Theoretical models in microbiology have a relative short but successful history. Research presented in this thesis explores the evolutionary origin of aging and the methods used to quantify syntrophic cooperation between microbial species that are distantly related. The mathematical and computational tools used in doing so are developed and discussed in detail. Microorganisms were long thought to be capable of immortality until recent evidence demonstrated otherwise. Theoretical models suggest that aging strategies sacrificing repair for segregation of damage have highest evolutionary fitness, but this is not reflected in nature. The model developed here corrects this view of aging through more realistic assumptions regarding repair. Many estimates of the rate of interspecies metabolite transfer are based on spatial point pattern statistics and assumptions regarding cell surface concentrations. These are shown to be very inaccurate, but proposed alternatives required greater parameterisation. The system is sensitive to difficulties in determining consumption affinity constants, an issue also raised by previous authors.

Microorganisms are the most ubiquitous, diverse, and functionally important organisms on Earth, yet their ecological patterns, and the underlying causative processes that determine their distributions over large spatial scales, remain poorly understood. Therefore, I test for macroecological patterns and processes within microbial communities. I use a combination of data generating approaches including meta-analysis, published sequence datasets and databases, and high-throughput sequencing, coupled with modern statistical methods. Firstly, I show that metagenomic sequencing, is superior to amplicon sequencing as a method of surveying microbial biodiversity, as it detected more diversity at all taxonomic levels. However, cost analysis shows that metagenomics is prohibitively expensive for macroecological studies, where many samples are required. I find that classic macroecological patterns, such as the distance-decay of similarity, are context-dependent and vary according to ecological context, and methodological differences. I therefore make recommendations for future analyses of spatial analyses of microbial communities. Furthermore, I show that whilst microbial communities may exhibit distance-decay relationships, they do not necessarily form biogeographic regions, highlighting a difference in the macroecology of micro- and macroorganisms. I build on this finding by showing that different regional microbial communities can show considerably different responses to the same environmental gradient, hinting that regional communities play an important role in determining microbial community composition at local scales. Finally, I investigate whether regional-scale climatic variables determine the distributions of microorganisms. I show that the climatic drivers and influence of these drivers varies strongly between microbial taxa, suggesting that different microbial taxa have very different macroecologies. I conclude that macroecological patterns in microorganisms may not be as general as in “macroorganisms”, and can be highly context-dependent, varying with taxon, regional metacommunity dynamics, or methodological choices. Careful consideration of these factors is therefore required when attempting to understand how microbial communities will respond to future environmental changes.

This study was designed to determine the regulatory network that controls expression from two \(Escherichia\) \(coli\) K-12 promoters, \(pyeaR\) and \(pogt\), during anaerobic growth. These promoters were identified from transcriptomic studies as being positively regulated by NarL independently of FNR, the master regulator of anaerobic respiration. Biochemical and genetic analyses presented in this study confirmed that expression from both the \(yeaR\) and \(ogt\) promoters is dependent upon NarL, which binds to a single site in the \(yeaR\) promoter and two sites in the \(ogt\) promoter. The nucleoid-associated protein, Fis, repressed transcription from both promoters, especially in rich medium, by binding to sites that overlap the NarL site, excluding the essential activator. Both promoters were more active in the absence of functional FNR. However, mutational analysis revealed that FNR does not bind to the \(yeaR\) promoter region, so this effect is indirect. How the absence of functional FNR might affect NarL-dependent nitrite signalling was investigated. The Ogt protein is known function as an O\(^6\)-alkyguanine methyltransferase. However, the functions of the gene products of \(yeaR-yoaG\) and another operon implicated in nitrosative stress management, \(hcp-hcr\), were unknown. Strains carrying a chromosomal \(yeaR-yoaG\) deletion were not more sensitive to nitric oxide or hydroxylamine compared with the parental strain, suggesting that the products of this operon are not essential for dealing with these toxic nitrogen species. Conversely, a strain deleted in \(hcp-hcr\) was shown to be slightly more sensitive to both nitric oxide and hydroxylamine, implicating Hcp and Hcr in nitrosative stress management.

