The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields. To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

This PhD thesis presents results of a research focussed on the evolution of phototrophy in the bacterial order Rhodobacterales with a special regard to its haloalkaliphilic representatives. The photoheterotrophic bacterium Rhodobaca barguzinensis alga05 was used as an organism of choice. Its phylogeny, genome organization, and metabolic potential was characterized. The main result of the thesis is that phototrophy is a genuine trait among the haloalkaliphilic representatives of the Rhodobacter-Rhodobaca group inside the Rhodobacterales clade.

<p>Historically, evolution has been studied either by looking at morphological traits in living organisms and the fossil record, or by using bioinformatics and comparative genomics. While highly useful for deducing evolutionary history, these approaches are not particularly well suited for studying the mechanisms of evolution. In order to address such issues, other methods are needed. Mathematical modelling is one of the most powerful options available, and it is the approach used in this thesis. By constructing models of biological systems, the work aims to resolve some of the many unresolved questions regarding evolutionary processes, such as how new genes evolve and how selection acts in fragmented populations. Some answers have been reached, and thus the thesis makes a small contribution to our overall understanding of evolution.</p><p>The creation of novel genes was studied both directly and by extension of an analogous system, which revolved around reversion of a frameshift mutant. The results pointed to gene amplification as a likely mechanism for both reversion of the frameshift mutant and creation of new genes.</p><p>Selection in fragmented populations is shown to be effective even when sub-populations, rather than individuals, are competing against each other. Modeling of a system of bacterial symbionts living in aphids indicates that, although the bacterial population within a single host is small and subject to rampant genetic drift, the bacterial population as a whole is regulated by selection on the host level. Thus, deleterious mutations do no accumulate and the population maintains its fitness over time.</p>

Historically, evolution has been studied either by looking at morphological traits in living organisms and the fossil record, or by using bioinformatics and comparative genomics. While highly useful for deducing evolutionary history, these approaches are not particularly well suited for studying the mechanisms of evolution. In order to address such issues, other methods are needed. Mathematical modelling is one of the most powerful options available, and it is the approach used in this thesis. By constructing models of biological systems, the work aims to resolve some of the many unresolved questions regarding evolutionary processes, such as how new genes evolve and how selection acts in fragmented populations. Some answers have been reached, and thus the thesis makes a small contribution to our overall understanding of evolution. The creation of novel genes was studied both directly and by extension of an analogous system, which revolved around reversion of a frameshift mutant. The results pointed to gene amplification as a likely mechanism for both reversion of the frameshift mutant and creation of new genes. Selection in fragmented populations is shown to be effective even when sub-populations, rather than individuals, are competing against each other. Modeling of a system of bacterial symbionts living in aphids indicates that, although the bacterial population within a single host is small and subject to rampant genetic drift, the bacterial population as a whole is regulated by selection on the host level. Thus, deleterious mutations do no accumulate and the population maintains its fitness over time.

Bacteria of the genus Bartonella infect the red blood cells of a wide range of wild and domestic mammals and are transmitted between hosts by blood-sucking insects. Although most Bartonella infections are asymptomatic, the genus contains several human pathogens. In this work, host adaptation and host switches in Bartonella have been studied from a genomic perspective, with special focus on the acquisition and evolution of genes involved in host interactions. As part of this study, the complete genome of B. grahamii isolated from a Swedish wood mouse was sequenced. A genus-wide comparison revealed that rodent-associated Bartonella species, which have rarely been associated with human disease, have the largest genomes and the largest number of host-adaptability genes. Analysis of known and putative genes for host interactions identified several families of autotransporters as horizontally transferred to the Bartonella ancestor, with a possible role both during early host adaptation and subsequent host shifts. In B. grahamii, the association of a gene transfer agent (GTA) and phage-derived run-off replication of a large genomic segment was demonstrated for the first time. Among all acquisitions to the Bartonella ancestor, the only well conserved gene clusters are those that encode the GTA and contain the origin of the run-off replication. This conservation, along with a high density of host-adaptability genes in the amplified region suggest that the GTA provides a strong selective advantage, possibly by increasing recombination frequencies of host-adaptability genes, thereby facilitating evasion of the host immune system and colonization of new hosts. B. grahamii displays stronger geographic pattern and higher recombination frequencies than the cat-associated B. henselae, probably caused by different lifestyles and/or population sizes of the hosts. The genomic diversity of B. grahamii is markedly lower in Europe and North America than in Asia, possibly an effect of reduced host variability in these areas following the latest ice age.

