Bacteria's ability to pass entire genes between one another, a process called Horizontal Gene Transfer (HGT), has a major impact on bacterial evolution. In an ongoing project at Harvey Mudd, computational methods have been used to catalogue the HGT events that have impacted a group of closely related bacteria. This thesis builds on that project, by improving our ability to identify gene families --- groups of genes in different strains that are related. Previously, similarity was measured only by comparing two genes' DNA sequences, ignoring their positions on the organism's DNA. Here, we leverage genes' relative position to make a better measurement of gene similarity. These improved similarity measurements will improve the existing pipeline's ability to identify HGT events.

The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. Results demonstrate the capture of a 30.8 kb SCCmec element on a conjugative plasmid for the first time, its transfer into both S. aureus and S. epidermidis recipients, and its excision from the plasmid with insertion in the orfX att site in recipients. The element was integrated into the plasmid by recombination between IS elements invariably present on all SCCmec types and pGO1/pSK41-like conjugative plasmids. These data explain the movement of SCCmec from reservoirs in commensal coagulase-negative staphylococci into different Staphylococcus aureus lineages using a ubiquitous conjugative plasmid that can transfer among staphylococci of different species and, thus, describes a mechanism for the environmental dissemination of methicillin resistance in nature.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The Hessian fly, Mayetiola destructor (Say), is a major pest of wheat, and is controlled mainly through deploying fly-resistant wheat cultivars. This study investigated five M. destructor populations collected from Texas, Louisiana, and Oklahoma, where infestation by Hessian fly has been high in recent years. Eight resistance genes including H12, H13, H17, H18, H22, H25, H26, and Hdic, were found to be highly effective against all tested M. destructor populations in this region, conferring resistance to 80% or more of plants containing one of these resistant genes. The frequency of biotypes virulent to resistant genes ranged from 0 to 45%. A logistic regression model was established to predict biotype frequencies based on the correlation between the percentages of susceptible plants obtained in a virulence test. In addition to the virulence test, the log-odds of virulent biotype frequencies were determined by a traditional approach to predict the logistic regression model. Characterization of a bacterial artificial chromosome (BAC) clone identified a gene encoding a protein with sequence similarity to bacterial levanases. Blast searching with the levanase-like protein identified 14 levanase/inulase-like genes or gene fragments. In this study, we determined the expression levels of these genes in different developmental stages and different tissues of 3-d old larvae of M. destructor. Sequence analysis revealed that six genes encode full length proteins, three were truncated at the 5’ end, and five truncated at the 3’ end. Sequences of putative proteins showed approximately 42% similarities to bacterial levanases or inulases, and 36% similarity to fungal levanases or inulases. No sequence similarities were found with any known animal or plant proteins. Comparative analysis of sequences among 14 levanase/inulase-like genes revealed that positions for intron/exon boundaries are conserved among different genes even though the length of each intron and exon varied among different genes. The expression patterns of the levanase/inulase-like genes were different among developmental stages and larval tissues of M. destructor. Interestingly, three genes presented alternative splicing bands in different developmental stages, and two genes exhibited splicing bands in different tissues of 3 d old M. destructor. This study would be useful for future studies of the characterization and function of levanase/inulase-like genes of these enzymes in plant-insect interactions.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.

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.

Os genes de Prolina racemase (PRACs) são enzimas intracelulares ou secretadas de T. cruzi (TcPRAC); essas enzimas estão envolvidas no metabolismo, diferenciação e virulência. Os genes PRAC foram identificados e caracterizados molecularmente em isolados representantes de toda a diversidade interespecífica de T. cruzi, T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini e T. lewisi. Além dessas espécies de tripanossomas restritas a mamíferos; homólogos de PRAC foram encontrados em tripanossomas de cobra (T. serpentis), crocodilo (T. grayi) e anuro (T. sp. 339). Análises filogenéticas e de sintenia entre homólogos de PRAC suportaram uma historia evolutiva totalmente congruente com as relações evolutivas previamente descritas dentro do gênero Trypanosoma. / Proline racemaces (PRACs) are intracellular or secreted enzymes of Trypanosoma cruzi (TcPRAC), implicated in metabolism, differentiation, virulence and the induction of nonspecific polyclonal B-lymphocyte in the host. We identified and molecularly characterized PRAC genes from isolates representing all intra-specific diversity (TcI-TcVI and Tcbat), T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli (isolates of lineages A-E), T. conorhini, and T. lewisi. In addition to these trypanosome species restricted to mammals, PRAC homologs were found in trypanosomes from snake (T. serpentis), crocodile (T. grayi) and anuran (T. sp 339). Phylogenetic and synteny analysis of PRAC homologs supported an evolutionary history totally congruent with the evolutionary relationships within the genus Trypanosoma.

