Metagenomic, metabolic and functional characterisation of polyextremophilic microbial consortia endogenous to acid mine drainage

The generation of extremely acidic and metal-contaminated effluent from mining can cause large scale environmental destruction, degradation of surrounding freshwater and soil and poses risks to both ecosystem and human health. Due to the chemistry of this synthetic ‘acid mine drainage’ (AMD), the diversity and function of microbial consortia native to such sites has been largely neglected. Firstly, in order determine the chemical composition of the acidic sediment, elemental analysis was performed, and compared to two non-extreme freshwater sites not impacted by mining- derived pollution. The acid mine drainage sediment (collected from a previous mined site in Central Scotland) demonstrated an extremely low pH, carbon levels reflective of oligotrophic conditions, elevated concentrations of heavy metals, and high sulphate levels. Taken together, these chemical characteristics demonstrated the abiotic conditions and nutritional scarcity associated with microbial proliferation in heavily contaminated systems. Subsequent metabolic profiling of sediment communities involving carbon-source degradation again demonstrated the acidophilic consortia’s more restricted diversity and reduced capacity to catabolise organic compounds compared to control environments and revealed significantly different carbon source utilisation patterns compared to copiotrophic and an additional oligotrophic microbial assemblage. Using a novel cultivation strategy, a member of the genus Thiomonas was successfully isolated, demonstrating the premise that extremophiles can be successfully recovered from their native conditions using non-conventional culture-based approaches. Further amplicon-based molecular analyses allowed elucidation of key taxonomic groups of microorganisms, and quantitation of their functional marker gene abundance. qPCR demonstrated the presence of methane cycling organisms via detection of the mcrA and pmoA genes, responsible for functions previously poorly, or not reported for, in members of acid mine drainage assemblages. Fungal presence was confirmed via quantification of the intergenic ITS2 region, expanding current knowledge on the biogeographical distribution of eukaryotes in low pH, synthetically derived effluents. Thereafter, the routes to community characterisation focused on metagenomic and metataxonomic approaches; allowing the elucidation of members of the acidophilic population and functional attributes which promote microbial survival in hazardous conditions. Long read DNA sequencing using Oxford Nanopore Technology successful allowed the reconstruction of metagenomic assembled genomes (MAGs); assemblies pertaining to organisms from the Pseudomonadota and Actinomycetota phyla were selected for further genomic analysis. Metabolic functions previously unreported for members of acid mine drainage communities were evident within the reassembled bacterial chromosomes. The highest quality assembly pertaining to a genome recovered from Metallibacterium scheffleri demonstrated multiple polyextremophilic adaptations, including resistance mechanisms to counteract acid influx into the cell. The Metallibacterium MAG sequence also demonstrated metabolic strategies employed by this taxon to survive in otherwise deleterious concentrations of pollutants, chiefly exposure to transition metals (via the presence of zitB, copA/B, cutA, merA and ATM-1 genes) and the metalloid arsenic (via arsB/C). Importantly, the Metallibacterium assembled genome also demonstrated more diverse carbon utilisation routes than previously reported, with three complete glycolytic pathways noted. Sulphur cycling capabilities previously contested for Metallibacterium were also evident within the content of the assembled chromosome. These results indicated Metallibacterium’s importance in underpinning biogeochemical function in acidic waste streams and the array of metabolic strategies that could exploited if using Metallibacterium as a future candidate for bioremediation. The Metallibacterium MAG also demonstrated an array of sulphur cycling mechanisms unreported in the previous literature. Additional MAGs relating to Acidiphilum and Mycobacterium are also presented; these MAGs also indicate novel functions, including extreme acid tolerance, autotrophy, and hydrocarbon degradation. Lastly, bioreactor-based experiments were employed to explore whether a specific community function (methanogenesis) could be established from sediment communities. A strong derivation of methanogenic function was achieved using copiotrophic communities. Methane output when using the acidophilic assemblage as a novel inocula was sporadic, however methane was detected in a single bioreactor. In this case, the archaeal community from Benhar Bing within the starting inocula appeared to withstand the bioreactor conditions and produced biogas (with over 65% methane detected within the headspace gas). Based on phylogenetic analysis of both the starting sample and the end point bioreactor samples following dismantling of the reactors (by pre-amplification of the archaeal 16S rRNA gene and subsequent analysis of the V4 region), it is likely that methanogens native to the extremely acidic conditions belong to the Thermoplasmatales which like their sister lineage, the Methanoplasmatales, may utilise a methylotrophic methanogenic pathway to conserve energy. Overall, acidophilic assemblages underpin ecological function in ecosystems degraded by anthropogenic mining activities and can promote pollutant mobility. Despite this, metagenomic results demonstrated that microbial species present can also aid in ecosystem recovery and can promote metal detoxication and the closure of metal redox cycles. Future work could continue to focus on methane cycling occurring in non-standard systems which have been overlooked regarding potential greenhouse gas emissions, which have likely been underestimated. Acidophilic assemblages hold an untapped metabolic repertoire that could be exploited in both the remediation of contaminated systems and potential low pH waste to resource biotechnologies

