Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2009.

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2009.

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen of a variety of grass genra and species, including Bromus tectorum, an exotic grass that has invaded many natural ecosystems of the U.S. Intermountain West. As a natural seed pathogen of B. tectorum, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing dormant B. tectorum seeds; however, few genetic resources exist for this fungus. Here, the genome assembly of a P. semeniperda isolate using 454 GS-FLX genomic and paired-end pyrosequencing techniques is presented. The total assembly is 32.5 Mb and contains 11,453 gene models greater than 24 amino acids. The assembly contains a variety of predicted genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, 454 sequence reads were used to identify single nucleotide polymorphisms between two isolates of P. semeniperda. In total, 20 SNP markers were developed for the purposes of recombination assesment of 600 individual P. semeniperda isolates representing 36 populations from throughout the U.S. Intermountain West. Although 17 of the fungal populations were fixed at all SNP loci, linkage disequilibrium was determined in the remaining 18 populations. This research demonstrates the effectiveness of the 454 GS-FLX sequencing technology, for de novo assembly and marker development of filamentous fungal genomes. Many features of the assembly match those of other Pyrenophora genomes including P. tritici-repentis and P. teres f. teres, which lend validity to our assembly. These findings present a significant resource for examining and furthering our understanding of P. semeniperda biology.

Infestações com Rhipicephalus microplus, o carrapato dos bovinos, causam enormes prejuízos econômicos para a pecuária. Os carrapatos estão desenvolvendo resistência aos carrapaticidas que, além dessa desvantagem, deixam resíduos em carne e leite. Vacinas anticarrapato representam uma alternativa sustentável de controle de infestações, mas as atualmente disponíveis têm efeitos parciais e transitórios. Surge, assim, a necessidade de identificar novos antígenos vacinais. Para alcançar esse objetivo este trabalho explora o fato de que bovinos apresentam fenótipos contrastantes e herdáveis de infestações que são específicos de certas raças. Além disso, o nível de imunidade do hospedeiro afeta a transcrição de genes de glândulas salivares do carrapato, órgão que produz proteínas que medeiam o parasitismo. A hipótese de trabalho é a que os diferentes níveis da imunidade anticarrapato do hospedeiro afetam, também, a composição salivar do parasita. Assim, em carrapatos alimentando-se em hospedeiros resistentes as proteínas que são cruciais ao parasitismo poderão estar ausentes ou deficientes na sua saliva e por isso os carrapatos não terminam sua refeição de sangue. A neutralização dessas mesmas proteínas pela imunidade humoral pode ter o mesmo efeito e por isso, essas proteínas constituem bons antígenos vacinais. Assim, o objetivo do trabalho foi identificar novos antígenos vacinais em saliva de fêmeas e glândulas salivares de ninfas, machos e fêmeas de carrapatos alimentados em hospedeiros resistentes e suscetíveis, bem como em larvas não alimentadas oriundas de ovos de fêmeas alimentadas nestes mesmos hospedeiros. Para isso, foram empregadas abordagens de sequenciamento de nova geração \"RNA-Seq\" (454) e abordagens proteômicas, como análise diferencial em gel (DIGE) e Western Blots (imunoproteoma) seguido de sequenciamento de massa, além da tecnologia de identificação de proteínas multidimensionais (ou Multidimensional Protein Identification Technology, MudPIT) para descrever o proteoma das glândulas salivares e da saliva de fêmeas. A análise transcriptômica resultou no sequencimanto de 1.999.086 reads que permitiu identificar e classificar 11.676 sequências codificadoras (CDS), muitas das quais (3.600 CDS) contêm peptídeo sinal que é indicativo de secreção, portanto podendo estar presente na saliva e Resumo Gustavo Rocha Garcia apresentar função importante na hematofagia. Por meio de MudPIT, identificamos 321 proteínas salivares diferentes, além de 126 proteínas no DIGE e 266 proteínas nos imunoproteomas. Muitas dessas proteínas podem ser consideradas antígenos potenciais por estarem associadas com a hematofagia/parasitismo, tais como proteases, nucleases, inibidores de proteases, peptídeos antimicrobianos, proteínas de fixação, entre outros, inclusive proteínas ainda não caracterizadas. A maioria dos genes codificantes dessas proteínas está mais expressa em carrapatos alimentados em hospedeiros suscetíveis, principalmente em carrapatos machos. Além disso, muitas dessas proteínas não são reconhecidas por soros bovinos, inclusive soros de bovinos infestados, embora soros de bovinos infestados e resistentes ao carrapato apresente a maioria das reatividades. O conjunto dos resultados sugere que em nível de proteína a composição da saliva também é afetada pelos diferentes níveis de imunidade dos hospedeiros, além de variar com o ciclo de vida do carrapato. Desse modo, concluímos que as estratégias de investigação empregadas foram satisfatórias para identificar um conjunto de antígenos salivares do carrapato R. microplus que representam proteínas alvos para compor vacinas multicomponentes anticarrapato. / Infestation with Rhipicephalus microplus, the cattle tick, causes huge economic losses to livestock. Ticks are developing resistance to acaricides that, besides this disadvantage, leave residues in meat and milk. The anti tick vaccines represent a sustainable alternative of the infestations control, but the currently available has partial and transient effects. Thus arises the need to identify new vaccine antigens. To achieve this goal, this work explores the fact that cattle exhibit contrasting phenotypes and inheritable of infestations that are specific to certain breeds. Furthermore, the level of immunity of the host affects gene transcription tick salivary gland, organ that produces proteins that mediate the parasitism. The working hypothesis is that different levels of anti tick immunity of host affect also the salivary composition of the parasite. So in ticks feeding on resistant hosts the proteins that are crucial to parasitism may be absent or deficient in their saliva, and by this the ticks do not finish your meal blood. The neutralization of these same proteins by humoral immunity can have the same effect and by this, these proteins are good vaccine antigens. So, the aim of the study was to identify new vaccine antigens in saliva from females and salivary glands of nymphs, males and females of ticks fed on resistant and susceptible hosts as well as in unfed larvae originating from eggs of females fed on these same hosts. To this, were employed sequencing approaches of new generation \"RNA-Seq\" (454) and proteomic approaches, such as differential analysis in gel (DIGE) and Western Blots (immunoproteomics) followed by sequencing mass, besides the Multidimensional Protein Identification Technology (MudPIT) to describe the proteome of the salivary glands and saliva of females. The transcriptomics analysis identified 11,676 coding sequences (CDS), many of which (3,600 CDS) contain predicted signal peptide indicative of secretion, therefore may be present in saliva and provide an important function in blood feeding. Through MudPIT, we identify 321 different salivary proteins, besides 126 proteins in DIGE and 266 proteins in immunoproteomics. Many of these proteins may be considered as potential antigens to be associated with the blood meal/ parasitism, such as proteases, nucleases, protease inhibitors, antimicrobial peptides, proteins of attachment, among Abstract Gustavo Rocha Garcia others, including proteins not yet characterized. Most of the genes encoding of these proteins are more expressed in ticks fed on susceptible hosts, especially in male ticks. Moreover, many of these proteins are not recognized by bovine sera, including sera from infested hosts, although sera from infested and resistant host to tick present the most reactivities. The overall results suggest that in protein level, the composition of saliva is also affected by the different levels of immunity of the host, besides vary with the tick life cycle. Thus, we conclude that the research strategies employed were satisfactory to identify a set of tick salivary antigens from R. microplus that represent target proteins for composing anti tick multicomponent vaccines.

