Loss of Genetic Redundancy in Reductive Genome Evolution

Biological systems evolved to be functionally robust in uncertain environments, but also highly adaptable. Such robustness is partly achieved by genetic redundancy, where the failure of a specific component through mutation or environmental challenge can be compensated by duplicate components capable of performing, to a limited extent, the same function. Highly variable environments require very robust systems. Conversely, predictable environments should not place a high selective value on robustness. Here we test this hypothesis by investigating the evolutionary dynamics of genetic redundancy in extremely reduced genomes, found mostly in intracellular parasites and endosymbionts. By combining data analysis with simulations of genome evolution we show that in the extensive gene loss suffered by reduced genomes there is a selective drive to keep the diversity of protein families while sacrificing paralogy. We show that this is not a by-product of the known drivers of genome reduction and that there is very limited convergence to a common core of families, indicating that the repertoire of protein families in reduced genomes is the result of historical contingency and niche-specific adaptations. We propose that our observations reflect a loss of genetic redundancy due to a decreased selection for robustness in a predictable environment

Trypanosoma cruzi genome plasticity and evolution

Trypanosoma cruzi, a protozoan from the Kinetoplastidae family, is the etiologic agent of Chagas disease, a major public health problem affecting mostly the poorest areas of Latin America. Due to the complex nature of the parasite’s genome it has been impossible to produce a complete reference genome sequence, thus hampering the implementation of post- genomic approaches to unveil the mechanisms of generation of antigenic variation and the identification of new drug targets. My doctoral studies have focused on the application of combined genome sequencing and computational methods to produce a complete reference T. cruzi genome sequence and perform comparative analyses to better understand the mechanisms that allow T. cruzi to evade the mammalian host immune system and to briskly adapt to novel environments. In paper I and II, different genome assembly strategies and second generation sequencing technologies were implemented to perform comparative analyses to identify elements of virulence between T. cruzi and two trypanosomatids that are non-pathogenic to humans: Trypanosoma cruzi marinkellei, a bat-restricted sub-species of the T. cruzi clade and the human avirulent species Trypanosoma rangeli. The studies reveal the expansion of T. cruzi- specific genomic traits specialised in the invasion of mammalian cells. In paper III, using third-generation, PacBio sequencing data it was possible to assemble the complete reference genome sequence of a Trypanosoma cruzi isolate from the DTU-I clade. This breakthrough allowed us - for the first time - to explore in detail the genome architecture of the subtelomeric areas where many parasite virulence factors are encoded. One of the most interesting discoveries was the overrepresentation of interspersed retrotransposons and microsatellites in tandem gene arrays coding for surface molecules, hinting at a retrotransposon-driven mechanism of recombination for generating new sequence variants. Whole genome sequencing of 35 T. cruzi DTU-I isolates, collected from different locations in the American continent, made possible to identify and characterise the mechanisms of adaptability employed by the parasite. Finally, paper IV analyses the mechanisms of genomic hybridisation in T. cruzi and the evolution over time of the hybrid offspring. The analysis revealed that during hybrid formation, the parasite integrates genetic material from each parental strains with the aid of retrotransposons and microsatellites, and the genome of these hybrid isolates moves quickly from a tetraploid to a diploid state. As a result, the hybrid strain has more genetic material, mostly in the subtelomeres, providing the parasite with a pool of new surface molecule genes with the potential to possibly increase its fitness in a new environment. In conclusion, the work presented here has advanced the understanding of parasite biology and provided a genomic resource to be exploited for the identification of drug targets and vaccine candidates

The Roles and Regulation of the Redundant Phenazine Biosynthetic Operons in Pseudomonas aeruginosa PA14