Adaptation and diversification of <i>Escherichia coli</i> K12 MC1000 in a complex environment

Complexiteit is inherent aan natuurlijke zowel als industriele habitats. Voorgaand wetenschappelijk werk heeft duidelijk het flexibele (genotypische en fenotypische) aanpassingsvermogen van microorganismen aan complexiteit laten zien. De meeste experimenten zijn echter onder relatief simpele (uniforme) omstandigheden verricht. Derhalve richtte het huidige onderzoek zich op bacteriele evolutie in complex groeimedium, waarbij de nadruk lag op de analyse van de mate van genetische / fysiologische diversifiering naar fitnessverhoging en nichedifferentiatie.De lange-termijn-aanpassingen (~1000 generaties) van E. coli K12 MC1000 in Luria-Bertani (LB) bouillon onder aerobe, wisselende en anaerobe condities werden geevalueerd. Verschillende genetische wegen resulteerden in aanpassingen en een aantal metabole routes waren geactiveerd. De veranderingen waren reproduceerbaar met betrekking tot geselecteerde functie, waarbij habitat de belangrijkste selector bleek. Een specific response werd waargenomen in de genen die betrokken waren bij het metabolisme van galactose (galR en galE). Daarbij werd een hoge mate van heterogeniteit gevonden tussen en binnen populaties. De verschillende fenotypische aanpassingen gaven ook aan dat parallele responses werden gestuurd door de verschillende genomen.De analyse van polymorfismen binnen een geevolueerde population toonde het bestaan van twee metabole and interactieve typen aan. Derhalve werd het voorkomen van additionele specifieke fenotypische eigenschappen (stress resistentie en metabole eigenschappen) bevestigd. De interactieve en stabiele coexistentie van deze vormen liet trade-offs in groei- en stress-eigenschappen tussen de vormen, en nicheverdeling, zien. De complexiteit van de habitat kan derhalve de vorming van aangepaste coexisterende vormen sturen.An inherent characteristic of natural as well as industrial environments is complexity. Scientific studies have revealed the flexible genetic and phenotypic capacities of microorganisms to cope with such complexity. However, most experiments have been conceptually simple, as they compare populations adapting to rather uniform environments. Therefore, the present work addressed bacterial evolution in a complex environment. The emphasis was on unraveling the level of diversification in respect of the genetic and physiological changes that the organism underwent, which allowed it to either acquire superior fitness or occupy a different niche.The long-term (~1000 generations) adaptive responses of E. coli K12 MC1000 in Luria-Bertani (LB) broth under aerobic, fluctuating and anaerobic conditions were evaluated. Several genetic solutions led to adaptation and a number of metabolic pathways were activated. Reproducibility of changes on genuine targets of selection was observed in parallel populations, suggesting a response triggered by medium. A specific response occurred in genes related to the metabolisms of galactose (galR and galE). Considerable heterogeneity was also found between and within populations. Differential phenotypic outcomes, suggested that parallel responses were affected by differing genomic backgrounds. Analysis of the polymorphisms in one evolved population revealed the existence of two main metabolic and interactive types. The emergence of additional specific phenotypic traits (stress resistance and metabolic properties) was confirmed. The interactive and stable coexistence of these forms revealed the presence of trade-offs and niche partitioning. The complexity of the environment has the potential to trigger the establishment of adapted and coexisting forms

Adaptation and diversification of <i>Escherichia coli</i> K12 MC1000 in a complex environment

