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The nutritional symbiosis between the wood feeding termite and its microbiota is an important model of biomass conversion. Recalcitrant wood is broken down to its basic components and is transformed into energy for the sustenance of the host termite. The microbial community is responsible for cellulose degradation, reductive acetogenesis and nitrogen fixation activities within the gut, and is composed of diverse bacteria, archaea and protists. A prominent taxon within the community is Treponema, a genus of helical motile bacteria. In the higher termite Nasutitermes takasagoensis treponemes are the dominant taxa with the potential of reductive acetogenesis, nitrogen fixation and cellulose degradation, they are joined by members of TG3 and Fibrobacteres and work synergistically in the utilisation of recalcitrant wood components. The genomes of these organisms were analysed using single cell genomics to assign putative functions to these uncultured bacteria and metatranscriptomics to uncover the contributions within the whole community. In the lower termite Hodotermopsis sjoestedti, treponemes are dominant taxa within the cellulolytic protist Eucomonympha. Here the treponeme has evolved an endosymbiotic lifestyle to sustain the host by reductive acetogenesis from H2 and CO2 to supply acetate and nitrogen fixation, and has lost its helical and motile characteristics. This elucidation was made possible again by use of single cell genomics and biochemical analyses. Treponemes from the lower termite Reticulitermes speratus were also sequenced and used in conjunction with other treponeme sequences from diverse environments to classify important genes associated with the environments they were isolated from. Virulence factors from human periodontal strains were the most important at distinguishing the environment they were associated with, whereas termite associated treponemes were categorised by central metabolic genes.

Background: Streptococcus pneumoniae is a significant human pathogen responsible for life-threating diseases such as pneumonia, septicaemia and meningitis. Of the nearly 100 distinct pneumococcal serotypes, pneumococcal serotype 1 is described as one of main causes of invasive disease worldwide. One highly enigmatic feature of serotype 1 resides in its being rarely detected in human nasopharyngeal specimens. Aim: The aim of this project was to compare serotype 1 lineage A (ST306) and C (ST615) with respect to their immunological and virulence properties using both in vitro and in vivo tools, as well as their differential gene expression profile. Methods: A series of in vitro experiments were performed to assess the ability of serotype 1 to adhere and invade epithelial cells, and to determine its ability to inhibit phagocytosis and gain insight into the mechanisms involved. In parallel, three in vivo standardized mouse models of pneumococcal infection were exploited to examine the virulence properties of ST306 and ST615 and their ability to colonise the nasopharyngeal tissue. Finally, RNA-seq analysis of in vitro cultures of ST306 and ST615 was carried out in an attempt to identify differential expression patterns. Results: ST615 serotype 1 was determined to be highly virulent, causing the death of 80-100% of infected mice by around 48h post-infection, while all mice infected with ST306 survived when using pneumococcal doses inductive of invasive pneumonia model. In a nasopharyngeal carriage mouse model, ST306 serotype 1 was shown to be able to establish colonisation persisting up to 28 day post-administration, although at a much lower density compared to ST615. While ST615 was capable of establishing nasopharyngeal colonization, clearance occurred earlier at day 14 compared to ST306. RNA gene expression analysis focused on virulence factors and critical biological functions determined that ST615 serotype 1 presented a profile consistent with its weak colonisation and its invasive properties. The genes associated with capsule synthesis were not differentially expressed between ST615 and ST306 but that other virulence factors such as psp, pavA, ply and cpbD were differentially expressed. Conclusions: Although ST306 and ST615 possesse a unique and virulent capsule, ST306 was not able to cause pneumonia to mice model.

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The ability of two virulent strains (505 and S100) and one avirulent strain (12S) of Corynebacterium fascians to degrade p-indolylacetic acid was examined using colorimetric, paper chromatographic and ultraviolet spectrophotometric methods. While strain 5100 did not degrade p-indolylacetic acid under any of the conditions studied, the decomposition of this substance by the other strains was found to be dependent on the pH of the medium. Cultures and buffered suspensions of strains 12S and 505, at acid pH values, degraded P-indolylacetic acid to an unidentified indole derivative, indole and anthranilic acid. Suspensions of strain 505, buffered at pH 7.0, did not degrade p-indolylacetic acid but with strain 12S, under the same conditions, the p-indolylacetic acid disappeared from the buffer supernatant without the accumulation of indolic breakdown products. It was found that the presence of p-indolylacetic acid in the suspending phosphate buffer, pH 7.0, enhanced the leakage of cellular material from strain 12S. The action of the three strains of Corynebacterium fascians on some other indole derivatives was also examined. Strains 12S and 505 degraded indolepropionic and indolebutyric acids to products similar to those found during the decomposition of p-indolylacetic acid by these strains. Strain 5100 did not attack indolepropionic acid but it degraded indolebutyric acid to p-indolylacetic acid. None of the strains were capable of degrading either indoleacetonitrile or indoleacetamide, in culture. Indole, however, was readily degraded by all three strains to anthranilic acid. The inability of the three strains to decompose skatole and the relatively slow degradation of indolealdehyde by strain 12S, indicated that these two substances were not intermediates in the degradation of IAA. An investigation was made of the use of paper chromatographic analysis of cell extracts for the differentiation of strains of Corynebacterium fascians and related organisms. It was found that while the pattern of material which reacted with ninhydrin was similar in all extracts, some distinct differences could be seen between virulent and avirulent strains of Corynebacterium fascians.