The opportunistic pathogen Pseudomonas aeruginosa has been well studied for its ability to cause nosocomial infections in immunocompromised patients. However, its pathogenicity is only one aspect of the biology that makes this bacterium one of the most versatile of its genus. Since its first description in 1885, P. aeruginosa has been known to produce colorful, small molecules called phenazines. These redox-active compounds were originally thought of as mere secondary metabolites or virulence factors that allow P. aeruginosa to infect plant and animal hosts. However, recently we have gained an appreciation for their diverse functions that directly benefit their producer: phenazines act as signaling molecules, regulate intracellular redox homeostasis and are implicated in iron uptake. As a result, phenazines also have dramatic effects on the structural development of multicellular communities of P. aeruginosa, generally referred to as biofilms. How phenazine production is regulated in response to environmental cues to allow for this functional diversity is still poorly understood. Pseudomonas aeruginosa produces at least five different phenazines, each of which have distinct chemical properties. The genes encoding the core phenazine biosynthetic enzymes are found in two redundant 7-gene operons. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode two sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid (PCA), the precursor for all other phenazine derivatives. Although the phz1 and phz2 operons are nearly identical (~98% similarity), they are differentially regulated. phz1 is regulated by quorum sensing (QS), while the factors controlling phz2 expression have not yet been identified. Furthermore, the contribution of phz2 to phenazine production is not fully understood. The phz2 operon is conserved among all P. aeruginosa species and we hypothesize that it may be vital to their ability to adapt to diverse environments. In this work, we have investigated the regulation of the phz2 operon and its contribution to colony biofilm development in P. aeruginosa PA14 (Chapter 2). We found that (1) phenazine production in biofilms is mediated exclusively through the phz2 operon, (2) phz2 expression is required for biofilm development and host colonization and (3) phz2 is regulated by quinolones, which are prominent signaling molecules in P. aeruginosa's QS system. We then investigated the roles of individual phenazines in colony development (Chapter 3) and the specificity of SoxR activation by redox active molecules (Chapter 4). We found that the effects of individual phenazines are not redundant and may be used in combination to modulate colony development. SoxR is a transcription factor that is activated by redox-active molecules including phenazines. Investigations into SoxR specificity showed that SoxR activation in non-enteric bacteria is tuned to specific redox potentials. Together, the findings presented in this thesis have expanded our knowledge about the role of phenazine production in biofilms and pathogenicity

Probing structure, function and dynamics in bacterial primary and secondary transporter-associated binding proteins

Substrate binding proteins (SBPs) are ubiquitous in all life forms and have evolved to perform diverse physiological functions, such as in membrane transport, gene regulation, neurotransmission, and quorum sensing. It is quite astounding to observe such functional diversity among the SBPs even when they are restricted by their fold space. Therefore, the SBPs are an excellent set of proteins that can reveal how proteins evolution novel function in a structurally conserved/constrained fold. This study attempts to understand the phenomenon of affinity and specificity evolution in SBPs by combining a set of biochemical, biophysical, and structural studies on the SBPs involved in translocation of substrates across the membrane using ATP-binding cassette (ABC) transporters and tripartite ATP-independent periplasmic (TRAP) transporters in gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa, respectively. Additionally, experimental, and computational methods were used in conjunction to highlight the variation in the dynamics of these SBPs. The results from this study highlight an intricate role of dynamics in complementing the structural alterations that are required for high-affinity ligand binding. Moreover, first ever neutron structure of a SBP was determined during my study to delineate the extensive network of water in the binding cavities of the SBPs that help stabilize larger substrates by forming water-mediated hydrogen bond interactions with the bound substrates. Furthermore, structures of two SBPs from T. maritima were determined in both substrate-free (apo) and substrate-bound (holo) forms and subsequently used for computational molecular dynamics simulation to determine the variation in dynamics due to substrate-binding. The novel TRAP SBP identified in P. aeruginosa was identified as a promiscuous binder of several tricarboxylic acid cycle (TCA) cycle intermediates. A total of six SBP structures were determined using X-ray crystallography and one SBP structure was determined using neutron crystallography. Finally, experimental neutron scattering was used to experimentally characterize the picosecond to nanosecond dynamics in SBPs and highlighted differences in the translational, rotational, and internal dynamical signatures of two SBP isoforms. Overall, the findings of this study can be broadly applied in biotechnology and biosensor development by artificially engineer affinity or specificity for a particular ligand

