Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.

Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step in genome annotation process which is needed, not only for the validation of predicted genes, but also for the identification of novel genes, especially those coding for non-coding RNA, which are missed by gene prediction programs. DNA sequence dependent structural properties such as DNA duplex stability, bendability and intrinsic curvature have been found to be associated with promoter regions in all domains of life. The work presented in this thesis focuses on the analysis of these structural features in the promoter regions of published prokaryotic transcriptome data. Furthermore, promoters were predicted using these structural features and their role in gene expression were studied. The organization of thesis is as follows. An overview of transcription machinery of prokaryotes, promoter architecture, available promoter prediction programs and sequence dependent structural features is presented in chapter 1. Chapter 2 describes the datasets and methods used in entire study. Structural features of promoters associated with primary and operon TSSs of H.pylori26695 genes and their orthologs (chapter 3) Promoter regions in genomic sequences from all domains of life show similar trends in their structural properties such as stability, bendability, curvature. This chapter dis-cuss the DNA duplex stability and bendability of various classes of promoter regions (based on the identification of different classes of transcription start sites, viz. primary, secondary, internal, operon TSSs etc, in transcriptome study) of Helicobacter pylori 26695 strain. It is found that the primary TSS and operon associated TSS promoters show significantly strong structural features in their promoter regions. DNA free energy based promoter prediction tool PromPredict has been used to annotate promoters of different classes and very high recall values (80%) are obtained for primary TSS. Orthologous genes from 10 different strains of H. pylori show conservation of structural properties in promoter regions as well as coding regions. PromPredict annotates promoters of orthologous genes with very high recall and precision values. DNA duplex stability of promoter region is conserved in the orthologous genes in 10 different strains of Helicobacter pylori genome. Sequence dependent structural features of promoters in prokaryotic transcriptome (chapter 4) Next-generation sequencing studies have revealed that a wide range of transcripts such as primary, internal, antisense and non-coding RNA, are present in the prokaryotic transcriptome and a large fraction of them are functionally involved in various regulatory activities. Identification of promoters associated with different transcripts is important for characterization of transcriptome. The current chapter discusses DNA sequence dependent structural properties like stability, bendability and curvature in the promoter region of six different prokaryotic transcriptomes (Helicobacter pylori, Anabaena, Synechocystis, Escherichia coli, Salmonella and Klebsiella). Using these structural features, promoters associated with different category of transcripts were predicted, which constitute an integral part of the transcriptome. Promoter annotation using structural features is fairly accurate and reliable as compared to motif-based approach since different category of transcripts show poor sequence conservation in the promoter region. Most importantly, it is universal in nature unlike sequence-based approach that is generally organism specific. Role of sequence dependent structural properties in gene expression in prokaryotes (chapter 5) DNA duplex stability, bendability and intrinsic curvature play crucial roles in the process of transcription initiation. Hence, in order to understand the relationship be-tween these structural features and gene expression, the relative differences in stability, bendability and curvature in the promoter regions of high and low expressed genes were studied. It is found that these features are relatively accentuated in the promoter regions associated with high gene expression as compared to low gene expression. Promoter regions associated with high gene expression are annotated more reliably using DNA structural features, compared to those for low gene expression. Sequence dependent structural properties in the promoter region of essential and non-essential genes of the prokaryotes (chapter 6) Essential genes are the minimal possible set of genes required for the survival of organism. These sets of genes can be identified by experiments such as single gene deletion and transposon mediated inactivation. Here, the analysis of DNA duplex stability and bendability in the promoter regions of essential and nonessential genes of prokaryotes is reported. It is found that the average free energy and bendability pro-files are distinct in the promoters regions of essential and nonessential genes. Whole genome promoter predictions using in-house program, PromPredict, for essential and nonessential genes has also been carried out. Chapter 7 present the summary and conclusion of the entire thesis work followed by future perspectives in the field. Optimization of PromPredict algorithm and updating PromBase with newly sequenced genomes (Appendix A) PromPredict is an in-house program, which is based on the relative stability of the DNA in flanking regions. It was found to perform well in predicting promoters across all organisms. In previous studies, it was observed that for organisms having low genomic GC content (<35%), promoter prediction resulted in low precision values, which indicates higher false positive rate. Threshold values of PromPredict algorithm were re-vised in order to optimize the algorithm with low false positive rate. PromBase is a comparative genomics database of microbial genomes. It stores different genomic and structural properties of the microbial genomes. It also displays the predictions obtained from PromPredict in a graphical as well as tabular format. Newly sequenced genomes were downloaded from NCBI and processed using in-house programs and added to the mysql database (back end of the PromBase). Stability profiles for predictions were also added for the RNA coding genes, earlier only profiles for protein coding genes were displayed. Comparative genomics of asymmetric gene orientation in prokaryotes (Appendix B) Transcription proceeds in 5’ to 3’ direction on the template strand, hence it provides directionality. Prokaryotic genomes show asymmetry in gene orientation on leading and lagging strands. The different phyla of prokaryotes were analyzed in terms of asymmetry in gene orientation. It is found that organisms belonging to a particular phyla known as “Firmicutes”, show high asymmetry in gene orientation, which are known to have different DNA polymerase systems for replication.

