The genomes of the hemiascomycetes Saccharomyces cerevisiae and Ashbya gossypii have been completely sequenced, allowing a comparative analysis of these two genomes, which reveals that a small number of genes appear to have entered these genomes as a result of horizontal gene transfer from bacterial sources. One potential case of horizontal gene transfer in A. gossypii and 10 potential cases in S. cerevisiae were identified, of which two were investigated further. One gene, encoding the enzyme dihydroorotate dehydrogenase (DHOD), is potentially a case of horizontal gene transfer, as shown by sequencing of this gene from additional bacterial and fungal species to generate sufficient data to construct a well-supported phylogeny. The DHOD-encoding gene found in S. cerevisiae, URA1 (YKL216W), appears to have entered the Saccharomycetaceae after the divergence of the S. cerevisiae lineage from the Candida albicans lineage and possibly since the divergence from the A. gossypii lineage. This gene appears to have come from the Lactobacillales, and following its acquisition the endogenous eukaryotic DHOD gene was lost. It was also shown that the bacterially derived horizontally transferred DHOD is required for anaerobic synthesis of uracil in S. cerevisiae. The other gene discussed in detail is BDS1, an aryl- and alkyl-sulfatase gene of bacterial origin that we have shown allows utilization of sulfate from several organic sources. Among the eukaryotes, this gene is found in S. cerevisiae and Saccharomyces bayanus and appears to derive from the alpha-proteobacteria. / Dissertation

<p>Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus <italic>Cladonia grayi</italic> and its green algal partner <italic>Asterochloris</italic> sp. This work uses the model lichen system <italic>Cladonia grayi</italic> and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.</p><p>Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.</p><p>In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from <italic>Cladonia grayi</italic> were shown to be functional by expression of the lichen genes in a strain of <italic>Saccharomyces cerevisiae</italic> in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. </p><p>In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of <italic>Cladonia grayi</italic> including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.</p> / Dissertation

Experimental evolution has been used to investigate both general and specific evolutionary processes. More recently, it has also been used to resolve protein-protein interactions. Viruses assemble through a series of protein-protein interactions which must remain more favorable than any competing off-pathway reaction. By constructing chimeric viruses with genes or segments of genes from another species, foreign elements are introduced into this system of assembly. Characterization of the resulting chimeras provides information about which proteins interact, the protein-protein interacting interface, the role of particular domains, and the importance of specific residues. Chimeric viruses often exhibit a reduction in fitness, as the foreign element is unable to interact as efficiently in the system as the native element. Through experimental evolution, mutations accumulate that affect interacting partners in the system, leading to a more optimal assembly pathway. The microviruses are well-characterized single-stranded (ss) DNA bacteriophages. They are divided into three clades, represented by φX174, G4, and α3. Incidences of horizontal gene transfer between microvirus clades are unusually rare and may be due to a complex assembly pathway with multiple stages: a foreign element has the potential to disrupt a multitude of morphogenetic steps. In this study, we exchanged major spike genes between the two microvirus species φX174 and G4, then monitored the evolutionary recovery. Results can be interpreted within this thermodynamic paradigm. Although the G4-φXG chimera could only form plaques at low temperature and exhibited reduced fitness, its evolutionary recovery was relatively straightforward. The other chimera, φX-G4G, could only form plaques when complemented with two wild-type φX174 genes. Isolating a complementation-independent chimera required the passaging of mutants through a series of different environments. The first selection yielded mutations of the largest effects. First, the truncation of a protein involved in DNA synthesis was recovered, resulting in a global decrease in gene expression. Next, a recombination event at the 3' end of the foreign gene resulted in a modification of the protein’s C-terminus. These mutations were subjected to further analysis to determine why they were so critical at this early stage of experimental evolution. Subsequent passages of the φX-G4G chimera eventually yielded viable strains, with additional mutations affecting stages of late assembly. Overall, results indicate how gene exchange can drastically affect flux through the pathway. When the system is initially perturbed, the process of experimental evolution allows the pathway to return to a normalized state. The mutations isolated during this recovery stage indicates how the flux was initially altered, and how it can be restored.