Dissertationen zum Thema „Microbial taxonomy“

Orientador: Hélia Harumi Sato
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Transglutaminase é uma enzima capaz de catalisar a formação de ligações cruzadas intra- e intermoleculares entre proteínas, peptídeos e aminas primárias por meio de ligações covalentes entre resíduos de lisina e glutamina. Desta forma, transglutaminase pode ser utilizada em diversos setores industriais para o desenvolvimento de novos produtos ou para a modificação de suas características. A linhagem B6 isolada de amostra de solo coletada na região do Estado de Minas Gerais foi identificada como tendo características morfológicas típicas de actinomicetos e pela análise da região 16S rRNA há colocou na subclasse Streptomyces próximo a linhagem Streptomyces angustmycinicus NBRC 3934T. A fim de aumentar a produção de transglutaminase (2,75 U/mL) pela linhagem Streptomyces sp. B6, o meio de fermentação foi submetido a processos de otimização. Como primeiro passo da otimização, o crescimento do micro-organismo e a produção da enzima foram estudados através de uma pré-seleção de fontes de carbono, nitrogênio e sais no meio de produção. Após as análises das diferentes fontes, um delineamento experimental do tipo Plackett-Burman foi utilizado para a seleção dos componentes do meio de cultivo que afetam a produção de transglutaminase. Os resultados do delineamento experimental indicaram que a produção de transglutaminase foi influenciada negativamente pela peptona bacteriológica e MgSO4.7H2O, positivamente pelo amido de batata, glicose, peptona de caseína e KH2PO4.7H2O e não foi influenciada pelo farelo de soja, considerando um nível de confiança de 95%. A concentração de amido de batata foi fixada no maior nível testado no planejamento Plackett-Burman devido à gelificação do meio de fermentação em concentrações maiores. Assim, os três fatores que influenciaram a produção de transglutaminase (glicose, peptona de caseína e KH2PO4.7H2O) foram otimizados para obter o máximo de produção da enzima utilizando delineamento composto central. Sob a condição otimizada, a qual continha 25 g/L de farinha de soja, 35 g/L de amido de batata, 5 g/L de glicose, 24,5 g/L de peptona de caseína e 8 g/L de KH2PO4.7H2O, a atividade enzimática atingiu 6,13 U/mL, apresentando 125% à mais de atividade em relação á obtida no meio antes da otimização. A transglutaminase microbiana produzida pela linhagem Streptomyces sp. B6 exibiu atividade ótima em 45°C e em pH de 6,5 e 11,0. A enzima manteve-se estável na faixa de pH 3,0-11,0 durante 60 minutos à 40°C durante 3 horas. A transglutaminase não foi inibida por Ca2+, Na+, Co2+, Mn2+, K+, Mg2+, Ba2+, EDTA, L-cisteína e glutationa na concentração de 5 mM, mas foi inibida na presença de Hg2+, Cu2+, Zn2+ e Fe2+ na concentração de 5mM. A linhagem Streptomyces sp. B6 é uma nova fonte de transglutaminase com características interessantes para aplicações biotecnológicas
Abstract: Transglutaminase is an enzyme capable of catalyzing the forming intra-and intermolecular cross-linking between proteins, peptides and primary amines by covalent bonds between lysine and glutamine residues. Thus, transglutaminase can be used in food processing industries to develop new products and modify their characteristics. The B6 strain was isolated from soil sample collected in the region state of Minas Gerais was identified as having morphological characteristics typical of the actinomycetes, and the 16S rRNA analysis placed it in the Streptomyces subclade, closely related to Streptomyces angustmycinicus NBRC 3934T. In order to increase the transglutaminase production (2.75 U/mL) from Streptomyces sp. B6 strain, the fermentation medium was subjected to optimization processes. In the first step of optimization, the micro-organism growth and enzyme production were studied through a pre-selection of carbon, nitrogen and salts sources in the culture medium. After analysis of different sources, the Plackett¿Burman experimental design was used for screening the components of the culture medium that affect the transglutaminase production. Results of the experiment indicated that production of transglutaminase was negatively influenced by bacteriological peptone and MgSO4.7H2O, positively influenced by potato starch, glucose, casein peptone and KH2PO4.7H2O and was not influenced by soybean meal, considering 95% of confidence level. The potato starch concentration was fixed at the highest level tested in Plackett¿Burman design due to gelation of the fermentation medium in higher concentrations. Thus, the three factors that influence the transglutaminase production (glucose, casein peptone and KH2PO4.7H2O concentrations) were optimized to obtain the maximum transglutaminase production using central composite design. Under the proposed optimized condition, which contained 25 g/L soybean meal, 35 g/L potato starch, 5 g/L glucose, 24.5 g/L casein peptone and 8 g/L KH2PO4.7H2O, the enzyme activity reached 6.13 U/mL, which was 125% more than the activity in relative obtained medium before optimization. The microbial transglutaminase produced by Streptomyces sp. B6 strain exhibited optimal activity at 45 oC and at pH 6.5 and 11.0. The enzyme remained stable in the pH range from 3.0 - 11.0 for 60 minutes and at 40 oC temperature for 3 hours. The transglutaminase was not inhibited by Ca2+, Na+, Co2+, Mn2+, K+, Mg2+, Ba2+, EDTA, L-cysteine and glutathione in concentration 5 mM, but was inhibited in the presence of Hg2+, Cu2+, Zn2+ and Fe2+ in concentration 5 mM. In conclusion, Streptomyces sp. B6 strain is a new source of transglutaminase with interesting features for biotechnological applications
Mestrado
Ciência de Alimentos
Mestre em Ciência de Alimentos

Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change.
M.S.
Soil is complicated, both in terms of its physical makeup and the organisms that live inside of it. Predicting changes in soil based on the addition of foreign material such as chemicals or biological waste is not an easy process, and whether or not it is even possible to reliably predict those changes is a matter of some dispute. This study is designed to illustrate that such changes can in fact be reliably and consistently predicted even with regard to the addition of complicated materials to the soil. In this study, specifically, the material in question is chicken litter. A mix of the bedding and waste produced by chickens, litter is commonly handled by composting and is added to soil in farms as a fertilizer rich in organic matter. It is possible to point at specific elements of the soil such as the chemistry and bacteria and see how it is changed with the addition of chicken litter, which allows us to determine the nature and extent of the change that chicken litter has on soil. This study is conducted on a larger scale than similar experiments conducted in the past, making it apparent that these relationships exist on a repeated basis. It is the object of this study to pave the way and make it easier for scientists in the future to determine these relationships in other unique contexts.

Background: MicrO is an ontology of microbiological terms, including prokaryotic qualities and processes, material entities (such as cell components), chemical entities (such as microbiological culture media and medium ingredients), and assays. The ontology was built to support the ongoing development of a natural language processing algorithm, MicroPIE (or, Microbial Phenomics Information Extractor). During the MicroPIE design process, we realized there was a need for a prokaryotic ontology which would capture the evolutionary diversity of phenotypes and metabolic processes across the tree of life, capture the diversity of synonyms and information contained in the taxonomic literature, and relate microbiological entities and processes to terms in a large number of other ontologies, most particularly the Gene Ontology (GO), the Phenotypic Quality Ontology (PATO), and the Chemical Entities of Biological Interest (ChEBI). We thus constructed MicrO to be rich in logical axioms and synonyms gathered from the taxonomic literature. Results: MicrO currently has similar to 14550 classes (similar to 2550 of which are new, the remainder being microbiologically-relevant classes imported from other ontologies), connected by similar to 24,130 logical axioms (5,446 of which are new), and is available at (http://purl.obolibrary.org/obo/MicrO.owl) and on the project website at https://github.com/carrineblank/MicrO. MicrO has been integrated into the OBO Foundry Library (http://www.obofoundry.org/ontology/micro.html), so that other ontologies can borrow and re-use classes. Term requests and user feedback can be made using MicrO's Issue Tracker in GitHub. We designed MicrO such that it can support the ongoing and future development of algorithms that can leverage the controlled vocabulary and logical inference power provided by the ontology. Conclusions: By connecting microbial classes with large numbers of chemical entities, material entities, biological processes, molecular functions, and qualities using a dense array of logical axioms, we intend MicrO to be a powerful new tool to increase the computing power of bioinformatics tools such as the automated text mining of prokaryotic taxonomic descriptions using natural language processing. We also intend MicrO to support the development of new bioinformatics tools that aim to develop new connections between microbial phenotypes and genotypes (i.e., the gene content in genomes). Future ontology development will include incorporation of pathogenic phenotypes and prokaryotic habitats.

With the advent of next generation sequencing there has been an explosion of the size of data that needs to be processed, where next generation sequencing yields basepairs of DNA in the millions. The rate at which the size of data increases supersedes Moores law therefore there is a huge demand for methods to nd meaningful labels of sequenced data. Studies of microbial diversity of a sample is one such challenge in the eld of metagenomics. Finding the distribution of a bacterial community has many uses for example, obesity control. Existing methods often resort to read-by-read classication which can take several days of computing time in a regular desktop environment, excluding genomic scientists without access to huge clusters of computational units. By using sparsity enforcing methods from the general sparse signal processing eld (such as compressed sensing), solutions have been found to the bacterial community composition estimation problem by a simultaneous assignment of all sample reads to a pre-processed reference database. The inference task is reduced to a general statistical model based on kernel density estimation techniques that are solved by existing convex optimization tools. The objective is to o er a reasonably fast community composition estimation method. This report proposes, clustering as a means of aggregating data to improve existing techniques run-time and biological delity. Use of convex optimization tools to increase the accuracy of mixture model parameters are also explored and tested. The work is concluded by experimentation on proposed improvements with satisfactory results. The use of Dirichlet mixtures is explored as a parametric model of the sample distribution where it is deemed that the Dirichlet is a good choice for aggregation of k-mer feature vectors but the use of Expectation Maximization is unt for parameter estimation of bacterial 16s rRNA samples. Finally, a semi-supervised learning method found on distance based classication of taxa has been implemented and tested on real biological data with high biological delity.
Nya tekniker inom DNA-sekvensering har givit upphov till en explosion pa data som nns att tillga. Nasta generations DNA-sekvensering generar baspar som stracker sig i miljonerna och mangden data okas i en exponentiell takt, vilket ar varfor det nns ett stort behov av ny skalbar metodik som kan analysera kvantitiv data for att fa ut relevant information. Den bakteriella artfordelning av ett provror ar en sadan problemst allning inom meta-genomik, vilket har era tillampningsomraden som exempelvis, studier av fettma. I dagslaget sa ar den vanligaste metoden for att fa ut artfordelningen genom att klassiera DNA-strangarna av bakterierna, vilket ar en tidskravande losning som kan ta upp emot ett dygn for att processera data med hog upplosning. En snabb och tillforlitlig losning skulle darfor tillata er forskare att ta del av nasta generations sekvensering och analysera dess data som i sin tur skulle ge upphov till mer innovation inom omradet. Alternativa losningar med inspiration fran signalbehandlig har hittats som nyttjar problemestallningens glesa natur genom anvandning av Compressed Sensing. Svar hittas genom att simultant tilldela strangar till en for-processerad referensdatabas. Problemstallningen har forenklats till en statistisk modell av provror med ickeparametrisk estimering for att implicit fa ut fordelningen av bakteriearter med hjalp av konvex optimering. Denna rapport foreslar anvandningen av klustrering for aggregering av data for att forbattra tillforlitligheten av svaren och minska tiden for berakning av dessa. Anvandningen av parametriska modeller, Dirichlet fordelningen, har utforskats dar rapporten har kommit fram till att antaganden for lampligheten av denna som ett medel att aggregera k-mer vektorer ~Ar rimliga men att parameterestimeringen med Expectation Maximization ej fungerar val i samband med Dirichlet och en omskrivning av parametern skulle behovas i vektorrymden som spans av 16S rRNA genen. Slutligen sa har distansbaserad tilldelning av bakterier testats pa data fran verklig biologisk kontext med valdigt hog noggranhet. ii

Three nontaxonomic indices; ATP/Chlorophyll a(ATP/Chla), ATP/ADP, and Chlorophyll a/Pheopigment (Chla/Pheo) were compared to the taxonomic measures of species diversity (d) and species richness as indicators of stress in aquatic environments. Field and laboratory microcosm responses of indigenous microbial communities exposed to municipal sewage treatment plant (STP) effluent were monitored. The STP effluent produced increased adenylate concentrations, ATP/ADP and ATP/Chla ratios, and decreased Chla, Chla/Pheo, d, and species richness relative to upstream reference communities. Nontaxonomic responses were consistent in four separate field tests.

Significant differences in responses were discernible in 3 d when communities were transferred from reference to polluted sites. Chla/Pheo decreased more rapidly than other measurements. The predictive capability of laboratory flowâ through microcosm tests was examined by simultaneously transferring communities from upstream reference sites to downstream field sites and to various dilutions of field effluent in the laboratory.

Master of Science

Orientadores: Helia Harumi Sato , Lara Durães Sette
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A transglutaminase (TGase) (EC 2.3.2.13; proteina-glutamina ?-glutaminiltransferase) é uma enzima capaz de catalisar reações de transferência de grupos acil utilizando resíduos de glutamina das ligações peptídicas de proteínas como doadores de grupos acil, e diversas aminas primárias como receptores. As ligações covalentes cruzadas entre inúmeras proteínas e peptídeos pela transglutaminase promovem mudanças nas propriedades de proteínas de alimentos. Por essa razão, a transglutaminase é amplamente utilizada nas indústrias de processamento de alimentos para o desenvolvimento de novos produtos e modificação de características como: viscosidade, capacidade emulsificante e valores nutricionais. Uma cepa de Actinomyceto, isolada de amostras de solo brasileiro, foi investigada taxonomicamente por uma combinação de técnicas moleculares e morfológicas, resultando na conclusão de que a cepa pertence ao gênero Streptomyces sp. A cepa, chamada de Streptomyces sp. CBMAI 837 produziu transglutaminase quando cultivada a 30°C por cinco dias, em agitador rotatório, no meio de fermentação otimizado, composto por: 0,2% KH2PO4, 0,1% MgSO4.7H2O, 2% farinha de soja, 2% amido de batata, 0,2% glicose, e 2% peptona, atingindo uma atividade enzimática de 1,37 U.mL-1. A transglutaminase foi purificada cerca de 5 vezes através de duas passagens cromatográficas sucessivas em uma coluna de filtração em gel Sephadex G-75, com 17% de recuperação. A purificação da proteína foi comprovada por homogeneidade eletroforética em SDS-PAGE. A massa molar da TGase foi estimada em cerca de 45 kDa. A transglutaminase de Streptomyces sp CBMAI 837, tanto na forma bruta quanto purificada, apresentou atividade enzimática ótima em pH 6,0-6,5, e em 35-40°C. Um segundo pico de atividade ótima foi observado em pH 10,0 na enzima no estado bruto. Ambas as formas da enzima foram estáveis na faixa de pH de 4,5 a 8,0 e até 45°C. A transglutaminase na forma bruta e purificada mostrou-se independente de íons cálcio, mas foi ativada na presença de K+, Ba2+, e Co2+; e inibida por Cu2+ e Hg2; o que sugere a presença de um grupo tiol no sítio ativo da enzima. A TGase purificada apresentou um Km de 6,37 mM e um Vmax de 1,70 U/mL, enquanto a enzima bruta apresentou Km de 6,52 mM e um Vmax de 1,35 U/mL para o substrato N-carbobenzoxi-L-glutaminil-glicina. A influência da transglutaminase de Streptomyces sp CBMAI 837 bruta, nas propriedades de géis ácidos de caseinato de sódio foi investigada, tendo como parâmetro géis preparados com a TGase comercial (Ajinomoto Inc.). Os géis com a enzima comercial tiveram um valor de módulo de elasticidade maior, porém, dependendo da concentração de proteína, estes foram menos deformáveis. Os géis com enzima bruta de Streptomyces sp. CBMAI 837 se mostraram muito mais rígidos e menos elásticos. Resultados da eletroforese indicaram que a enzima comercial promoveu a formação de polímeros de proteínas de massa molecular mais alta do que a enzima de Streptomyces sp. CBMAI 837. Os testes de microscopia eletrônica de varredura e da capacidade de retenção de água mostraram que características particulares de cada um dos géis poderiam estar associadas ao tipo específico de interação promovida por cada uma das amostras enzimáticas testadas