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Transposable elements (TEs) are DNA regions that can move within and between genomes, causing great impact on the host organisms. The Tc1-Mariner superfamily stands out for being, probably, the DNA transposons superfamily with greater distribution in nature, being ubiquitous in eukaryotes. In part of this work, we characterize elements of the mariner family in Neotropical drosophilids, which were obtained through amplification with degenerated primers. The primers were designed for the catalytic domain region of mariner transposase allowing amplification of a wide range of mariner-like sequences. A sum of twenty-three species have mariner-like sequences belonging to three subfamilies (mellifera, mauritiana and irritans). These elements present a patchy distribution and incongruences with the host phylogeny, suggesting horizontal transmission events between drosophilids and even between drosophilids and species of other families, subfamilies and orders. Moreover, some sequences present open reading frames, conserved catalytic motifs and evidence for the action of a strong purifying selection, which suggest yhat they originated from active elements. In another part of the work, we characterize Paris-like elements (belonging to Tc1 family), through searches in the twelve Drosophila genomes available. These searches, enabled us to find five new Paris-like elements (one in D. ananassae, one in D. pseudoobscura, one in D. persimilis, one in D. mojavensis and one in D. willistoni), with a copy number ranging from two to seven. Three species have putatively active elements. The evolutionary analysis of these elements suggests that they have envolved through vertical transmission associated with some events of stochastic loss in the analysed species. / Elementos transponíveis (TEs) são regiões do DNA que podem se mover dentro e entre genomas, causando grande impacto na evolução dos organismos. A Superfamília Tc1-Mariner se destaca por ser, provavelmente, a superfamília de transposons de DNA com maior distribuição na natureza, sendo ubíqua em eucariotos. Em parte desse trabalho, caracterizamos elementos da família mariner em drosofilideos Neotropicais para os quais obtivemos amplificação com primers degenerados. Os primers foram construídos na região do domínio catalítico da transposase de mariner o que permite amplificar uma ampla gama de sequências relacionadas à mariner. Um total de 23 espécies apresentou sequências relacionadas à mariner pertencentes a três subfamílias (mellifera, mauritiana e irritans). Esses elementos apresentaram uma distribuição descontínua e incongruências com a filogenia das espécies hospedeiras, o que sugere eventos de transmissão horizontal entre drosofilideos e, até mesmo entre drosofilideos e espécies de outra família, superfamília e ordem. Além disso, algumas sequências apresentaram um quadro aberto de leitura, os motivos catalíticos conservados e uma forte seleção purificadora atuando, o que sugere que esses elementos sejam provenientes de elementos ativos. Em outra parte do trabalho, caracterizamos as sequências relacionadas ao elemento Paris (pertencentes à família Tc1), com buscas nos doze genomas de Drosophila disponíveis. Nessas buscas,foram encontrados cinco novos elementos relacionados à Paris (um em D. ananassae, um em D. pseudoobscura, um em D. persimilis, um em D. mojavensis e um em D. willistoni), com um número de cópias variando de dois a sete. Três espécies apresentaram elementos potencialmente ativos. A análise evolutiva desses elementos sugere que estão sendo mantidos por transmissão vertical, com alguns eventos de perda estocástica nas espécies analisadas.