Fitoplasmas são organismos procarióticos desprovidos de parede celular, parasitas intracelulares obrigatórios de floema e patogênicos às plantas. Numerosas doenças associadas a este tipo de agentes patogênicos têm sido frequentemente relatadas em espécies cultivadas e plantas daninhas. No presente estudo, sintomas tipicamente induzidos por fitoplasmas, tais como clorose e avermelhamento foliar, redução no tamanho das folhas, diminuição dos órgãos florais, superbrotamento de ramos e declínio da planta, foram observados em pomares de fruteiras de caroço como pessegueiro e nectarineira. Além disto, em campos de couve-flor, plantas daninhas conhecidas como mostarda-do-campo também manifestaram sintomas que pareciam ter sido provocados por fitoplamas, expressados por superbrotamento de ramos finos e redução no tamanho de folhas e flores. Assim, o presente trabalho teve por objetivos detectar, identificar e classificar a nível molecular os possíveis fitoplasmas associados às plantas sintomáticas citadas anteriormente, utilizando as técnicas de PCR, análise de RFLP virtual e o método de classificação on-line denominado de iPhyclassifier. Para tanto, o DNA das amostras das fruteiras de caroço e da erva daninha foi extraído com o auxílio de um kit comercial ou através do protocolo 2X CTAB. A detecção dos fitoplasmas foi conduzida por duplo PCR com os primers universais R16mF2/mR1 e SN910601/SN011119 na primeira reação e com o par R16F2n/R2 na segunda reação. Para a mostarda, a identificação dos fitoplasmas também foi realizada através de duplo PCR com os primers e R16(III)F2/R16(III)R1 específicos para fitoplasmas do grupo 16SrIII. A aplicação de PCR com primers universais permitiu a detecção consistente desses agentes nos tecidos das plantas sintomáticas de pessegueiro, nectarineira e mostarda-do-campo pela amplificação de um fragmento genômico de 1250 pb, correspondente ao gene 16S rRNA. Para todos os hospedeiros analisados, os produtos amplificados por duplo PCR com os primers universais foram purificados, clonados em Escherichia coli e submetidos ao sequenciamento. A análise de RFLP virtual e iPhyclassifier permitiram classificar os fitoplasmas encontrados em mostarda nos grupos 16SrIII e 16SrVII. Os fitoplasmas detectados na mostarda-do-campo, foram definitivamente classificados como representantes dos subgrupos 16SrIII-B, 16SrIII-J e 16SrIII-U e 16SrVII-B. Em relação aos fitoplasmas encontrados no pessegueiro e nectarineira, foi possível classificar os fitoplasmas nos grupos 16SrI e 16SrIII, sendo definitivamente classificados como representantes dos subgrupos 16SrI-B em pessegueiro, e em nectarineira, classificados em 16SrI-B e 16SrIII-J. / Phytoplasmas are prokaryotic organisms devoid of cell wall, obligate intracellular phloem parasites and pathogenic to plants. Numerous diseases associated with this type of pathogen have been frequently reported in cultivated species of plants and weeds. In this present study, symptoms typically induced by phytoplasmas, such a yellowing an reddeling leaves, small leaves and flowers, \"witches\' broom\" appearance on terminal new bud growth, and death in plants, were observed in orchards of stone fruit trees such as peach and nectarines. In addition, in fields of cauliflower, weeds known as mustard-field also exhibited symptoms that appeared to have been caused by phytoplasmas, expressed by thin branches and reduced leaf and flower size. The objective of this present study was to detect, identify and classify at molecular level the phytoplasmas possibly associated with the symptomatic plants. PCR techniques, virtual RFLP analysis and the online method known as iPhyclassifier were used. Total DNA was extracted using a commercial kit or through the CTAB protocol. The detection of phytoplasmas was conducted by nested PCR with the universal primers R16mF2/mR1 and SN910601/SN011119 in the first reaction and R16F2n/R2 pair in the second reaction. For mustard-field, the identification of the phytoplasmas was also performed through nested PCR with the primers R16(III) F2/R16(III) R1, which are specific to detection of 16SrIII group phytoplasmas. The application of PCR with universal primers allowed the consistent detection of these agents in the tissues in symptomatic plants of peach, nectarine and mustard-field by the amplification of a 1250 bp genomic fragment, corresponding to the 16S rRNA gene. For all hosts analyzed, the products amplified by nested PCR with the universal primers were purified, cloned in Escherichia coli and sequenced. The analysis of virtual RFLP and iPhyclassifier allowed to classify the phytoplasmas found in mustard in the 16SrIII and 16SrVII groups. The phytoplasmas detected in mustard-field were definitively classified as representatives of the subgroups 16SrIII-B, 16SrIII-J and 16SrIII-U and 16SrVII-B. The phytoplasmas found in the peach and nectarine were classified within the 16SrI and 16SrIII groups, as representatives of the subgroups 16SrI-B for peach and 16SrI-B and 16SrIII-J for nectarine.