Identification and characterization of Chlamydia trachomatis type III secretion substrates

Chlamydiae are a large group of Gram-negative obligate intracellular bacteria that only grow within a membrane-bound vacuole in eukaryotic host cells. All Chlamydiae share a unique biphasic developmental cycle, in which the non-replicative elementary bodies (EBs) invade host cells and remain restricted within the bacterial vacuole, known as inclusion. Soon after invasion, EBs develop into replicative RBs that grow and divide by binary fission. Later in the cycle, RBs undergo a secondary differentiation into EBs, which are released from the host cell to initiate new rounds of invasion.(...

Infections, Symbiosis, Immunity and Adaptation

Evolution has been shaping the genetic structure of populations across generations, using mutation and recombination, migration, drift and selection to create and/or corrode variation. The array of traits presented by individuals in a population is dependent on several factors, such as their heritability or the genetic pool available to the adaptive process. Additionally, the multitude of complex relationships within and between species creates another level of complexity that can compromise the pinpointing of the contributing factors and their relative weight to such changes. As so, understandably, disentangling the factors that influence the course of evolution in natural populations is of extreme importance but also of great difficulty. (...

Probabilistic Reconstruction and Comparative Systems Biology of Microbial Metabolism

With the number of sequenced microbial species soon to be in the tens of thousands, we are in a unique position to investigate microbial function, ecology, and evolution on a large scale. In this dissertation I first describe the use of hundreds of in silico models of bacterial metabolic networks to study the long-term the evolution of growth and gene-essentiality phenotypes. The results show that, over billions of years of evolution, the conservation of bacterial phenotypic properties drops by a similar fraction per unit time following an exponential decay. The analysis provides a framework to generate and test hypotheses related to the phenotypic evolution of different microbial groups and for comparative analyses based on phenotypic properties of species. Mapping of genome sequences to phenotypic predictions -such as used in the analysis just described- critically relies on accurate functional annotations. In this context, I next describe GLOBUS, a probabilistic method for genome-wide biochemical annotations. GLOBUS uses Gibbs sampling to calculate probabilities for each possible assignment of genes to metabolic functions based on sequence information and both local and global genomic context data. Several important functional predictions made by GLOBUS were experimentally validated in Bacillus subtilis and hundreds more were obtained across other species. Complementary to the automated annotation method, I also describe the manual reconstruction and constraints-based analysis of the metabolic network of the malaria parasite Plasmodium falciparum. After careful reconciliation of the model with available biochemical and phenotypic data, the high-quality reconstruction allowed the prediction and in vivo validation of a novel potential antimalarial target. The model was also used to contextualize different types of genome-scale data such as gene expression and metabolomics measurements. Finally, I present two projects related to population genetics aspects of sequence and genome evolution. The first project addresses the question of why highly expressed proteins evolve slowly, showing that, at least for Escherichia coli, this is more likely to be a consequence of selection for translational efficiency than selection to avoid misfolded protein toxicity. The second project investigates genetic robustness mediated by gene duplicates in the context of large natural microbial populations. The analysis shows that, under these conditions, the ability of duplicated yeast genes to effectively compensate for the loss of their paralogs is not a monotonic function of their sequence divergence

Investigating the functional and evolutionary significance of Group B Sox genes in arthropods

