To fulfil global energy demand and to mitigate economical, geopolitical and ecological challenges associated with fossil fuel utilisation, the energy sector is moving towards greater use of sustainable and environmentally friendly energy sources, including biofuels. The ideal transport biofuel would be hydrocarbons that are identical to fossil petroleum. However, to date characterised hydrocarbon biosynthetic pathways include a decarbonylation or decarboxylation reaction, which involves the loss of one carbon resulting in odd-numbered carbon chain hydrocarbons. This carbon loss decreases carbon efficiency for alkane production, which reduces microbial fuel economic competitiveness. Therefore, it is key that new pathways for alkane production are identified. The sulphate-reducing bacteria genus Desulfovibrio was previously reported to synthesise even-numbered carbon chain alkanes, which suggests an alternative route for alkane production without carbon loss. This investigation aimed to verify Desulfovibrio alkane biosynthesis and characterise the possible synthetic pathway. Ten Desulfovibrio strains, representing seven species, were screened for alkane synthesis using isotopically labelled growth media. The ability to produce alkanes within the Desulfovibrio genus was confirmed and was shown to be strain-specific under a set of culture conditions. The biogenic alkanes detected were octadecane (C18), nonadecane (C19) and eicosane (C20), with a predominance of even-numbered carbon chain alkanes. Fatty acid analysis of Desulfovibrio strains showed an alkane biosynthetic pathway was unlikely to involve a decarbonylation or decarboxylation step. A novel hypothesis was therefore proposed that alkane biosynthesis by Desulfovibrio follows a metabolic route, which has not previously been characterised, involving a series of reduction reactions from the fatty acid pool. The characterisation of the putative Desulfovibrio hydrogenation pathway for alkane biosynthesis was undertaken via a target-directed genome mining approach. The genomic DNA of nine Desulfovibrio spp. was purified, sequenced, de novo assembled and annotated. Seven of these nine genomes are unpublished to date. No homologs of previously characterised alkane biosynthetic enzymes from bacteria were in silico identified in the genomes and proteomes of alkane producing Desulfovibrio spp., suggesting that Desulfovibrio alkane biosynthetic pathway is likely to be catalysed by currently uncharacterised enzymes. The 16S rRNA-based phylogeny of Desulfovibrio spp. supported the hypothesis that the Desulfovibrio alkane biosynthetic pathway was acquired by a common ancestral strain via horizontal gene transfer. The ability of Desulfovibrio to produce alkanes was therefore hypothesised to be due to the presence of recruited genes encoding enzymes involved in alkane synthesis. A comparative genomic analysis intersecting six-alkane producing and four non-alkane producing Desulfovibrio genomes resulted in the in silico identification of 33 hypothetical proteins considered with high confidence to be exclusive to alkane producing Desulfovibrio strains. A novel hypothetical Desulfovibrio alkane biosynthetic pathway was proposed involving a V-type ATPase, an uncharacterised protein, named as a putative reductase in this investigation, and a putative methyltransferase, which were predicted to be exclusive to alkane producing Desulfovibrio spp. The inorganic phosphates resulting from the ATP hydrolysis catalysed by the V-type ATPase would be involved in a reaction with fatty alcohols to form alkyl phosphates, which are putative activated intermediates required for the hydrogenation route from fatty alcohols to alkanes. The putative reductase and the methyltransferase, predicted to share similar structural features with known alkane-binding proteins, would subsequently reduce alkyl phosphates to alkanes and to iso-alkanes respectively. Empirical investigation of the candidate molecular basis function in Desulfovibrio alkane biosynthesis was undertaken. The Desulfovibrio alkane biosynthetic pathway remains to be fully characterised.

Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up-regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv.

Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up-regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv.

Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up-regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate