D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

Este projeto teve como objetivo a construção de biosensores para a detecção de compostos monoaromáticos do grupo BTEX a partir dos componentes da via de degradação de compostos monoaromáticos codificada no plasmídeo TOL de Pseudomonas putida, mais precisamente: o promotor Pu e a proteína reguladora XylR, que ativa o promotor Pu após a ligação ao efetor monoaromático. Um objetivo secundário foi a verificação da existência de sequências reguladoras desconhecidas a montante do promotor Pu, construindo três variantes com fragmentos de Pu que se estendem por diferentes comprimentos a montante do promotor (Pu202 pb, Pu396 pb e Pu802 pb), cuja existência foi sugerida em trabalho anterior do laboratório. O promotor Pu foi ligado ao gene indicador para fosfatase alcalina isolado de E. coli. Todos os componentes das três variantes de biossensores foram clonados com sucesso. A construção de um dos plasmídeos de biossensoramento com a variante mais curta de Pu (Pu202) foi concluída. / Monoaromatic compounds are mainly responsible for the contamination of areas, this is because they are components of gas and the fuel stations most of accidents sites. Such compounds are highly toxic and, in between non-polar compounds, present high solubility and vapor pressure which assists them in dispersion at groundwaters and soil. This work aim to develop a biosensor based on alkaline phosphatase indicator gene under the regulation of the Pu promoter and its regulatory protein, XylR, that is activated by monoaromatic. Moreover, this work will continue a previous project of the research group that indicated a possible regulatory region not described for Pu, that hypothesis will be tested by producing different plasmids biosensors with varying sizes of Pu (202 bp, 396 bp and 802 bp). All biosensor fragments were purified and cloned on pGem T Easy and biosensor with Pu 202 pb was produced. Next goals are finishing others biosensors assemble and perform induction tests.

Os manguezais são ecossistemas complexos tipicamente encontrados na interface entre a terra e o mar. Apesar de sua grande importância ecológica estes ambientes estão em risco, devido à proximidade de áreas com elevada exposição a poluentes, como hidrocarbonetos policíclicos aromáticos (HPAs) liberados em derramantos de petróleo. Em nosso trabalho nós exploramos a diversidade e abundância taxonômica e funcional de bactérias em três manguezais localizados sob diferentes estágios de preservação no Estado de São Paulo. Os resultados mostraram que a concentração total de HPAs nos sedimentos foram diferentes entre todos os pontos de amostragem, com o sedimento de manguezal apresentando as concentrações mais elevadas. Genes <font face=\"Symbol\">a-ARHDs foram encontrados em todos os locais de amostragem, revelando a presença de enzimas envolvidas no metabolismo do bifenilo, naftaleno, dibenzofurano, 3-fenilpropanoato e benzeno. A PCR em tempo real demonstrou um maior número de cópias do gene <font face=\"Symbol\">a-ARHD e 16S rRNA nas áreas contaminadas. A análise das seqüências obtidas pelo sequenciamento do gene 16S rRNA mostrou estruturas de comunidades distintas para todas as amostras. O filo mais frequente foi Proteobacteria e o número de Unidades Taxonômicas Operacionais (OTUs) detectadas nas amostras da área preservada foi superior às daárea contaminada. A análise das seqüências obtidas para o pirosequenciamento do gene bph indicou um maior número de Famílias Proteicas Operacionais (FPOs) no ambiente com atividade antrópica. Em conclusão, os resultados permitiram acessar e identificar uma extensa diversidade de bactérias, incluindo os genes ARHD, 16S rRNA e bph. Tais dados podem oferecer novas abordagens para melhorar a recuperação de tais ambientes. / Mangroves are complex ecosystems typically found at the interface between land and sea. Despite its great ecological importance, these environments are at risk due to the proximity of areas with high exposure to pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) released by oil spill. In our work we explore the diversity and taxonomic and functional abundance of bacteria in three mangroves under different stages of preservation located in the state of São Paulo. The results showed that the total concentration of PAHs in sediments were different between all the sampling sites, with mangrove sediment having the higher concentrations. <font face=\"Symbol\">a-ARHDs genes were found in all the sampling sites, revealing the presence of enzymes involved in the metabolism of biphenyl, naphthalene, dibenzofuran, 3-phenylpropanoate and benzene. The real-time PCR demonstrated an increased number of copies of the <font face=\"Symbol\">a-ARHD and 16S rRNA genes in the contaminated areas. The analysis of the sequences obtained by pyrosequencing of 16S rRNA gene showed distinct communities structures for all samples. The phylum Proteobacteria was more frequent and the number of Operational Taxonomic Units (OTUs) detected in pristine samples area was higher than contaminated area. The analysis of the sequences obtained for pyrosequencing of bph gene indicated a greater number of Operational Protein Families (OPFs) in the environment with human activity. In conclusion, the results allow to access and identify a wide variety of bacteria, including ARHD genes, 16S rRNA and bph. Such data may provide new approaches for improving the recovery of such environments.