Mupirocin (Pseudomonic acid A) has long been used against Methicillin Resistant Staphylococcus aureus MRSA, yet bacteria have developed resistance, threatening future use. Structurally similar to mupirocin is thiomarinol A, a natural compound produced by the marine bacterium Pseudoalteromonas spp, which possesses stronger antibacterial activities. However, it differs from mupirocin by four distinct differences and among these are extra 4-hydroxylation and joining to pyrrothine. Studying these differences should enhance our understanding of the molecular assembly and biosynthesis machinery. Complementation and mutagenesis studies identified the tmuB gene to be responsible for the 4-hydroxylation as a final tailoring step. In vivo and in vitro studies on purified TmuB revealed that it can hydroxylate diverse pseudomonic acids but is inhibited by molecules with an 8-hydroxyl group, which primarily affects catalysis rather than binding. Molecular modelling plus docking and mutagenesis provides increased understanding of both TmuB potential to modify other substrates and how mupirocin activity can be modulated by 4-hydroxylation. This study also expressed holA, purified its gene product, a non-ribosomal polypeptide synthetase (NRPS), and assayed its activity by pyrophosphate release. It presents a proposed pathway for pyrrothine biosynthesis catalysed by HolA, which exhibits the unusual ability to join two cysteine molecules by a single NRPS module.

Anammox (anaerobic ammonium oxidation) is an environmentally significant process with great importance for global biogeochemical cycles. This process is mediated by a unique suite of phylogenetically distinct chemolithoautotrophic bacteria which demonstrate novel physiological and metabolic characteristics. However, despite the importance of these organisms, there is still much which is poorly understood about them, specifically the diversity and distribution of these bacteria and their controlling environmental factors. Furthermore, genomic studies and observations from the field suggest that anammox bacteria may have a far greater metabolic diversity than previously thought, suggesting that the current understanding of these organisms is incomplete. This study aimed to elucidate these aspects of the ecology of anammox bacteria in estuarine and OMZ (oxygen minimum zone) environments. A clear community shift was observed in estuarine environments from Ca. Brocadia spp. dominated freshwater sites to Ca. Scalindua spp. dominated marine sites. The OMZ was dominated by Ca. Scalindua spp. though diversity within this clade was observed between organisms in the upper oxycline and those within the core of the OMZ. Microcosm experiments amended with organic substrates suggested that some anammox organisms (namely Ca. Brocadia spp., Ca. Jettenia spp. and Ca. Kuenenia spp.) may have the ability to assimilate carbon directly from organic substrates such as dimethylamine and urea. However, these data were inconclusive and further investigations are required to prove or disprove the hypothesis that anammox bacteria can utilise organic substrates. Nevertheless, this study improves the understanding of the ecology of anammox organisms in estuarine and OMZ environments, providing an unprecedented depth of data as to the diversity and distribution and unique insights into potentially novel metabolic capabilities of these organisms.

The population dynamics of carbon-starved E. coli K12 cultures was investigated. It was found that less cell lysis occurred when cells were previously grown in low glucose concentrations. Exponential-phase cells grown previously in 0.05% (w/v) glucose had survival rates comparable with their stationary-phase counterparts, suggesting that the rate of growth is more important in determining the outcome of starvation than the phase of batch culture growth. Long-termstarved cells (18-24 months) showed very little protein, DNA and RNA synthesis. Methionine was shown to alter the de novo synthesis protein profiles of longterm- starved cells and growth was seen to occur in the presence of methionine. This suggests that radio-labelling of proteins with 35S-methionine in these cells should be interpreted with care as the cells have been subjected to a nutrient upshift. Radio-labelling of proteins with 3H-leucine did not have the same effect. The ATP content of cells during prolonged incubation was shown to decrease in the first 48 hours incubation, increase until 5-7 days incubation then decrease after 7-8 days. After 13 days a slow, steady increase occurred. The ATP content of cells incubated for 16 days was higher than that of 48 hour-incubated cells. The physiology of long-term-starved cells was investigated with respect to their permeability to routine bacteriological stains ( e.g. DAPI, saffranin, Geimsa) and it was found that very few of these dyes were able to penetrate the cells, indicating that a decrease in cell permeability may be an important factor in survival as is seen in endospores of Bacillus species and swarmer cells of Rhodomicrobium vannielii and Caulobacter crescentus. Resistance of long-term starved cells to heat and biocide challenge was increased in comparison with exponential- and short-term (48 hour) stationary-phase cells and the resistance to biocides was shown to be retained through subsequent generations. Examination of the nucleoids of long-term-starved cells revealed that a more condensed form was present in cultures incubated for over 14 days, suggesting that dehydration of the DNA had occurred, similar to the situation found in endospores of Bacillus species and suggestive of dormancy. Analysis of outer-membrane proteins and lipopolysaccharide of long-term-starved cells showed that alterations occurred to the surface of the cells and it was demonstrated that hydrophobicity changes occurred. Hydrophobicity reached a maximum after 48 hours incubation then subsequently declined between days 2 and 3 which corresponded with an increase in cell numbers. Cell surface hydrophobicity was shown to be a potential method for separating heterogeneous, carbon-starved populations into homogeneous subpopulations. The data suggest that E. coli produces a dormant survival cell type which is morphologically and physiologically distinct from the parent cell.