Complexiteit is inherent aan natuurlijke zowel als industriele habitats. Voorgaand wetenschappelijk werk heeft duidelijk het flexibele (genotypische en fenotypische) aanpassingsvermogen van microorganismen aan complexiteit laten zien. De meeste experimenten zijn echter onder relatief simpele (uniforme) omstandigheden verricht. Derhalve richtte het huidige onderzoek zich op bacteriele evolutie in complex groeimedium, waarbij de nadruk lag op de analyse van de mate van genetische / fysiologische diversifiering naar fitnessverhoging en nichedifferentiatie.De lange-termijn-aanpassingen (~1000 generaties) van E. coli K12 MC1000 in Luria-Bertani (LB) bouillon onder aerobe, wisselende en anaerobe condities werden geevalueerd. Verschillende genetische wegen resulteerden in aanpassingen en een aantal metabole routes waren geactiveerd. De veranderingen waren reproduceerbaar met betrekking tot geselecteerde functie, waarbij habitat de belangrijkste selector bleek. Een specific response werd waargenomen in de genen die betrokken waren bij het metabolisme van galactose (galR en galE). Daarbij werd een hoge mate van heterogeniteit gevonden tussen en binnen populaties. De verschillende fenotypische aanpassingen gaven ook aan dat parallele responses werden gestuurd door de verschillende genomen.De analyse van polymorfismen binnen een geevolueerde population toonde het bestaan van twee metabole and interactieve typen aan. Derhalve werd het voorkomen van additionele specifieke fenotypische eigenschappen (stress resistentie en metabole eigenschappen) bevestigd. De interactieve en stabiele coexistentie van deze vormen liet trade-offs in groei- en stress-eigenschappen tussen de vormen, en nicheverdeling, zien. De complexiteit van de habitat kan derhalve de vorming van aangepaste coexisterende vormen sturen.An inherent characteristic of natural as well as industrial environments is complexity. Scientific studies have revealed the flexible genetic and phenotypic capacities of microorganisms to cope with such complexity. However, most experiments have been conceptually simple, as they compare populations adapting to rather uniform environments. Therefore, the present work addressed bacterial evolution in a complex environment. The emphasis was on unraveling the level of diversification in respect of the genetic and physiological changes that the organism underwent, which allowed it to either acquire superior fitness or occupy a different niche.The long-term (~1000 generations) adaptive responses of E. coli K12 MC1000 in Luria-Bertani (LB) broth under aerobic, fluctuating and anaerobic conditions were evaluated. Several genetic solutions led to adaptation and a number of metabolic pathways were activated. Reproducibility of changes on genuine targets of selection was observed in parallel populations, suggesting a response triggered by medium. A specific response occurred in genes related to the metabolisms of galactose (galR and galE). Considerable heterogeneity was also found between and within populations. Differential phenotypic outcomes, suggested that parallel responses were affected by differing genomic backgrounds. Analysis of the polymorphisms in one evolved population revealed the existence of two main metabolic and interactive types. The emergence of additional specific phenotypic traits (stress resistance and metabolic properties) was confirmed. The interactive and stable coexistence of these forms revealed the presence of trade-offs and niche partitioning. The complexity of the environment has the potential to trigger the establishment of adapted and coexisting forms

Functional Genomics of the Insect-Vector Symbiont, Sodalis glossinidius

Animal- (AAT) and human African trypanosomiasis (HAT) is endemic within sub-Saharan Africa and is caused by Trypanosoma spp. parasites vectored by biting tsetse flies. The facultative secondary symbiont, Sodalis glossinidius, has been controversially implemented in increased parasite establishment in tsetse. As the role of S. glossinidius in tsetse is not fully understood within the literature, the research presented here aimed to utilise a functional genomics approach to elucidate S. glossinidius functionality from a genetic context. Initial phenotypic-level in vitro media screening experiments confirmed S. glossinidius heterotrophy and revealed higher growth levels in the presence of glucose: S. glossinidius was unable to grow in a minimal salts medium (M9) devoid of a sufficient organic carbon source, and showed higher growth values in glucose-positive M9 variations compared to equivalent glucose-negative counterparts. This glucose utilisation was also observed with better growth between a complex medium rich in glucose (Mitsuhashi and Maramorosch Insect Medium) versus one with lower concentrations (Schneider’s Insect Medium). Subsequent genotypic-level transposon-directed insertion site sequencing (TraDIS) library selection experiments supported S. glossinidius glucose utilisation with essential gene candidacy in glycolysis, gluconeogenesis and the pentose phosphate pathway. These results, in combination within essentiality in the citric acid cycle, a wide range of carbon source metabolism pathways, and virulence-associated genes (Omp porins, flagellar components and type III secretion system constituents), experimentally confirm the sequence-inferred literature consensus that S. glossinidius has retained a functional repertoire more aligned with free-living organisms. Many of the essential gene candidates were pseudogenes, which when considered with the literature evidence that S. glossinidius is actively maintaining a core pseudogene set across lineages, experimentally supports the theory that symbionts in early stages of genome degradation associated with the free-living to symbiont lifestyle switch preference pseudogene retention. The novel TraDIS library presented here provides the currently missing tool for subsequent targeted functionality in vivo experiments, aimed at fully understanding the S. glossinidius role in the tsetse system