Group B Sox genes play a critical developmental role in both vertebrates and insects. Within the model species Drosophila melanogaster, two SoxB genes, Dichaete and SoxNeuro, have been shown to act as ‘master regulators’ in the early development of the central nervous system. SoxB genes have only been characterised in a handful of arthropod species thus far, with most work to date focusing on drosophilids. The purpose of this investigation was twofold. First, I set out to resolve the phylogenetic origins of arthropod SoxB genes, as mutually exclusive models explaining their emergence are still contested. I have identified and annotated the SoxB of several invertebrate taxa. In total, my investigation includes 24 different metazoan taxa, and represents the largest investigation of arthropod SoxB phylogeny to date. In light of this research, I have proposed a new model of SoxB evolution which resolves the conflicting elements of the two primary competing models. Second, to study the evolution of SoxB in terms of functional conservation/divergence, I selected the emerging model organism Tribolium castaneum to draw a comparative analysis with Drosophila melanogaster. I first began by characterising the spatiotemporal expression patterns of SoxNeuro mRNA in early Tribolium embryos using whole mount in situ hybridisation, and examined published Dichaete expression patterns in the context of central nervous system development in T. castaneum. Using these data, I draw a comparison to the expression profiles of Dichaete and SoxNeuro orthologues in Drosophila melanogaster and other species. I have found that both Dichaete and SoxNeuro expression patterns in the developing central nervous system are remarkably well-conserved across species. I also attempted to characterise genome-wide binding for both Dichaete and SoxNeuro proteins in Tribolium in what would have represented the first genomic investigation of its kind in this emerging species. Using a tethered DNA adenine methyltransferase (Dam) enzyme for both SoxNeuro and Dichaete, I hoped to characterise the genomic loci with which each protein interacts within the beetle genome (a technique known as DamID). Unfortunately, these last set of experiments have proved unsuccessful, despite several attempts which have made use of different promoters, different DNA enrichment methodologies, and tackling unforeseen DNA contamination issues. Nevertheless, the troubleshooting experiments that I have carried out will pave the way for further genomic experiments in Tribolium, easing the establishment of genomic research in this emerging organism.Medical Research Counci

Studies on the protein biology of the plant pathogen, Candidatus Liberibacter solanacearum.

Characterisation determined the protein was folded and not functional. Solution structural analysis found that CLso adenyltransferase/IMP cyclohydrolase exists as a monomer in solution, which contrasts to the dimer observed in other bacterial homologues. Dimerisation is required for activity of the enzyme and the oligomeric state of the enzyme was determined to account for the lack of activity in CLso adenyltransferase/IMP cyclohydrolase. Chaperone co-expression resulted in the isolation of an additional five CLso proteins: serralysin (virulence factor), dehydrorhamnose epimerase, dehydrorhamnose reductase (L- rhamnose biosynthesis), pyruvate kinase (glycolysis) and dihydrodipicolinate synthase (lysine biosynthesis), which are hypothesised to be required for the organisms’ survival since gene expression studies show high bacterial expression when in infected psyllids and in planta. Characterisation of purified dihydrodipicolinate synthase and pyruvate kinase showed that both enzymes were active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial homologues. The establishment of a robust method for the expression of CLso proteins allows for the structural and kinetic characterisation of dihydrodipicolinate synthase, a key enzyme as it catalyses the committed step and is a potential antimicrobial target. The structure of the CLso dihydrodipicolinate synthase was solved in complex with the substrates: pyruvate, pyruvate and succinic semi-aldehyde (an analogue of (S)-aspartate semialdehyde) and with the allosteric inhibitor, lysine. Structural analysis showed that there was little difference in the CLso dihydrodipicolinate synthase compared with the Agrobacterium tumefaciens orthologue. Kinetic analysis of the enzyme reports constants that suggest a high binding affinity for substrates. It was proposed that CLso dihydrodipicolinate synthase has evolved a high binding affinity for its substrates in response to nutrient limitation, which could occur as a result of the intracellular lifestyle of the organism. Overall, this work provides new data that enriches our understanding of CLso biology and the relationship of the organism with the psyllid host and provides a method for future characterisation of CLso proteins, enabling new research and drug discovery programmes to study and manage the pathogenicity of the organism