Infestações com Rhipicephalus microplus, o carrapato dos bovinos, causam enormes prejuízos econômicos para a pecuária. Os carrapatos estão desenvolvendo resistência aos carrapaticidas que, além dessa desvantagem, deixam resíduos em carne e leite. Vacinas anticarrapato representam uma alternativa sustentável de controle de infestações, mas as atualmente disponíveis têm efeitos parciais e transitórios. Surge, assim, a necessidade de identificar novos antígenos vacinais. Para alcançar esse objetivo este trabalho explora o fato de que bovinos apresentam fenótipos contrastantes e herdáveis de infestações que são específicos de certas raças. Além disso, o nível de imunidade do hospedeiro afeta a transcrição de genes de glândulas salivares do carrapato, órgão que produz proteínas que medeiam o parasitismo. A hipótese de trabalho é a que os diferentes níveis da imunidade anticarrapato do hospedeiro afetam, também, a composição salivar do parasita. Assim, em carrapatos alimentando-se em hospedeiros resistentes as proteínas que são cruciais ao parasitismo poderão estar ausentes ou deficientes na sua saliva e por isso os carrapatos não terminam sua refeição de sangue. A neutralização dessas mesmas proteínas pela imunidade humoral pode ter o mesmo efeito e por isso, essas proteínas constituem bons antígenos vacinais. Assim, o objetivo do trabalho foi identificar novos antígenos vacinais em saliva de fêmeas e glândulas salivares de ninfas, machos e fêmeas de carrapatos alimentados em hospedeiros resistentes e suscetíveis, bem como em larvas não alimentadas oriundas de ovos de fêmeas alimentadas nestes mesmos hospedeiros. Para isso, foram empregadas abordagens de sequenciamento de nova geração \"RNA-Seq\" (454) e abordagens proteômicas, como análise diferencial em gel (DIGE) e Western Blots (imunoproteoma) seguido de sequenciamento de massa, além da tecnologia de identificação de proteínas multidimensionais (ou Multidimensional Protein Identification Technology, MudPIT) para descrever o proteoma das glândulas salivares e da saliva de fêmeas. A análise transcriptômica resultou no sequencimanto de 1.999.086 reads que permitiu identificar e classificar 11.676 sequências codificadoras (CDS), muitas das quais (3.600 CDS) contêm peptídeo sinal que é indicativo de secreção, portanto podendo estar presente na saliva e Resumo Gustavo Rocha Garcia apresentar função importante na hematofagia. Por meio de MudPIT, identificamos 321 proteínas salivares diferentes, além de 126 proteínas no DIGE e 266 proteínas nos imunoproteomas. Muitas dessas proteínas podem ser consideradas antígenos potenciais por estarem associadas com a hematofagia/parasitismo, tais como proteases, nucleases, inibidores de proteases, peptídeos antimicrobianos, proteínas de fixação, entre outros, inclusive proteínas ainda não caracterizadas. A maioria dos genes codificantes dessas proteínas está mais expressa em carrapatos alimentados em hospedeiros suscetíveis, principalmente em carrapatos machos. Além disso, muitas dessas proteínas não são reconhecidas por soros bovinos, inclusive soros de bovinos infestados, embora soros de bovinos infestados e resistentes ao carrapato apresente a maioria das reatividades. O conjunto dos resultados sugere que em nível de proteína a composição da saliva também é afetada pelos diferentes níveis de imunidade dos hospedeiros, além de variar com o ciclo de vida do carrapato. Desse modo, concluímos que as estratégias de investigação empregadas foram satisfatórias para identificar um conjunto de antígenos salivares do carrapato R. microplus que representam proteínas alvos para compor vacinas multicomponentes anticarrapato. / Infestation with Rhipicephalus microplus, the cattle tick, causes huge economic losses to livestock. Ticks are developing resistance to acaricides that, besides this disadvantage, leave residues in meat and milk. The anti tick vaccines represent a sustainable alternative of the infestations control, but the currently available has partial and transient effects. Thus arises the need to identify new vaccine antigens. To achieve this goal, this work explores the fact that cattle exhibit contrasting phenotypes and inheritable of infestations that are specific to certain breeds. Furthermore, the level of immunity of the host affects gene transcription tick salivary gland, organ that produces proteins that mediate the parasitism. The working hypothesis is that different levels of anti tick immunity of host affect also the salivary composition of the parasite. So in ticks feeding on resistant hosts the proteins that are crucial to parasitism may be absent or deficient in their saliva, and by this the ticks do not finish your meal blood. The neutralization of these same proteins by humoral immunity can have the same effect and by this, these proteins are good vaccine antigens. So, the aim of the study was to identify new vaccine antigens in saliva from females and salivary glands of nymphs, males and females of ticks fed on resistant and susceptible hosts as well as in unfed larvae originating from eggs of females fed on these same hosts. To this, were employed sequencing approaches of new generation \"RNA-Seq\" (454) and proteomic approaches, such as differential analysis in gel (DIGE) and Western Blots (immunoproteomics) followed by sequencing mass, besides the Multidimensional Protein Identification Technology (MudPIT) to describe the proteome of the salivary glands and saliva of females. The transcriptomics analysis identified 11,676 coding sequences (CDS), many of which (3,600 CDS) contain predicted signal peptide indicative of secretion, therefore may be present in saliva and provide an important function in blood feeding. Through MudPIT, we identify 321 different salivary proteins, besides 126 proteins in DIGE and 266 proteins in immunoproteomics. Many of these proteins may be considered as potential antigens to be associated with the blood meal/ parasitism, such as proteases, nucleases, protease inhibitors, antimicrobial peptides, proteins of attachment, among Abstract Gustavo Rocha Garcia others, including proteins not yet characterized. Most of the genes encoding of these proteins are more expressed in ticks fed on susceptible hosts, especially in male ticks. Moreover, many of these proteins are not recognized by bovine sera, including sera from infested hosts, although sera from infested and resistant host to tick present the most reactivities. The overall results suggest that in protein level, the composition of saliva is also affected by the different levels of immunity of the host, besides vary with the tick life cycle. Thus, we conclude that the research strategies employed were satisfactory to identify a set of tick salivary antigens from R. microplus that represent target proteins for composing anti tick multicomponent vaccines.

Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling and growth of flax (Linum usitatissimum L.). However, limited information is available regarding the symbiotic association between flax and AMF in field environments. A study was conducted to survey AMF communities colonizing flax grown in Saskatchewan. Additionally, field and growth chamber studies investigated the impact of AMF inoculation on nutrient uptake and growth of flax. Eighteen commercial flax fields were surveyed to assess mycorrhizal colonization of flax and to assess the impact of agricultural practices and soil abiotic factors on AMF activity. The flax root-associated AMF communities were explored using a 454 sequencing method, together with microscopic-based measurements of root AMF colonization and soil spore density. High levels of root colonization were detected in most flax fields. Of the 222 AMF operational taxonomic units (OTUs) identified in flax roots, 181 OTUs clustered as Funneliformis-Rhizophagus, 19 as Claroideoglomus, 14 as Paraglomus, six as Diversisporales and two as Archaeospora. Results suggest that tillage influenced the composition of AMF communities colonizing flax, and reduced relative AMF abundance and species richness. Additionally, AMF community characteristics were related to soil abiotic factors such as pH, EC, available phosphorus and nitrogen. Field experiments were conducted over two years (two sites per year) using a commercial AMF inoculant applied at three rates (0, 1X, and 2X the recommended rate) with or without P fertilizer (16.8 kg ha-1). The response of flax cultivars to AMF inoculation was examined in a growth chamber experiment. In addition, 454 sequencing was employed to examine the impact of AMF inoculation on root-associated AMF communities. Under field conditions, only one site showed increased root colonization with AMF inoculation. Flax responded to AMF inoculation differently under different field conditions. At the two sites with intermediate initial soil P level, evidence of increased above-ground biomass and plant nutrient uptake with AMF inoculation was observed. However, such an effect was not detected when P fertilizer was combined with the inoculation. At a low P site and an irrigated site, P application accounted for all of the increases in plant nutrient uptake and biomass of flax, whereas no responses to AMF inoculation were detected. The 454 sequencing revealed different inoculation-induced changes in the diversity and composition of root-associated AMF communities between sites, which was possibly related to different field environments and native AMF communities. In the growth chamber, AMF inoculation resulted in general increases of plant nutrient uptake among cultivars, but only one cultivar showed enhanced biomass with inoculation. The diversity of AMF communities colonizing different flax cultivars was generally reduced by AMF inoculation. Community composition shifted under AMF inoculation, and the shifts appeared to be cultivar specific. These results suggested that benefits of AMF inoculation in flax production are limited and currently not predictable, and the degree of response is likely dependent on a myriad of soil and environmental conditions.