The development of efficient hemi-autotrophic carbon fixation in Escherichia Coli

Carbon fixation is a process vital to any life and as by far its most prevalent variant, the Calvin Benson Bassham (CBB) cycle is vital to virtually all known terrestrial life. Mostly occurring in plants, it uses light energy to sequester atmospheric carbon dioxide (CO2) and convert it into biomass. As the most inefficient natural carboxylation process and source of most biomass documented, even a small increase of its performance could have vast downstream effects. Such a development could assimilate the abundantly available atmospheric CO2 while generating minimal amounts of waste for any biosynthesized product. The Escherichia coli bacterium was previously shown to functionally express the CBB cycle upon the addition of phosphoribulokinase (PRK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further knock-outs severed its energetic metabolism from the carbon metabolism resulted CO2-dependent biomass accumulation. This carbon fixation is driven by the energy independently generated in the TCA cycle from a supply of pyruvate. This unique, split metabolism was dubbed hemi-autotrophy. The hemi-autotrophic strain of E. coli serves as a model organism for the CBB cycle, but lacking any of the difficulties of light-dependent or multi-cellular organisms. A pyrophosphate-dependent 6-phosphofructokinase (PFP) originating from Methylococcus capsulatus Bath was characterised as catalyzing three reactions of the typical CBB cycle. Where PRK completes its catalysis with a dependency on energy-carrier adenosine triphosphate (ATP), PFP was shown to complete this reaction with the less energetic pyrophosphate (PPi) that is partially generated in its FBPase and SBPase-equivalent reactions. Successful integration of this synthetic CBB cycle would conserve 33% of all ATP expended in the native CBB cycle. The hemi-autrophic E. coli strain’s unique culturing requirements proved challenging but methods with increased dependability were established. Transformations without the relief of these conditions remain elusive, requiring pre-cultures in rich media and heterotrophic metabolism. The consecutive sub-culturing of the strain to increase its hampered growth characteristics resulted in mild improvements. Despite observing modest culturing characteristic and a relatively high chromosomal mutation rate, the strain did not demonstrated an increase in transformation efficiency. The attempted replacements of the plasmid-encoded prkA by pfp did not result in hemi-autotrophic growth in any of its constructs, despite modulation of their expression. Troubled by high mutation rates, it remains unknown whether the expression range of the significantly less efficient PFP was sufficient or if the cytoplasmic availability of PPi remained below its functionally required concentration. The putative H+-pyrophosphatase pump (HPP), natively expressed as the second gene in the pfp-hpp operon, remains uncharacterised but its co-expression did not manage to compensate for this deficiency either. Though native fbp was successfully knocked-out, the essential inorganic pyrophosphatase gene of E. coli remains. Thorough analysis of the components in the CBB system led to several design improvements and pathway modelling indicates the proposed synthetic CBB cycle is a viable alternative to its natural variant. Thermodynamic feasibility of the synthetic pathway was confirmed and kinetic analysis also predicted it to perform at reduced efficiencies while still indicating culture viability. Growth rates approximating those of the hemi-autotrophic strain were produced in a kinetic model of the central carbon metabolism while incorporating minimal assumptions. Modifying it to support the synthetic CBB cycle suggested its viability at a nominal reduction of growth, while suggesting further directions of research for the system

The development of efficient hemi-autotrophic carbon fixation in Escherichia Coli

Carbon fixation is a process vital to any life and as by far its most prevalent variant, the Calvin Benson Bassham (CBB) cycle is vital to virtually all known terrestrial life. Mostly occurring in plants, it uses light energy to sequester atmospheric carbon dioxide (CO2) and convert it into biomass. As the most inefficient natural carboxylation process and source of most biomass documented, even a small increase of its performance could have vast downstream effects. Such a development could assimilate the abundantly available atmospheric CO2 while generating minimal amounts of waste for any biosynthesized product. The Escherichia coli bacterium was previously shown to functionally express the CBB cycle upon the addition of phosphoribulokinase (PRK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further knock-outs severed its energetic metabolism from the carbon metabolism resulted CO2-dependent biomass accumulation. This carbon fixation is driven by the energy independently generated in the TCA cycle from a supply of pyruvate. This unique, split metabolism was dubbed hemi-autotrophy. The hemi-autotrophic strain of E. coli serves as a model organism for the CBB cycle, but lacking any of the difficulties of light-dependent or multi-cellular organisms. A pyrophosphate-dependent 6-phosphofructokinase (PFP) originating from Methylococcus capsulatus Bath was characterised as catalyzing three reactions of the typical CBB cycle. Where PRK completes its catalysis with a dependency on energy-carrier adenosine triphosphate (ATP), PFP was shown to complete this reaction with the less energetic pyrophosphate (PPi) that is partially generated in its FBPase and SBPase-equivalent reactions. Successful integration of this synthetic CBB cycle would conserve 33% of all ATP expended in the native CBB cycle. The hemi-autrophic E. coli strain’s unique culturing requirements proved challenging but methods with increased dependability were established. Transformations without the relief of these conditions remain elusive, requiring pre-cultures in rich media and heterotrophic metabolism. The consecutive sub-culturing of the strain to increase its hampered growth characteristics resulted in mild improvements. Despite observing modest culturing characteristic and a relatively high chromosomal mutation rate, the strain did not demonstrated an increase in transformation efficiency. The attempted replacements of the plasmid-encoded prkA by pfp did not result in hemi-autotrophic growth in any of its constructs, despite modulation of their expression. Troubled by high mutation rates, it remains unknown whether the expression range of the significantly less efficient PFP was sufficient or if the cytoplasmic availability of PPi remained below its functionally required concentration. The putative H+-pyrophosphatase pump (HPP), natively expressed as the second gene in the pfp-hpp operon, remains uncharacterised but its co-expression did not manage to compensate for this deficiency either. Though native fbp was successfully knocked-out, the essential inorganic pyrophosphatase gene of E. coli remains. Thorough analysis of the components in the CBB system led to several design improvements and pathway modelling indicates the proposed synthetic CBB cycle is a viable alternative to its natural variant. Thermodynamic feasibility of the synthetic pathway was confirmed and kinetic analysis also predicted it to perform at reduced efficiencies while still indicating culture viability. Growth rates approximating those of the hemi-autotrophic strain were produced in a kinetic model of the central carbon metabolism while incorporating minimal assumptions. Modifying it to support the synthetic CBB cycle suggested its viability at a nominal reduction of growth, while suggesting further directions of research for the system

Modelling tools and methodologies for rapid protocell prototyping

The field of unconventional computing considers the possibility of implementing computational devices using novel paradigms and materials to produce computers which may be more efficient, adaptable and robust than their silicon based counterparts. The integration of computation into the realms of chemistry and biology will allow the embedding of engineered logic into living systems and could produce truly ubiquitous computing devices. Recently, advances in synthetic biology have resulted in the modification of microorganism genomes to create computational behaviour in living cells, so called “cellular computing”. The cellular computing paradigm offers the possibility of intelligent bacterial agents which may respond and communicate with one another according to chemical signals received from the environment. However, the high levels of complexity when altering an organism which has been well adapted to certain environments over millions of years of evolution suggests an alternative approach in which chemical computational devices can be constructed completely from the bottom up, allowing the designer exquisite control and knowledge about the system being created. This thesis presents the development of a simulation and modelling framework to aid the study and design of bottom-up chemical computers, involving the encapsulation of computational re-actions within vesicles. The new “vesicle computing” paradigm is investigated using a sophisticated multi-scale simulation framework, developed from mesoscale, macroscale and executable biology techniques