Elemental stoichiometry defines a critical understanding of the relationship between nutrient availability and usage throughout different levels of the biological community. We found there is a link between available phosphorus (P), cellular phosphorus, and nematode development as postulated by the growth rate hypothesis (GRH). I predicted that in a P-poor environment, cellular RNA concentrations would be lower than they are in P-rich environment, and thus the 18s rRNA expression level will have reduced. To most efficiently regulate the uptake of limited P, I predicted that nematodes in P-poor environments would decrease the number of copies of the 18s rRNA gene in their genome. I measured life history traits as well as rRNA gene expression and gene copy number. We found that elemental stoichiometry predicts evolutionary changes consistent with the Growth Rate Hypothesis. We sequenced and assembled a draft genome of P. murrayi. Although we expected to find genes responsible for stress tolerance, we hypothesized that in response to strong selection pressure associated with living in a simplified ecosystem, over time the genome of P. murrayi should have undergone significant decay (gene loss) relative to species in ecosystems structured more strongly by biotic interactions. We found significantly fewer genes in P. murrayi. To compare patterns of gene expression between two highly divergent Antarctic nematode species, we sequenced and assembled the transcriptomes of S. lindsayae and P. murrayi. Under laboratory conditions at 4˚C, S. lindsayae had significantly lower rates of gene expression but expressed a significantly larger number of genes. We speculate that the differences in gene expression are correlated with life history traits (developmental rates) while the differences in the number of genes expressed can be explained by their different genetic systems (S. lindsayae is amphimictic, P. murrayi is parthenogenic) and the soil environments to which they are adapted. Since we previously showed that differences in available P content can influence the evolution of gene expression via gene copy number, and that this ultimately influences growth rate, we wondered how much of this response is driven by genetics versus how strongly these patterns are driven by temperature. To better understand this, we maintained wild type populations of P. murrayi in P-rich and P-poor conditions at 5˚C, 10˚C and 15˚C in the laboratory for over 40 generations and sequenced the transcriptomes prepared from each treatment group. We found that nutrient levels played an important role in gene expression when the temperature is optimal for P. murrayi culturing and that temperature is more important in gene expression when the available P is limited. This work underscores the utility of using principles of elemental stoichiometry coupled with genomic and transcriptomics research tools to make and test predictions about life history evolution. The results of my work also inform inferences about the ways in which nutrient availability also drives the organization of trophic interactions and ultimately ecosystems.

Once thought to be an evolutionary dead-end, hybridization is now being detected in genomes across kingdoms, perhaps even playing an integral role in evolution. In chapter 1, I investigated the potential influences of hybridization on the evolution of a group of vagrant, asexual species in the Rhizoplaca melanophthalma species group. I sequenced the mitochondrial and nuclear genomes of 55 specimens and found well-supported nuclear phylogenies of both datasets. There were, however, multiple instances of discordance between the mitochondrial and nuclear trees, which can be caused by hybridization. PhyloNet and ABBA-BABA also detected widespread hybridization among this group. In chapter 2, I shifted to the Holarctic clade of lichen-forming fungi in Xanthoparmelia to characterize gene tree conflict and investigate the potential for hybrization. Here, I used three different tests for hybridization that account for incomplete lineage sorting "ABBA-BABA, PhyloNet, and SplitsTree" as well as PhyParts to characterize gene tree conflict. Like the Rhizoplaca species group, widespread hybridization was detected in the Holarctic clade despite robust phylogenies. My research underscores the value of investigating hybridization when studying species boundaries and evolutionary history.

Yes / A central challenge in microbial community ecology is the delineation of appropriate units of biodiversity, which can be taxonomic, phylogenetic, or functional in nature. The term ‘community’ is applied ambiguously; in some cases, the term refers simply to a set of observed entities, while in other cases, it requires that these entities interact with one another. Microorganisms can rapidly gain and lose genes, potentially decoupling community roles from taxonomic and phylogenetic groupings. Trait-based approaches offer a useful alternative, but many traits can be defined based on gene functions, metabolic modules, and genomic properties, and the optimal set of traits to choose is often not obvious. An analysis that considers taxon assignment and traits in concert may be ideal, with the strengths of each approach offsetting the weaknesses of the other. Individual genes also merit consideration as entities in an ecological analysis, with characteristics such as diversity, turnover, and interactions modeled using genes rather than organisms as entities. We identify some promising avenues of research that are likely to yield a deeper understanding of microbial communities that shift from observation-based questions of ‘Who is there?’ and ‘What are they doing?’ to the mechanistically driven question of ‘How will they respond?’