A method for growing Azotobacter vinelandii under conditions of optimal aeration has been devised such that the culture could be sampled frequently so allowing growth and metabolism to be followed continuously. At medium culture densities a discontinuity was observed in the culture properties of both strains used for comparison throughout this work. This discontinuity was attributed to oxygen starvation of the cells and the different properties of the exponentially-growing cultures before and after this change have been noted. The intra- and extra-cellular pools of nitrogen compounds were examined. The presence of 3,4 dihydropyridazinone-5-carboxylic acid could not be confirmed but paper chromatography on its own was clearly inadequate to settle the status of hydrazine derivatives in fixation. The effect of artificial extracellular nitrogen pools on growth aml fixation ras examined using cultures in various states of training with respect to the substrate. The results were confirmed and expended using isotopically labelled ammonium and gaseous nitrogen. Fixation and ammonium uptake occur simultaneously in trained cultures: the implications cf this are discussed rind it is proposed that free ammonium can no longer be regarded as an obligatory intermediate in the fixation process. Urea is metabolised by the same routes as ammonium though its hydrolysis is not effected by a simple exogenous urease. Though L-asparagine and, to a lesser extent, L-glutamine are used Pr partial nitrogen sources, these effects are of minor importance. The amides, in common with L-aspartic, acid, L-glutamic acid and L-alanine, stimulate fixation (per unit cellular material synthesised) by quantities of up to 7O of its normal value. The excess nitrogen thus fixed is excreted either as peptides or in the form of more of the added amino acid itself. These results are Interpreted in terms of specific transport mechanisms and interference with structural peptide synthesis, which is assumed to take place in the cell envelope. The results are fitted into an overall scheme proposed to explain the fate of gaseous nitrogen in the metabolism of Azotobacter vinelandii.

The opportunistic pathogen Clostridium difficile is the most common cause of antibiotic-associated diarrhoea, with severity of disease ranging from mild diarrhoea to fulminant pseudomembranous colitis and death. It poses a major burden on healthcare providers, costing millions of pounds each year due to ward closures, isolation measures and prolonged illness. The current antibiotic therapy for C. difficile infection is effective, but high rates of relapse lead to ongoing misery for patients and spiralling costs for healthcare providers. Novel therapeutics for C. difficile are therefore desperately sought, and the antibiotic-induced nature of the disease has led to interest in development of non-antibiotic therapies. Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. Following the discovery that the Sortase A enzyme of Staphylococcus aureus is essential for pathogenesis, sortase inhibitors are under investigation as novel therapeutics. Being ubiquitous in gram-positive bacteria, it is likely that other gram-positive pathogens require sortase enzymes for their pathogenesis and may be targets for development of sortase inhibitors. This work describes a characterisation of the sortase enzyme of C. difficile. To provide evidence for a role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB. To provide structural insight into the catalytic mechanism of the C. difficile sortase, an active site mutant was crystallised and its structure solved to 2.55Å by X-ray diffraction. The wall-linked protein CD0386 was also crystallised and subject to successful test diffraction. Analyses of SrtB reaction products by chromatography and mass spectroscopy indicate that the enzyme cleaves an SPKTG peptide motif and catalyses a transpeptidation reaction with a component of the C. difficile peptidoglycan.

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No description available.

Bioprocesses performance is monitored using microbial growth kinetics models. However most of them are empirical and unstructured ignoring molecular and transcriptional interactions thus failing in accurate prediction. Pseudomonas putida mt-2, which harbours the TOL plasmid, is a strain of great biotechnological potential. M-xylene and toluene are commonly utilised by TOL pathway while toluene enables chromosomal ortho-cleavage pathway activation. Herein, the transcriptional kinetics of TOL and key ortho-cleavage promoters which control substrate bioconversion resulting in biomass formation was consistently studied. Thus, revealing the interconnection of the two pathways and promoters’ specific expression patterns. The experimental observations lead to a dynamic model coupling transcriptional regulation to microbial growth kinetics by providing upstream quantitative information. This model enables adequate predicition capability of substrate utilisation and biomass growth under a wide range of initial conditions. However in nature it is uncommon for bacteria to degrade a sole substrate. Therefore P. putida mt-2 cells induction with succinate-toluene, m-xylene-toluene mixtures is studied. The transcriptional kinetics revealed promoters’ bi-modal expression pattern and carbon catabolite repression regardless of the growth conditions. Transcriptional regulation upon entry of m-xylene-toluene mixture was modelled resulting in a mechanistic microbial growth kinetic model development which accurately predicts substrate(s) utilisation and biomass growth patterns. The current double substrate microbial growth kinetic model can more accurately predict the macroscopic phenomena compared to the Monod, Monod-type and competitive enzymatic interaction models.