O efeito de alterações climáticas sobre os micro-organismos ainda é incerto, pois pouco se conhece sobre as espécies que habitam regiões extremas como o gelo, solo antártico, e o solo permanentemente congelado (permafrost). O permafrost tem como característica a preservação de material biológico por milhões de anos, servindo como fonte para estudos de evolução e biogeografia de micro-organismos. O objetivo deste trabalho foi estudar a diversidade microbiana em amostras de gelo, solo exposto por recuo de geleira e permafrost polares, e a diversidade funcional do gene alcano monoxigenase (alk). Métodos independentes de cultivo baseados no gene 16S rRNA foram utilizados, como DGGE, clonagem e pirossequenciamento. As geleiras da Ilha Rei George (Península Antártica) e do Pólo Sul Geográfico possuem cerca de 3.104 cél./mL e são compostas por micro-organismos diferentes, com predominância dos Filos Proteobacteria, Actinobacteria, Firmicutes e Cyanobacteria, muitos dos quais já descritos em outros ambientes criogênicos. O solo em frente à geleira Baranowski apresenta uma estrutura de comunidade diferente do gelo. O solo exposto por recuo de geleira apresenta uma sucessão ecológica, com predominância de heterotróficas durante todo o processo. Fixadores de nitrogênio no solo foram compostos por cianobactérias no início, e por Rhodopseudomonas e Rhodobacter no final da sucessão. Estes resultados foram melhor observados com o pirossequenciamento. As mudanças observadas podem estar relacionadas ao aumento de K, Mg+, NH4+, NO3- e/ou CO2 detectados após 15-20 anos de exposição do solo. A comunidade de permafrosts varia com o local e a idade de congelamento (de 5.000 a 8 milhões de anos). O gene alkM foi detectado em permafrosts do Ártico com 3 milhões de anos, e o gene alkB em amostras do Ártico com 15.000 e 120.000 anos, e em solos modernos da Antártica. Alguns clones indicam que podem representar novos genes para alcano monoxigenases. As contribuições deste projeto abrangem os objetivos do Ano Polar Internacional (IPY 2007-2009), sobretudo na avaliação da ecologia microbiana da Antártica. / The effect of climate changes on microorganisms is still unclear, because little is known about the species that inhabit the extreme regions as the glacial ice, antarctic soils and the permanently frozen soil (permafrost). The permafrost is able to preserve the sedimented biological materials by thousands or even millions of years, being an important source for microbiological studies. The objective was to study the microbial diversity in cryogenic samples: glacial ice, soil exposed by glacial retreat and polar permafrosts, as well as to study the functional diversity of alkane monooxygenase genes (alk) in the permafrost. Cultivationindependent methods based on the 16S rRNA gene were used, as DGGE, clone library and 454 Pyrosequencing. Analysis of the King George Island (Antarctic Peninsula) glaciers and the South Pole ice revealed about 3x104 cells/mL each, and different micro-organisms were detected, predominantly members from Proteobacteria, Actinobacteria, Firmicutes and Cyanobacteria, many of which already described in other cryogenic environments. The soil in front of the Baranowski Glacier has a different community structure compared with the ice. Soils exposed by glacier retreat revealed an ecological succession, and heterotrophic bacteria occurred all through the process. Nitrogen-fixing populations were composed by cyanobacteria at the early stages, and shifted to Rhodopseudomonas and Rhodobacter in the older soils. The observed changes may be related to an increase of K, Mg+, NH4 +, NO3- and/or CO2, detected after 15-20 years of soil exposure. The community of permafrosts varies by location and age (5,000 - 8 millions of years). The alkM gene was detected in old Arctic permafrosts (3 millions of years), while alkB genes were found on Arctic samples from 15,000 to 120,000 years, and in Antarctic modern soils. Some of these clones may represent new alk genes. The contributions of this project covers the goals of the International Polar Year (IPY 2007-2009), particularly in assessing the microbial ecology of Antarctica.