Abstract: Transglutaminase (EC 2.3.2.13; protein-glutamine ?-glutaminyltransferase) is an enzyme that catalysis an acyl transfer reaction using protein or peptide-bond glutamine residues as acyl donors and several primary amines as receptors. The covalent cross-links between a number of proteins and peptides introduced by transglutaminase promote modification of the functional properties of the food proteins. Therefore, transglutaminase are widely used by food-processing industries for the purpose of new product development, modification of the product properties such as viscosity, emulsification foaming and nutritional values. An actinomycete strain, isolated from Brazilian soil, was taxonomically investigated using a combination of molecular and morphological basedmethods, resulting on the conclusion that the strain belongs to the genus Streptomyces sp. The strain, named Streptomyces sp. CBMAI 837, produce transglutaminase when cultivated at 30°C for 5 days at 200 rpm in a rotatory shaker, on the optimized fermentation medium composed of 0.2% KH2PO4, 0.1% MgSO4.7H2O, 2% soybean flour, 2% potato starch, 0.2% glucose, and 2% peptone, with a enzymatic activity of 1.37 U.mL-1. The enzyme purification was performed by of two successive chromatographies on Sephadex G-75 columns with yields of 48% and 17%, respectively. The protein purification was successfully achieved to electrophoretical homogeneity on SDS-PAGE. The molecular mass of the MTGase was estimated to be about 45 kDa. The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0-6.5 pH range and at 35-40°C. A second maximum of activity was observed at pH 10.0 on the crude Streptomyces sp. enzyme. Both forms of transglutaminase were stable over the pH range from 4.5 to 8.0 and up to 45°C. The activities of all the TGase samples were independent of Ca+2 concentration, but they were elevated in the presence of K+, Ba2+, and Co2+ and inhibited by Cu2+ and Hg2+, which suggests the presence of a thiol group in the TGase¿s active site. The purified enzyme presented Km of 6.37 mM and Vmax of 1.7 U/mL, while the crude enzyme demonstrated Km of 6.52 mM and Vmax of 1.35 U/mL. The influence of the transglutaminase on acid-gel properties was studied. Texture parameters showed that the commercial TGase (Ajinomoto Inc.) gels had greater values of elasticity modulus and could promote the formation of more elastic and soft food systems, while addition of the crude TGase of Streptomyces sp. CBMAI 837 to the gel led to the formation of more rigid and less elastic gels. The electrophoresis showed that the commercial TG enzyme in this system promoted higher molecular mass protein polymers than the enzyme from Streptomyces sp. CBMAI 837. Microscopy and water holding capacity (WHC) observations showed that all the gel characteristics could be associated to specific interactions promoted by each TGase tested
Doutorado
Doutor em Ciência de Alimentos

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Atualmente, existe um crescente interesse em explorar diversos habitats, a fim de revelar a biodiversidade microbiana, incluindo as leveduras. Tal diversidade ainda não acessada guarda a descoberta de novas espécies para ciência, provavelmente muitas das quais com potencial para aproveitamento em processos biotecnológicos. Com o objetivo de explorar e conservar a diversidade de fungos, o Central de Recursos Microbianos da UNESP (CRM – UNESP) mantém em seu acervo várias estirpes de leveduras isoladas de ecossistemas diversos, sendo alguns deles pouco explorados. No início deste trabalho sabíamos que muitas das leveduras depositadas no acervo do CRM – UNESP não estavam totalmente caracterizadas tanto em nível taxonômico, quanto em relação