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Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925 / Biomedical Sciences

<p>Plants use an array of photoreceptors to measure the quality, quantity, and direction of light in order to respond to ever-changing light environments. Photoreceptors not only determine how and when individual plants complete their life cycles, but they also have a profound and long-term macroevolutionary influence on species diversification. Despite their significances, very little is known about photoreceptors across plants as whole, and we lack a comprehensive view of photoreceptor evolution. </p><p> In my dissertation, I investigate the origin and evolution of three of the most prominent photoreceptor gene families in plants: phytochromes, phototropins and neochromes. Using newly available transcriptomic and genomic data, I completed the first in-depth survey of these photoreceptor families across land plants, green algae, red algae, glaucophytes, cryptophytes, haptophytes, and stramenopiles. </p><p> Phytochromes are red/far-red photoreceptors that play essential roles in seed germination, seedling photomorphogenesis, shade-avoidance, dormancy, circadian rhythm, phototropism, and flowering. Here, I show that the canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte green algae plus land plants), and I identify the most likely sequence whereby the plant phytochrome structure evolved from its ancestral phytochrome. Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts, and Selaginella apparently possess a single phytochrome gene copy, whereas independent gene duplications occurred within mosses, lycopods, ferns, and seed plants, leading to diverse phytochrome families in these clades. My detailed phylogeny encompasses all of green plants and enables me to not only uncover new phytochrome lineages, but also to make links to our current understanding of phytochrome function in Arabidopsis and Physcomitrella (the major model organism outside of flowering plants). Based on this robust evolutionary framework, I propose new hypotheses and discuss future directions to study phytochrome mechanisms.</p><p> Phototropins are blue-light photoreceptors that regulate key adaptive physiological responses, including shoot-positive phototropism, root-negative phototropism, chloroplast accumulation/avoidance, stomatal opening, circadian rhythm, leaf expansion, and seedling elongation I show that phototropins originated in the common ancestor of Viridiplantae (all green algae [charophytes, chlorophytes, prasinophytes] plus land plants). Phototropins repeatedly underwent independent duplications in all major plant lineages (mosses, lycopods, ferns and seed plants), except for liverworts and hornworts, where phototropin is a single-copy gene. Following each major duplication event, phototropins subsequently differentiated in parallel, resulting in two specialized (yet partially overlapping) functional forms that primarily mediate either low- or high-light responses. My gene phylogeny further suggests that phototropins have co-evolved with phytochromes, as is evident from their molecular interactions and strikingly similar gene duplication patterns. I hypothesize that the co-evolution of phototropins with phytochromes, together with their subsequent convergent functional divergences in phototropic responses, contributed to the success of plants in adapting to diverse and heterogeneous habitats.</p><p> Neochromes are chimeric photoreceptors that, by fusing phytochrome and phototropin modules into a single protein, are able to use both red/far-red and blue light to modulate phototropic responses. Neochromes were first discovered in ferns, and the evolution of neochromes was implicated as a key innovation that facilitated fern diversification under the low-light angiosperm canopies. Despite its significance from an evolutionary standpoint, the origin of neochromes has remained a mystery. Here I present the first evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in my large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 MYA, long after the split between the two plant lineages (at least 400 MYA). By analyzing the draft genome of the Anthoceros punctatus hornwort, I also discovered a novel phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was horizontally transferred to ferns, where it may have played a significant role in the diversification of modern ferns. </p><p> In summary, my studies identified the molecular origins of phytochromes, phototropins and neochromes, and reconstructed their respective evolutionary histories. This new framework for photoreceptor evolution will stimulate new research linking ecology, evolution, and photochemistry to understand how plants adapt to variable light environments.</p> / Dissertation

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions. / NO AFRIKAANS SUMMARY AVAILABLE

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.