Made available in DSpace on 2017-07-21T19:59:45Z (GMT). No. of bitstreams: 1 Fernanda Gravina.pdf: 2149838 bytes, checksum: e6a1a4c198175fce83c0ce2c98c81a23 (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Microorganisms are essential components in the maintenance of biogeochemical cycles and ecosystems. In agricultural environments, continuous use of herbicides to minimize the loss in productivity could damage growth inibition of micro-organisms. One cause of this inhibition is increased production of ROS (reactive oxygen species), such as superoxide radicals (O2 -) and hydroxyl (OH-) and hydrogen peroxide (H2O2), causing oxidative stress. The magnitude of this stress can be conditioned by the chemical group and mode of action of the herbicide. The cell responses against ROS involve an increase in the expression of enzymes such as superoxide dismutase and catalase, responsible for the dismutation of O2- and H2O2, respectively. The objective of this study was to evaluate the relationship between the effect of herbicides with different modes of action and answers of superoxide dismutase (SOD) isoenzymes in the bacterium Escherichia coli K-12 bacteria. For this, 2,4-D, paraquat and atrazine herbicides were used, and knockout strains of E. coli K-12 strains carrying mutations in the gene encoding Mn-SOD (sodA) and Fe-SOD (sodB). Herbicides were shown to be capable of promoting the imbalance of redox potential, increasing the production of H2O2 and malondialdehyde (MDA) above the rates observed in controls and differently between strains. Exposed to paraquat, which indices in vivo redox cycling, strains of E. coli wt and ΔsodB showed increased production of H2O2, and increase in the Mn-SOD activity, probably as a result from activation of the SoxR, which promotes the transcription of the gene sodA. In ΔsodA, the toxicity rates with paraquat were not higher than the control, indicating a possible regulation of the Fe-SOD expression by OxyR transcriptional factor. Our results indicated that deletion of genes encoding SOD enzymes originated patterns of antioxidative responses, according to the tested periods of time, regardless the herbicides. To ΔsodB, the damage was minor in time 9pm; but in ΔsodA, the damage was minor in time 5pm, demonstrating the important role of these genes in the defense against oxidative stress in different stages of growth. Cellular responses demonstrated despite the observed toxicity indices, the strains were able to grow at rates close to those verified in control, including an increase in the fitness value in ΔsodA, which indicates a wide plasticity of responses, and a potential for a quick fitting of E. coli K-12. Extending this hypothesis, considering an environment containing a toxic molecule, such as an herbicide, and a bacterium having a polymorphic system for SOD, if a mutation appears in a gene coding for isoforms, it may show an increase in cell viability and still maintain a functional antioxidative enzyme. We suggest that E. coli K-12, a strain created in a laboratory, and with probably low survivability in a natural environment, developed mechanisms of in vitro herbicide tolerance, even without previous selection. This phenotypic plasticity model might be found in other bacteria of agricultural land, with high turnover of cultures and intense use of herbicides, which could cause a considerable impact on the diversity and functionality of soil microbiota. / Microrganismos representam componentes essenciais na manutenção dos ciclos biogeoquímicos e de ecossistemas. Em ambientes agrícolas, o uso contínuo de herbicidas para minimizar a perda na produtividade pode acarretar danos inibindo o crescimento em microrganismos. Uma das causas desta inibição é o aumento da produção de ERO (espécies reativas de oxigênio), como os radicais superóxido (O2-) e hidroxila (OH-), e o peróxido de hidrogênio (H2O2), causando estresse oxidativo. A magnitude deste estresse pode ser condicionada pelo grupo químico e pelo modo de ação do herbicida. As respostas celulares contra ERO envolvem o aumento na expressão de enzimas como superóxido dismutase e catalase, responsáveis pela dismutação de O2- e H2O2, respectivamente. O objetivo deste trabalho foi avaliar a relação entre o efeito de herbicidas com diferentes modos de ação e as respostas das isoenzimas de superóxido dismutase (SOD) na bactéria Escherichia coli K-12. Assim, foram utilizados os herbicidas 2,4-D, atrazina e paraquat, e linhagens mutantes de E. coli K-12 nocauteadas para os genes codificantes das enzimas Mn-SOD (sodA) e Fe-SOD (sodB). Os herbicidas mostraram-se capazes de promover o desequilíbrio do potencial redox, aumentando a produção de H2O2 e malondialdeido (MDA), acima das taxas observadas nos controles e de forma distinta entre as linhagens. Expostas ao paraquat, o qual origina ciclismo redox in vivo, as linhagens de E. coli wt e ΔsodB apresentaram aumento na produção de H2O2, além de aumento na atividade de Mn-SOD, provavelmente como consequência da ativação do SoxR, o qual promove a transcrição do gene sodA. Em ΔsodA, os índices de toxicidade com o paraquat não foram maiores que o controle, indicando uma possível regulação da expressão da enzima Fe-SOD pelo fator transcricional OxyR. Nossos resultados indicaram que a deleção dos genes codificantes para SOD provocaram padrões nas respostas antioxidativas, de acordo com os tempos testados, independentemente dos herbicidas. Para ΔsodB, os danos foram menores no tempo de 9h; já em ΔsodA, os danos foram menores no tempo de 5h, demonstrando a importância do papel destes genes na defesa contra o estresse oxidativo em diferentes fases de crescimento. As respostas celulares demonstraram que, apesar dos índices de toxicidade observados, as linhagens foram capazes de crescer em taxas próximas às verificadas no controle, incluindo ainda um aumento do valor adaptativo em ΔsodA, o que indica uma plasticidade ampla de respostas e um potencial de adaptação rápida. Ampliando esta hipótese, considerando um ambiente contendo uma molécula tóxica, e uma bactéria que possua um sistema polimórfico para SOD, caso ocorra uma mutação no gene para uma de suas isoformas, ela poderá aumentar a viabilidade celular e ainda manter uma enzima antioxidativa funcional. Sugere-se que E. coli K-12, uma linhagem desenvolvida em laboratório e provavelmente com baixa capacidade de sobrevivência em ambiente natural, apresenta mecanismos de tolerância in vitro a herbicidas, mesmo sem seleção prévia. Tal modelo de plasticidade fenotípica poderia ser encontrado em outras bactérias de solo agrícola com alta rotatividade de culturas e intenso uso de herbicidas, podendo causar um impacto considerável na diversidade e funcionalidade desta microbiota.