Made available in DSpace on 2015-03-05T13:53:45Z (GMT). No. of bitstreams: 0 Previous issue date: 3 / Universidade do Vale do Rio dos Sinos / A expressão dos genes em procariotos é desencadeada quando a enzima RNApolimerase interage com uma região adjacente ao gene, chamada de promotor, onde se encontram os principais elementos regulatórios do processo de transcrição. Apesar do crescente avanço das técnicas experimentais em biologia molecular, caracterizar e identificar um número significante de promotores, presentes em um dado genoma, continua sendo uma tarefa demorada e cara. Abordagens in silico são bastante utilizadas para reconhecer essas regiões em procariotos. Entretanto, além do alto número de falsos positivos obtidos, elas enfrentam a inexistência de um número adequado de promotores conhecidos para identificar padrões conservados entre as espécies. Logo, um método criterioso e confiável para predizêlos em qualquer organismo procariótico ainda é um desafio. Esta dissertação propõe um protocolo de uso de hidden Markov models (HMMs) que emprega Estimação de Limiar de Decisão (ELD) e Análise de Discriminação (AD) neste problema. Quatro espécie / Gene expression on prokaryotes initiates when the RNA-polymerase enzyme interacts with DNA regions called promoters. In these regions are located the main regulatory elements of the transcription process. Despite the improvement of in vitro techniques for molecular biology analysis, characterizing and identifying a great number of promoters on a genome is a complex task. In silico approaches are usually employed to recognize theses regions on prokaryotes. Nevertheless, the main drawback is the absence of a large set of promoters to identify conserved patterns among the species. Hence, a in silico method to predict them on any species is a challenge. This work proposes a protocol to use hidden Markov models (HMMs) methodology with Decision Threshold Estimation and Discrimination Analysis on this problem. Four prokaryotic species are investigated (Escherichia coli, Bacillus subtilis, Helicobacter pylori e Helicobacter hepaticus). The influence of different aspects in the recognition and prediction are examined:

<p>Seagrass beds cover large intertidal and subtidal areas in coastal zones around the world and they are subjected to a wide variety of anthropogenic influences, such as nutrient enrichment due to sewage seepage. This study was undertaken to address specific questions focusing on whether near shore tropical seagrasses that receive a constant influx of groundwater nutrient inputs, would exhibit a higher productivity and to what extent epiphytic algae reflect the impacts of nutrient inputs. An additional aspect of study was to determine the prevalence of “acid zones” in tropical seagrasses. The productivity of the seagrasses <i>Cymodocea rotundata</i>,<i> Thalassia hemprichii</i> and <i>Thalassodendron ciliatum</i> was compared in two sites along the Kenyan coast; Nyali (a high nutrient site) and Vipingo (a low nutrient site). Of the three seagrasses <i>T. hemprichii</i> showed the most distinct differences with higher growth and biomass in the nutrient rich site whereas the growth of <i>C. rotundata</i> was similar in the two sites. A high epiphytic cover was found on the shoots of <i>T. ciliatum</i> found in the high nutrient site Nyali.