This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

Les diatomées sont des algues microscopiques qui contribuent à hauteur de 25% environ à la production primaire planétaire. Les diatomées sont très souvent entourées d’une flore bactérienne, avec laquelle de nombreuses interactions ont été documentées. Les génomes de diatomées contiennent par ailleurs de nombreux gènes dont l’origine prédite est bactérienne.Nous avons étudié Asterionella formosa, une diatomée pennée présente dans de nombreux lacs et cours d’eau à l'aide de données omiques. L’utilisation de la métagénomique a permis de reconstruire 30 génomes bactériens, utilisés pour prédire d'éventuelles interactions avec la diatomée. Le séquençage de la sous-unité 16S de l’ARN ribosomique a montré que les différentes espèces bactériennes avaient une abondance variable au cours des phases de croissance de la diatomée, et que certaines étaient plus souvent au contact de la diatomée que libres dans le milieu. Le génome d'A. formosa a ensuite été séquencé à l'aide de la technologie Pacbio et comparé à ceux d'espèces proches. Enfin, l’impact des bactéries sur les diatomées a été abordé sous l’angle de l’évolution et des transferts horizontaux de gènes, qui ont été prédits à partir des données transcriptomiques d’une centaine de diatomées marines.Ce travail représente une première étape dans l'étude de la communauté bactérienne associée à A. formosa. Des expériences complémentaires incluant l’utilisation de transcriptomique ou métabolomique sont maintenant envisageables. Les données collectées et/ou analysées dans ce travail contribuent d'ores et déjà à l’effort global de caractérisation génomique des diatomées. / Diatoms are ubiquitous microalgae that contribute approximately 25% to the primary production worldwide. Many interactions, either positive, neutral or negative, have been documented between diatoms and bacteria. Diatom genomes also harbor numerous genes of putative bacterial origin.We are studying Asterionella formosa, a freshwater pennate diatom. We characterized the community using a combination of omics and laboratory techniques. We reconstructed of the genome of the diatom as well as 30 individual genomes from co-cultured bacterial species and investigated metabolisms that could support diatom-bacteria interactions. 16S rRNA sequencing revealed that the abundance of some bacterial species was highly variable over the course of A. formosa growth. Some species seemed preferentially attached to the diatom while others were mainly free-living. Then, the reference sequence of the A. formosa genome was improved by additional long-read (Pacbio) sequencing. Last, relationships between diatoms and bacteria were investigated at a broader evolutionary scale, by looking at horizontal gene transfers using transcriptomic data of a hundred marine diatoms.This work is a first step in the study of the dynamic and complex bacterial community associated with the diatom A. formosa. The accurate identification and the reconstruction of the genome of these bacteria will enable further in silico predictions based on metabolic networks and new omics experiments using transcriptomic or metabolomic. This work already contributes to a global effort to study diatoms by the means of genomics.

Os genes de Prolina racemase (PRACs) são enzimas intracelulares ou secretadas de T. cruzi (TcPRAC); essas enzimas estão envolvidas no metabolismo, diferenciação e virulência. Os genes PRAC foram identificados e caracterizados molecularmente em isolados representantes de toda a diversidade interespecífica de T. cruzi, T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini e T. lewisi. Além dessas espécies de tripanossomas restritas a mamíferos; homólogos de PRAC foram encontrados em tripanossomas de cobra (T. serpentis), crocodilo (T. grayi) e anuro (T. sp. 339). Análises filogenéticas e de sintenia entre homólogos de PRAC suportaram uma historia evolutiva totalmente congruente com as relações evolutivas previamente descritas dentro do gênero Trypanosoma. / Proline racemaces (PRACs) are intracellular or secreted enzymes of Trypanosoma cruzi (TcPRAC), implicated in metabolism, differentiation, virulence and the induction of nonspecific polyclonal B-lymphocyte in the host. We identified and molecularly characterized PRAC genes from isolates representing all intra-specific diversity (TcI-TcVI and Tcbat), T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli (isolates of lineages A-E), T. conorhini, and T. lewisi. In addition to these trypanosome species restricted to mammals, PRAC homologs were found in trypanosomes from snake (T. serpentis), crocodile (T. grayi) and anuran (T. sp 339). Phylogenetic and synteny analysis of PRAC homologs supported an evolutionary history totally congruent with the evolutionary relationships within the genus Trypanosoma.

The historic relationship of species and genes are traditionally depicted using trees. However, not all evolutionary histories are adequately captured by bifurcating processes and an increasing amount of research is devoted towards using networks or network-like structures to capture evolutionary history. Lateral gene transfer (LGT) is a previously controversial mechanism responsible for non tree-like evolutionary histories, and is today accepted as a major force of evolution, particularly in the prokaryotic domain. In this thesis, we present models of gene evolution incorporating both LGTs and duplications, together with efficient computational methods for various inference problems. Specifically, we define a biologically sound combinatorial model for reconciliation of species and gene trees that facilitates simultaneous consideration of duplications and LGTs. We prove that finding most parsimonious reconciliations is NP-hard, but that the problem can be solved efficiently if reconciliations are not required to be acyclic—a condition that is satisfied when analyzing most real-world datasets. We also provide a polynomial-time algorithm for parametric tree reconciliation, a problem analogous to parametric sequence alignment, that enables us to study the entire space of optimal reconciliations under all possible cost schemes. Going beyond combinatorial models, we define the first probabilistic model of gene evolution incorporating a birth-death process generating duplications, LGTs, and losses, together with a relaxed molecular clock model of sequence evolution. Algorithms based on Markov chain Monte Carlo (MCMC) techniques, methods from numerical analysis, and dynamic programming are presented for various probability and parameter inference problems. Finally, we develop methods for analysis of cancer progression, a biological process with many similarities to the process of evolution. Cancer progresses by accumulation of harmful genetic aberrations whose patterns of emergence are graph-like. We develop a model of cancer progression based on trees, and mixtures thereof, that admits an efficient structural EM algorithm for finding Maximum Likelihood (ML) solutions from available cross-sectional data. / QC 20100812