from 23 heterocytous cyanobacteria were obtained and phylogenetic tree from these sequences and sequences available in the GenBank was constructed. Relationships between traditional taxonomy and phylogenetic clusters were discussed.

D.Phil. (Chemistry) / Please refer to full text to view abstract

Avian tuberculosis remains a significant economic problem for the poultry industry in many countries. An improved understanding of the interaction between host and mycobacteria is important in designing more efficient vaccine strategies. This study investigated the immune responses to M. avium (Ma.) strains isolated from different sources, Ma strains 1-2 isolated from chickens, Ma.3-5 from cattle and Ma.6-8 from humans, using human cells in vitro and avian cells in vitro and in vivo. The responses were characterised by expression levels of pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6 and chemokines CXCLi1 (K60) and CXCLi2 (IL-8) and nitric oxide production. A differential immune response was observed between the strains in terms of induction of cytokine and chemokine production in both human THP-1 and avian HD11 cells. With the exception of the avian strains 1 and 2, all strains induced increases in pro-inflammatory cytokines in THP1 cells. Two of the 3 bovine strains also had this effect in HD11 cells but the other strains had little effect. Use of specific inhibitors indicated that in THP-1 cells the extracellular regulating kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were important in inducing pro-inflammatory cytokines whereas the p38 pathway did not appear to be involved particularly in regulating TNF-α and IL-1β production. The results also indicate an interaction between the mitogen activated protein kinases (MAPK) pathways studied in regulation of cytokine production in HD11 cells. Although layer chickens were resistant to extensive M. avium multiplication in the tissues, significant expression of pro-inflammatory cytokines was observed in the liver of infected birds towards the end of the period of observation of 21 days as granulomata had started to appear, characterised by lymphocyte accumulation. In conclusion, the findings of this study provide further understanding of the host-mycobacteria interaction and that different strains of M. avium might behave differentially regardless of their source of isolation.

Philosophiae Doctor - PhD / This thesis presents significant advances into the microbial ecology of hypolithic communities in two hyperarid deserts. Deserts account for one fifth of the Earths total surface area. These zones differ substantially in terms of climate, geomorphology, hydrology and vegetation. Desert biomes are, however, generally depauperate with particularly with respect to macroorganisms. Hypoliths, photosynthetic microbial assemblages associated with quartz rocks, are widely distributed in hot and cold desert environs where they may represent a large fraction of the standing biomass and mediate key ecosystem processes, including nutrient cycling. However, important questions regarding their (i) development (ii) community structure and assembly patterns and (iii) functional structure remain unaddressed. Here, molecular tools (T-RFLP, clone libraries and pyrosequencing) and multivariate data analyses were used to address these questions. This study presents evidence of species recruitment in the development of hypolithic communities in the Namib Desert. Hypolithic bacterial communities were compared at a fine scale (10 m radius). Multivariate analysis of T-RFLP-derived data showed that hypolithic and open soil communities were structurally distinct. Applying the ecological concept of ‘indicator species’, 6 and 9 indicator lineages were identified for hypoliths and soil, respectively. Hypolithic communities were dominated by cyanobacteria affiliated to Pleurocapsales, whereas actinobacteria were prevalent in the open soil. These results are consistent with the concept of species sorting and suggest that the underside of the quartz rocks provide conditions suitable for the development of discrete and demonstrably different microbial assemblages.However, strong evidence for neutral assembly processes was found, as almost 90% of the taxa present in the hypoliths were also detected in the open soil. All together, these results suggest that hypolithons do not develop independently from microbial communities found in the surrounding soil, but selectively recruit from local populations.The bacterial community structure and assembly patterns in hypolithons from Miers Valley (Antarctica) were investigated. Previous studies in this valley have identified three morphologically distinct hypolithic community types: cyanobacteria dominated(Type I), fungus dominated (Type II) and moss dominated (Type III). The bacterial composition of surface soils and hypolithic communities were shown to be clearly and robustly distinct, using T-RFLP analysis. Moreover, the bacterial assemblages were similar in Type II and Type III hypolithons and clearly distinct from those foundin Type I. Using16S ribosomal RNA gene (rRNA) 454 pyrosequencing,Proteobacteria were shown to be the most important bacterial component of all three types of hypolithic communities. As expected, Cyanobacteria dominated Type I hypolithons, whereas Actinobacteria dominated Types II and III hypolithons. Using a probabilistic dissimilarity metric and random sampling, deterministic processes were demonstrated to be relatively more important in shaping the structure of the bacterial community found in Type II and Type III hypolithons. Taken together, these results suggest that hypolithic development favors a sequential pathway with Type II hypolithons serving as an intermediate development state between Type I and Type In a more in depth analysis of the diversity patterns of key nutrient cycling genes in Antarctic Miers Valley edaphic communities, genes coding for carbon fixation (greenand red-like cbbL), nitrogen fixation (nifH), nitrification (amoA) and denitrification(nirK and nirS), were targeted. Multivariate analysis (PERMANOVA) showed that hypolithic and open soil communities were functionally distinct. Type I hypoliths were functionally more diverse than soils, suggesting higher potential for enzymatic activities. Taxonomic structure (derived from 16S rRNA data) showed congruence with functional traits (genes involved in C and N cycling). Redundancy analysis suggested that chemical variables (S, F, and NO3) were important structuring forces in the different communities. Taken together, the results suggest that stochastic processes such as dispersion cannot override the influence of environmental factors on functional diversity patterns.

High-resolution lithostratigraphic data from rock sequences known as the Indidura Formation near Parras de La Fuente, Coahuila, NE Mexico, led to achieve a significant improvement of our knowledge of that Formation. The results of this study indicate for the first time that the sequence at Parras de La Fuente developed from the deposition of calcareous cyanobacterial microspheroids that accumulated under perennial blooms during the Late Cenomanian through the Middle Turonian. Multi-proxy analyses included sedimentological, petrographical, scanning electron microscopy, stable isotope, trace element geochemistry, and paleontological data. The combined results allowed the correlation of δ13C and anomalies in Mo, V, and Cr with the abundance and predominance of calcareous cyanobacterial microspheroids, which were the main suppliers of the carbonate components and the organic matter throughout deposition of the Indidura Formation in the Parras de la Fuente area, under dysoxic/anoxic conditions. Conspicuous interbeds of dark and light-gray laminated marly calcilutites, and dark-gray marlstones that characterize the stratigraphic sequence formed in response to external forcing climatic factors of millennial-scale Milankovitch cycles (ca. 20 ka precession). At the microscopic level, the prominent dark and light-gray laminae were formed during cycles similar to the 10 to 15 years solar irradiance maximum, and represent alternating periods of high and low calcareous cyanobacterial microspheroids productivity.