Amostras de biofilme de biomassa suspensa de tanque de aeração de lodo ativado, de córrego contaminado com esgoto e de vários pontos de planta de tratamento de água foram analisadas com relação à presença de bactérias indicadoras e patogênicas. Nos testes presuntivos foram detectados todos os microrganismos-alvo (Coliformes, Salmonella spp, Klebsiella spp., Staphylococcus spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., Clostridium spp., Shigella spp., Aeromonas spp., Campylobacter spp. e Legionella spp.), porém nos testes confirmatórios por PCR e teste bioquímico somente as espécies E. coli, Salmonella enterica subsp. Enterica serovar Typhi, K. pneumoniae, V. cholerae, A. hydrophila, e L. pneumophila foram confirmadas para algumas cepas selecionadas dos testes presuntivos. S. flexneri foi somente confirmada por teste bioquímico e S. aureus somente por PCR. Nenhuma amostra de C. jejuni foi confirmada por nenhum dos testes. Estes resultados demonstram que os meios seletivos para testes presumptivos não se mostraram confiáveis uma vez que muitas amostras presuntivas não foram confirmadas por PCR ou teste bioquímico específico. Estes resultados demonstram o potencial de biofilmes como reservatório de patógenos. / Biofilm samples from activated sludge reactors, surfaces from water treatment plants and water samples from a creek contaminated with raw sewage were analyzed for the presence of microbial indicator bacteria and pathogens. All target organisms (Coliforms, Salmonella spp, Klebsiella spp., Staphylococcus spp., Pseudomonas spp., Enterococcus spp., Vibrio spp., Clostridium spp., Shigella spp., Aeromonas spp., Campylobacter spp. and Legionella spp.) were detected by using presumptive testing media. Only a small proportion of positive colonies from presumptive tests were confirmed as pathogenic strains of E. coli, Salmonella enterica subsp. Enterica serovar Typhi, K. pneumoniae, V. cholerae, A. hydrophila, and L. pneumophila in confirmatory testing by selective PCR and biochemical tests. S. flexneri was only confirmed in biochemical tests, S. aureus only by PCR and no colony of C. jejuni was confirmed positive by either PCR or biochemical testing. Presumptive media are therefore not safe means for assessing pathogen load in biofilm samples. These results demonstrate the importance of microbial biofilms as reservoirs for microbial pathogens.