BACKGROUND:Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.RESULTS:Here we present an 'A to Z' protocol for obtaining complete human mitochondrial (mtDNA) genomes - from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).CONCLUSIONS:All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual 'modules' can be swapped out to suit available resources.

<p>Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, <italic>Cladonia grayi</italic>, and one from a cyanolichen, <italic>Peltigera praetextata</italic>). Non-photobiont bacterial genes for nitrogen fixation were not detected in the <italic>Cladonia</italic> thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.</p> / Dissertation

Marine micro-organisms are important components of various biogeochemical cycles, complex food webs and ecological niches. Metagenomic sequencing can provide rapid profile of metabolic activities within the sponge and resident microbes. However, the study of metatranscriptomes from sponges using high throughput sequencing technology has only recently begun. Through this study we isolated, characterized and compared metatranscriptome profiles of Axinella corrugata host and sponge-specific microbial communities using 454 pyrosequencing technology. Four cDNA libraries (two eukaryotic and two prokaryotic) were generated from Axinella corrugata sponge samples collected in December 2009 and May 2010, and were characterized to a) reveal which metabolic genes were actively expressed and b) reveal possible interactions between the sponge and its microbial symbionts. The techniques used for isolation of mRNA and cDNA normalization also helped in optimization of whole-transcriptome amplification. More than 130,000 ESTs were generated for the two seasonal sponge samples and the metagenomic data sets were analyzed using bioinformatics tool, MG-RAST. Several stress-related transcripts were found which can increase our understanding of sensitivity of the sponge to changes in physical parameters in nature. The involvement of the sponge and its microbial consortia is depicted through actively expressed nitrogen and sulfur metabolism genes. Novel genes involved in several functional pathways may be discovered upon further studying hypothetical genes found across all four metagenomic data sets. Metatranscriptomic data sheds light on the functional role of microbes within the sponges and the extent of their involvement in sponge metabolism. 16S rRNA analysis was also carried out using genomic DNA of the same samples, to better elucidate the bacterial taxa abundance in the sponge. This study provides a profile of active mRNA trancripts in Axinella corrugata which include eukaryotic as well as prokaryotic sequences. The data analysis of this research provides new information at the cross-disciplinary interface between molecular biology and computational science.

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.