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Their parasitic lifestyle has lead them to lose a great number of genes and, thus, biochemical pathways capacities, but these reductive processes have been often offset by the acquisition of several genes by means of horizontal gene transfer (HGT). First, in this thesis, we will describe the complete genomes of Encephalitozoon hellem and Encephalitozoon romaleae. Both species also were found to harbor a number of protein-coding genes absent in other microsporidia, which products assembled complete metabolic pathways. All these genes are functionally related to DNA and folate metabolism, and all appear to have been acquired from HGT events from different eukaryotic and prokaryotic donors. Interestingly in E. romaleae genes involved in de novo synthesis of folate are all pseudogenes, highlighting the transient nature of transferred genes. Secondly, we took a closer look at the ploidy and sexual status of Encephalitozoon cuniculi, a vertebrate pathogen, by mapping Illumina sequence reads against the genomes of four strains of this species. We identified the presence of low level of heterozygosity in all strains investigated; a feature that revealed the diploid nuclear state of the species. This reductive intra-individual genetic diversity could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. Overall, the work presented in this thesis resulted in a much greater understanding of the genome evolution of a medically and economically important group of parasites.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Benzalkonium chlorides (BACs) are widely used, broad-spectrum disinfectants and frequently detected in the environment, even at toxic levels for life. Since such disinfectants can induce broad resistance capabilities, BACs may fuel the emergence of antibiotic resistance in the environment. A substantial body of literature has reported that exposure to BACs causes antibiotic resistance; yet, other studies suggest that the resistance linkage is rare, unsystematic, and/or clinically insignificant. Accordingly, whether or not disinfectant exposure mediates antibiotic resistance and, if so, what molecular mechanisms underlie the resistance link remains to be clearly elucidated. Further, understanding how microbial communities degrade BACs is important not only for alleviating the possible occurrence of antibiotic resistance but also reducing the potential risks to environmental and public health. An integrated strategy that combines metagenomics, metatranscriptomics, genetics, and traditional culture-dependent approaches was employed to provide novel insights into these issues. The integrative approach showed that a microbial community exposed to BACs can acquire antibiotic resistance through two mechanisms: i) horizontal transfer of previously uncharacterized efflux pump genes conferring resistance to BACs and antibiotics, which were encoded on a conjugative plasmid and co-selected together upon BACs and ii) selective enrichment of intrinsically multi-drug resistant organisms. Further, a microbial community adapts to BAC exposure via a variety of mechanisms, including selective enrichment of BAC-degrading species and amino acid substitutions and horizontal transfer of genes related to BAC resistance and degradation. The metatranscriptomic data suggests that the BAC-adapted microbial community metabolized BACs by cooperative interactions among its members. More specifically, Pseudomonas nitroreducens cleaved (i.e., dealkylated) BACs, metabolized the alkyl chain (the dealkylated product of BACs), and released benzyldimethylamine (the other product of BACs), which was further metabolized by other community members (e.g., Pseudomonas putida). Collectively, this study demonstrates the role of BACs in promoting antibiotic resistance and advances current understanding of a microbial community degrading BACs. The results of this work have important implications for (appropriate) usage of disinfectants and for assessing, predicting, and optimizing biological engineering processes treating BAC-bearing waste streams.

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.