Multicellular organisms are not single individuals but carry a complex natural microflora with them. This complex's diversity and function can be considered a distinct ecosystem. Traditional methods of isolation and identification miss >90% of the actual diversity. This study uses the gut microflora of the Madagascar hissing roach, Gromphadorhina portentosa, as a model to examine this ecosystem. Isolated cultured bacteria were used to establish methods for identifying members of the microflora based on ribosomal RNA sequences. Universal primers for Eubacterial, Archaeal, and Eukaryotic 16s/18s rRNA were then used for PCR amplification of total DNA isolated from gut contents. Sequences from isolates were compared using BLAST, ClustalW, and other programs to recognize the isolates' identities and place them using a phylogenetic tree analysis. Eubacterial, Archaeal, and Eukaryotic organisms were found present in the hissing roach gut which can serve as a model ecosystem since it houses Eubacterial, Archaeal, and Eukaryotic organisms.

The health of the northern hardwood forest in the southern Appalachian Mountains of Tennessee, North Carolina, and Virginia has gained attention from the media and environmental stakeholders due to a purported decline in forest health at higher elevations. This project examined lead (Pb) and calcium (Ca) concentrations in growth rings of an important northern hardwood species, American beech (Fagus grandifolia Ehrh.) at Mount Rogers and Whitetop Mountain, Virginia and attempted to examine concentration relationships with stem growth patterns. Dominant and codominant trees were sampled from 16 research plots at two elevations. Tree cores were crossdated, divided into sections of 10-yr periods, and analyzed using atomic absorption spectroscopy. Lead concentrations correlated negatively with ring width. Elevation and aspect were significantly associated with the Pb concentration, while Ca concentrations were only associated with aspect. Tree core samples taken from higher elevation plots contained higher Pb concentrations than samples collected from lower elevation plots, while the northwest and southwest aspects contained significantly higher amounts of Pb and Ca. Both Pb and Ca concentrations increased during the 1860s and again during the mid-1900s.

Diarrheal diseases are a global public health burden, killing 1.8 million people annually. Diarrhea disproportionately affects children and those in poverty. Most diarrheal cases can be prevented through safe drinking water, basic hygiene and/or sanitation measures, with drinking water interventions having the most impact on reducing diarrheal disease. There is no generally agreed-upon field method for determining biosand water filter effectiveness that is usable in low-resource communities. A pilot study was conducted of potential field use indicators, including the Colilert coliform presence/absence (P/A) test, hydrogen sulfide, alkalinity, hardness, pH, and fluorescently labeled latex microspheres. The study included both laboratory and field testing. The Colilert P/A test had the highest correlation to the United States Environmental Protection Agency standard method (IDEXX Quanti-trays), but more data are needed before making a recommendation. This study adds to understanding about evaluation of biosand water filters and provides preliminary data to address the need for a field use indicator for biosand water filters.

More than 270,000 km of rivers and streams are impaired due to fecal pathogens, creating an economic and public health burden. Fecal indicator organisms such as Escherichia coli are used to determine if surface waters are pathogen impaired, but they fail to identify human health risks, provide source information, or have unique fate and transport processes. Statistical and machine learning models can be used to overcome some of these weaknesses, including identifying ecological mechanisms influencing fecal pollution. In this study, canonical correlation analysis (CCorA) was performed to select parameters for the machine learning model, Maxent, to identify how chemical and microbial parameters can predict E. coli impairment and F+-somatic bacteriophage detections. Models were validated using a bootstrapping cross-validation. Three suites of models were developed; initial models using all parameters, models using parameters identified in CCorA, and optimized models after further sensitivity analysis. Canonical correlation analysis reduced the number of parameters needed to achieve the same degree of accuracy in the initial E. coli model (84.7%), and sensitivity analysis improved accuracy to 86.1%. Bacteriophage model accuracies were 79.2, 70.8, and 69.4% for the initial, CCorA, and optimized models, respectively; this suggests complex ecological interactions of bacteriophages are not captured by CCorA. Results indicate distinct ecological drivers of impairment depending on the fecal indicator organism used. Escherichia coli impairment is driven by increased hardness and microbial activity, whereas bacteriophage detection is inhibited by high levels of coliforms in sediment. Both indicators were influenced by organic pollution and phosphorus limitation.