The hypothesis of horizontal gene transfer of a hybrid gene from bacteria to the filamentous Ascomycota was tested using constrained phylogenetic analyses and tests of topological congruence. Results suggest that the hybrid gene was acquired from bacteria by a single transfer before the radiation of the Leotiomyceta. The phylogenetic relationship among Nimbya and Embellisia was investigated using both morphological and molecular data. Examination of conidia morphology revealed that Nimbya and Embellisia comprise two and four distinct morphological groups, respectively. Molecular analyses support all morphological groups of Nimbya and Embellisia and reveal that both genera are polyphyletic. Results suggest the circumscription of these genera is based upon convergent morphological characters. To further understand the evolutionary relationship among Alternaria and closely related genera, ten protein-coding genes were sequenced across 176 species. Three genes possessed significant substitution saturation and two other genes did not possess sufficient phylogenetic signal to assess relationships among the asexual Alternaria. The remaining five loci revealed strong support for asexual Alternaria and the order of divergence among eight asexual Alternaria species-groups. The current polyphyly of Alternaria was resolved using morphological and molecular data. Morphological examination revealed that most members of the infectoria clade (sexual Alternaria) produce diagnostic colony characters on dichloran rose bengal yeast extract sucrose agar (DRYES) and weak potato dextrose agar (WPDA) that are fundamentally different as compared to other small-spored Alternaria species. These data also revealed that all members of the infectoria clade produce arachnoid vegetative hyphae with multiple primary conidiophores, whereas other small-spored Alternaria species do not. Phylogenetic analyses revealed that the sexual infectoria clade clusters with other sexual genera phylogenetically distant to the asexual Alternaria. Lastly, the validity of taxonomy and the phylogenetic relationship among three small-spored Alternaria species was examined. Total ortholog comparisons and whole-genome comparisons revealed that the DNA sequence of A. alternata ATCC 11680 has a higher percent similarity to A. tenuissima EGS 34-015 than to A. alternata EGS 34-016. This suggests that these two isolates share a more recent common ancestor and A. alternata EGS 34-016 is more distantly related.

Recent studies suggest that horizontal gene transfer (HGT) plays an important role in niche adaptation in some eukaryotes and may be a major evolutionary force in unicellular lineages. One subcategory of HGT is endosymbiotic gene transfer (EGT), which is characterized by a large influx of genes from endosymbiont to host nuclear genome and is a critical step in the establishment of permanent organelles, such as plastids. The dinoflagellates are a diverse group of mostly marine eukaryotes that have a propensity for both HGT and plastid endosymbiosis. Many dinoflagellates are predators and can acquire both genes and plastids from prey, blurring the distinction between HGT and EGT. Here, I measure genome mosaicism in dinoflagellates to investigate how HGT has impacted gene innovation and plastid endosymbiosis in this group. Because analysis of HGT depends on accurate phylogenetic trees, I first assessed the sensitivity of automated phylogenomic methods to variation in taxon sampling due to homolog selection parameters. Using methods based on this analysis, I showed that a large amount of HGT has occurred in dinoflagellates, particularly from bacterial donors. Further, I demonstrated that the dinoflagellate Alexandrium tamarense has the largest number of genes gained relative to related eukaryotes using ancestral gene content reconstruction. Additionally, dinoflagellates have lost several ubiquitous eukaryotic metabolic genes, but missing genes have been functionally replaced by xenologs from many evolutionarysources. Other transferred genes are involved in diverse functions. These results suggest that dinoflagellate genomes are heavily impacted by HGT. Also, I investigated the timing and consequences of HGT in plastid endosymbiosis. Using the dinflagellate Dinophysis acuminata, a mixotrophic species that sequesters and maintains prey plastids, I identified plastid-targeted proteins that function in photosystem stabilization and metabolite transport. Dinophysis acuminate may be able to extend the useful life of the stolen plastid by protecting the photosystem and replacing damaged transporters. Phylogenetic analyses showed that genes are derived from multiple sources indicating a complex evolutionary history involving HGT. Dinophysis acuminate can acquire both genes and plastids from prey, which suggests that HGT could play an important role in plastid acquisition during the earliest stages of this transition.

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.

Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.

Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations. In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions. Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations. In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components. Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%.