Horizontal gene transfer (HGT) is now indisputably the predominant driving force, if not the sole force, behind speciation and the evolution of novelty in bacteria. Of all mechanisms of horizontal gene transfer (HGT), conjugation, the contact-dependent plasmid-mediated transfer of DNA from a bacterial donor to a recipient cell, is probably the most universal. First observed between bacteria, conjugation also mediates gene transfer from bacteria to yeast, plant and even animal cells. The range of environments in which bacteria naturally exchange DNA has not been extensively explored. The interior of the animal cell represents a novel and potentially medically relevant environment for gene transfer. Since most antibiotics are ineffective inside mammalian cells, our cells may be a niche for the evolution of resistance and virulence in invasive pathogens. Invading bacteria accumulate in vacuoles inside human cells, protected from antibiotics. Herein, I demonstrate the ability of intracellular Salmonella typhimurium to meet and exchange plasmid DNA by conjugation within animal cells, revealing the animal intracellular milieu as a permissive environment for gene exchange. This finding evokes a model for the simultaneous dissemination of virulence and antibiotic resistance within a niche protected from both antibiotics and the immune system and extends the variety of environments in which bacteria are known to exchange genes. Unlike conjugation between bacteria, conjugation between bacteria and eukaryotic cells requires the import of transferred DNA into the nucleus before the transferred genes can be expressed and inherited. Plant-cell nuclear transformation by the conjugation system of the Agrobacterium tumefaciens Ti plasmid is believed to be mediated by nuclear localization sequences (NLSs) carried within the proteins that accompany the T-DNA during transfer. Whether NLSs are equally important for transmission of other conjugative plasmids to eukaryotic cells is unknown. Herein, I demonstrate nuclear localization potential within the putative conjugative escort protein TraI of the IncPa plasmid RP4. In contrast, MobA, the putative escort protein from the IncQ plasmid RSF1010, lacked any clear nuclear localization potential. It is therefore likely that specific nuclear localization signals within conjugative proteins are not essential for nuclear transformation per se, although they may assist in efficient plasmid transmission.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008. / Bibliography: p. 257-274. / Literature review -- General materials and methods -- Characterisation of strain collection -- Distribution of integrons and gene cassettes in pseudomonas -- Genomic context of pseudomonas integrons -- Evolutionary analysis of pseudomonas spp. integrons 199 -- Final discussion -- Appendix -- References. / Integrons associated with mobile genetic elements have played a central role in the emergence and spread of multiple antibiotic resistance in many pathogenic bacteria. However, the discovery of integrons in the chromosomes of diverse, non-pathogenic bacteria suggests that integrons have a broader role in bacterial evolution. The Pseudomonas stutzeri species complex is a well studied model for bacterial diversity. Members of the complex are genetically closely related, but sub-taxa are not able to be defined by exclusively shared sets of phenotypic characters. Rather, on the basis of total DNA:DNA similarity, Ps. stutzeri strains have been divided into 17 different groups (termed genomovars). Two Ps. stutzeri strains have been found to contain Chromosomal Integrons (CIs). This thesis involved exploration of the hypothesis that a CI was present in the common ancestor of the Ps. stutzeri species complex and assessed the impact of integrons on diversity across all Pseudomonads. The history and significance of integrons is discussed in Chapter 1 as part of a literature review, and general materials and methods are provided in Chapter 2. Chapters 3 - 6 comprise the sections in which data generated during my PhD project are presented. A comprehensive analysis of the relationships between the strains being analysed is presented in Chapter 3. In Chapter 4, results of PCR and hybridisation screening for integrons across the strain collection are presented. In Chapter 5 the recovery of additional integrons and in depth sequence analysis of the recovered integrons are described. Finally, Chapter 6 contains statistical analyses of integron-associated genes and Chapter 7 contains a final discussion the most significant findings. Twenty-three Pseudomonas spp. strains were screened for the presence of integrons. All but three were found to contain integron-like sequences; however, most integron sequences recovered contained inactivated core integrons. viii Despite having a chromosomal locus, integrons in Pseudomonas were found to have properties indicative of frequent horizontal transfer. Evidence was also obtained which suggests that integrons have been acquired at the same locus on multiple independent occasions. This has not been observed in other families of chromosomal integrons and suggests that the loci at which integrons in Pseudomonas are found are hotspots for recombination. / Mode of access: World Wide Web. / xiii, 274 p. ill