</p><p>Morphological and genetic characterization of bacterial and cyanobacterial epiphytes showed specific associations of nitrogen fixing cyanobacteria on the seagrass <i>C. rotundata</i> in the low nutrient site (Vipingo). At this site, shoots of <i>C. rotundata</i> had a higher C:N ratio compared to shoots in the high nutrient site (Nyali) indicating that the association with nitrogen fixing cyanobacteria is a strategy, for this species, to meet its nutrient needs. Bacterial epiphytes belonging to the group Cytophaga-Flavobacteria-Bacteroides (CFB) were found on <i>T. ciliatum</i> and <i>T. hemprichii</i> from the two sites. CFB bacteria are characteristic of waste water, particularly from livestock farming areas, thereby confirming seepage of groundwater from surrounding catchment areas. These prokaryotic associations were specific for the different seagrasses and it appears that the establishment of epiphytic associations may not be a random encounter but a specific association that meets specific needs.</p><p>The seagrass <i>T. ciliatum</i> in the high nutrient site had an abundance of macroalgal epiphytes and the impact of the epiphytic coverage was assessed using Pulse Amplitude Modulated (PAM) fluorometry. The photosynthetic activity of seagrass parts that were covered by epiphytes was suppressed but the productivity of the whole shoot was not significantly reduced. In the nutrient rich site, epiphytes were found to contribute up to 45% of the total estimated gross productivity, during the SE monsoon season, while epiphytic contribution in the nutrient poor site, was 8%. Epiphytic abundance and contribution to productivity decreased during the NE monsoon. The photosynthetic activity of <i>T. ciliatum</i> shoots was similar in the two study sites with shoots in the nutrient rich site growing faster. <i>T. ciliatum</i>, in the low nutrient site, invested in the development of below ground root tissue which may indicate the development of a strategy to gain access to pore water nutrient pools.</p><p>Carbon uptake strategies of eight tropical seagrasses were re-evaluated to determine how common the “acid zone” mechanism is among tropical seagrasses. Six of the eight species studied showed photosynthetic inorganic carbon (Ci) acquisition based on carbonic anhydrase catalysed HCO₃^- to CO₂ conversions within an acidified diffusion boundary layer (“acid zone”). <i>Cymodocea serrulat</i>a appeared to maintain its carbon uptake by extracellular carbonic anhydrase catalysed CO₂formation from HCO₃^- without the need for acidic zones, whereas, <i>Halophila ovalis</i> appeared to have a system in which H⁺ extrusion may be followed by HCO₃^--H⁺ co-transport into the cells. These findings indicate that competition for carbon, between the host seagrass species and epiphytes, could determine seagrass-epiphyte associations.</p>

Seagrass beds cover large intertidal and subtidal areas in coastal zones around the world and they are subjected to a wide variety of anthropogenic influences, such as nutrient enrichment due to sewage seepage. This study was undertaken to address specific questions focusing on whether near shore tropical seagrasses that receive a constant influx of groundwater nutrient inputs, would exhibit a higher productivity and to what extent epiphytic algae reflect the impacts of nutrient inputs. An additional aspect of study was to determine the prevalence of “acid zones” in tropical seagrasses. The productivity of the seagrasses Cymodocea rotundata, Thalassia hemprichii and Thalassodendron ciliatum was compared in two sites along the Kenyan coast; Nyali (a high nutrient site) and Vipingo (a low nutrient site). Of the three seagrasses T. hemprichii showed the most distinct differences with higher growth and biomass in the nutrient rich site whereas the growth of C. rotundata was similar in the two sites. A high epiphytic cover was found on the shoots of T. ciliatum found in the high nutrient site Nyali. Morphological and genetic characterization of bacterial and cyanobacterial epiphytes showed specific associations of nitrogen fixing cyanobacteria on the seagrass C. rotundata in the low nutrient site (Vipingo). At this site, shoots of C. rotundata had a higher C:N ratio compared to shoots in the high nutrient site (Nyali) indicating that the association with nitrogen fixing cyanobacteria is a strategy, for this species, to meet its nutrient needs. Bacterial epiphytes belonging to the group Cytophaga-Flavobacteria-Bacteroides (CFB) were found on T. ciliatum and T. hemprichii from the two sites. CFB bacteria are characteristic of waste water, particularly from livestock farming areas, thereby confirming seepage of groundwater from surrounding catchment areas. These prokaryotic associations were specific for the different seagrasses and it appears that the establishment of epiphytic associations may not be a random encounter but a specific