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound.

No description available.

Detailed steroid hormone profiling of human follicular fluid has paved the way for research into the modulation of human sperm by physiological concentrations of follicular fluid steroid hormones. Synthetic human follicular fluid (shFF), a novel steroid hormone analogue of human follicular fluid, based upon local data, was prepared consisting of 14 different steroid hormones including progesterone. Exposure of human spermatozoa ( > 2000 cells) to shFF stimulus at physiological and standard laboratory temperatures resulted in a rapid biphasic elevation in [Ca2+]i characterised by an initial transient Ca2+ influx immediately followed by a sustained elevation of [Ca2+]i for the duration of shFF exposure. A significant increase in the percentage of acrosome-reacted spermatozoa was observed in shFF-treated sperm (P < 0.05) however, this was significantly lower than the % AR in spermatozoa treated with progesterone alone (P < 0.01). With regards to shFF-induced sperm kinesis, a significant reduction in selected sperm motility parameters was observed 5 minutes post-incubation with shFF (P < 0.05). The study of shFF-induced chemotaxis revealed a chemokinetic effect characterised by a significant inhibition of sperm migration up a gradient of shFF (P < 0.05), possibly due to ‘hyperactivated trapping’. We conclude that the high concentration of progesterone (13.5µM) present in the shFF mixture is likely to be responsible for the biphasic sperm [Ca2+]i influx characteristic of a progesterone stimulus. However, the data obtained from the sperm kinesis and AR experiments leads us to hypothesize that the other steroid hormones present in the shFF mixture exert antagonistic effects on progesterone-mediated physiological responses in human spermatozoa.

The activities of competing wood decay fungi bring about the decomposition of deadwood. The rate of decomposition is determined by the community composition, which is shaped by competitive interactions. Fungi compete with one another for territory and resources within 3 dimensional space via the production of an arsenal of inhibitory chemical compounds, yet interspecific interactions and chemical warfare within communities have never before been studied in 3 dimensional resources. This thesis, therefore, aims to determine how interactions cause metabolic processes to change, and, by the use of novel 3 dimensional systems, how those processes are affected by increased species diversity and spatial heterogeneity. Overarching hypotheses which have driven investigations are: (1) secondary metabolism and interaction outcome are inherently linked, both of which are affected by environmental conditions, (2) changes to spatial distributions and increased species diversity cause interaction outcomes and community dynamics to alter, and (3) changes to metabolic strategies for antagonism and resource utilisation will reflect alterations to community dynamics. Transitive interactions in pair wise 2 dimensional systems often became intransitive in species richer 3 dimensional systems, and the extent of patch fragmentation determined the length of coexistence of individuals within communities. Production of secondary metabolites comprising the fungal chemical weaponry, namely extracellular lignocellulolytic enzymes and volatile organic compounds (VOCs), was significantly different between 2 and 3 dimensional systems, between systems of varied species diversity, and between different spatial distributions of fungi, reflecting the observed changes to community dynamics. Furthermore, the production of intracellular metabolites which function in the biosynthesis pathways of antimicrobial compounds and agents of decay, was affected by species diversity, spatial distributions and territory fragmentation. Additionally, secondary metabolism and interaction outcome were found to be inherently linked, and affected by abiotic conditions. Specifically, interaction outcomes changed under different temperatures, and when wood was pre colonised for different lengths of time. Changes to the production of both enzymes and VOCs reflected the different outcomes, as either the cause or effect of outcome changes. These conclusions not only confirm the thesis hypotheses, but also demonstrate the importance of environmental conditions, species diversity and spatial dynamics to fungal community dynamics and forest ecosystem functioning.