association that meets specific needs. The seagrass T. ciliatum in the high nutrient site had an abundance of macroalgal epiphytes and the impact of the epiphytic coverage was assessed using Pulse Amplitude Modulated (PAM) fluorometry. The photosynthetic activity of seagrass parts that were covered by epiphytes was suppressed but the productivity of the whole shoot was not significantly reduced. In the nutrient rich site, epiphytes were found to contribute up to 45% of the total estimated gross productivity, during the SE monsoon season, while epiphytic contribution in the nutrient poor site, was 8%. Epiphytic abundance and contribution to productivity decreased during the NE monsoon. The photosynthetic activity of T. ciliatum shoots was similar in the two study sites with shoots in the nutrient rich site growing faster. T. ciliatum, in the low nutrient site, invested in the development of below ground root tissue which may indicate the development of a strategy to gain access to pore water nutrient pools. Carbon uptake strategies of eight tropical seagrasses were re-evaluated to determine how common the “acid zone” mechanism is among tropical seagrasses. Six of the eight species studied showed photosynthetic inorganic carbon (Ci) acquisition based on carbonic anhydrase catalysed HCO3- to CO2 conversions within an acidified diffusion boundary layer (“acid zone”). Cymodocea serrulata appeared to maintain its carbon uptake by extracellular carbonic anhydrase catalysed CO2 formation from HCO3- without the need for acidic zones, whereas, Halophila ovalis appeared to have a system in which H+ extrusion may be followed by HCO3--H+ co-transport into the cells. These findings indicate that competition for carbon, between the host seagrass species and epiphytes, could determine seagrass-epiphyte associations.

CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.

The general goal of this project was to use computational biology to understand signal transduction mechanisms in prokaryotes. Its specific focus was to characterize the cytoplasmic domain of methyl-accepting chemotaxis proteins (MCP_CD), a protein domain central to the function of chemotaxis, the most complex signaling network in prokaryotes. Chemotaxis enables cells to sense and respond to multiple external and internal stimuli by actively navigating to an optimal environment. MCP_CD is a central part of this circuit, but its coiled coil structure is difficult to analyze using traditional tools of computational biology. In this project, a new method for analysis of the domain was developed and used to gain insight into its function and evolution. Research advance 1: Characterization of the MCP_CD protein domain. Before this work, MCP_CD was known to have two distinct functional regions: the signaling region that activates the histidine kinase CheA and the methylation region where adaptation enzymes CheB and CheR store information about recent stimuli. The result of this project is classification of ~2000 MCP_CDs into twelve subfamilies. The unique mechanism of evolution of the domain has been clarified and precise boundaries of the adaptation and signaling regions determined. A new functional region, the flexible bundle subdomain, was identified and its contribution to the signaling mechanism elucidated by analysis of conserved sequence features. Conserved and variable sequence features in the adaptation and signaling subdomains led to a better understanding of the evolutionary history of the adaptation mechanism and of alternative higher-order arrangements of receptors within the membrane. Research advance 2: Development of a sensor / kinase correlation algorithm to couple diverse MCP_CD and kinase subfamilies. The receptor diversity discovered in this work is complemented by diversity in the kinases with which they interact. In this work, an algorithm was developed to associate receptor / kinase pairs which facilitated understanding of the function and evolution of chemotaxis. Research advance 3: Development of Cheops, a database of chemotaxis pathways. The Cheops (Chemotaxis operons) database presents the results of the sensor / kinase correlation algorithm and the information about receptor and kinase diversity in an integrated and intuitive way.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 141-152). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 141-152). Also available on the Internet.