ao potencial biotecnológico que poderiam apresentar. Portanto, o presente estudo foi desenhado para caracterizar e identificar taxonomicamente leveduras depositadas no CRM – UNESP, bem como selecionar estirpes que produzem enzimas extracelulares degradadoras de polissacarídeos como amilase, celulase, xilanase, pectinase e ligninase. Usando uma abordagem polifásica, um total de 340 isolados de leveduras foi identificado, sendo que 71,2% compreendem 43 taxa de ascomicetos e os restantes 28,8% foram classificados em 27 taxa de basidiomicetos. O estudo também levou à descoberta de 8 prováveis novas espécies. Baseado nesta constatação, a classificação taxonômica e análise filogenética foi realizada para duas espécies anamórficas de ascomicetos e uma espécie teleomórfica de basidiomiceto. A descrição destas três espécies é apresentada neste estudo. Os resultados demonstraram que Wickerhamiella kiyanii FB1-1DASPT e W. pindamonhangabaensis H10YT pertencem à clade Wickerhamiella da ordem Saccharomycetales...
In recent time, there has been an increasing interest in exploring diverse ecological habitats in order to reveal the yeast biodiversity. The increased awareness in the biotechnological potentials of yeasts has also spurred attempts to search for new species with novel biotechnological capabilities. Aiming to explore and conserve the fungal diversity from various ecosystems, the UNESP – Central for Microbial Resources (UNESP – CMR) harbors various strains of ecologically diverse yeasts isolates, some of which were yet to be identified. Therefore, this study was designed to identify and characterize some yeasts from the UNESP – MRC and to select strains possessing extracellular plant polysaccharide degrading enzymes namely amylase, cellulase, xylanase, pectinase and ligninase. Using a polyphasic approach, a total of 340 strains were identified. Taxonomic classification grouped 71.2% of these isolates into 43 ascomycetous taxa while the remaining 28.8% were classified in 27 basidiomycetous taxa. The study also led to the discovery of 8 putative new species. As a result, we classified two anamorphic species in the Ascomycota and one teleomorphic species in the Basidiomycota. In this study we provide the description of both species. Our results demonstrated that the two ascomycetous species proposed as Wickerhamiella kiyanii FB1-1DASPT and W. pindamonhangabaensis H10YT belong to the Wickerhamiella clade of the Saccharomycetales (Saccharomycetes) while the basidiomycetous species proposed as Bulleromyces texanaensis ATT064T belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Agaricomycotina). In order to show the significance of intraspecific diversity in yeasts, in one of our studies, we subjected 11 strains, (including the type strain CBS 8960T) of Hannaella kunmingensis... (Complete abstract click electronic access below)

Orientador: Fernando Carlos Pagnocca
Coorientador: André Rodrigues
Banca: Lara Durães Sette
Banca: Vanderlei Gerlado Martins
Banca: Paula Benevides de Morais
Banca: Aline Silva
Resumo: Atualmente, existe um crescente interesse em explorar diversos habitats, a fim de revelar a biodiversidade microbiana, incluindo as leveduras. Tal diversidade ainda não acessada guarda a descoberta de novas espécies para ciência, provavelmente muitas das quais com potencial para aproveitamento em processos biotecnológicos. Com o objetivo de explorar e conservar a diversidade de fungos, o Central de Recursos Microbianos da UNESP (CRM - UNESP) mantém em seu acervo várias estirpes de leveduras isoladas de ecossistemas diversos, sendo alguns deles pouco explorados. No início deste trabalho sabíamos que muitas das leveduras depositadas no acervo do CRM - UNESP não estavam totalmente caracterizadas tanto em nível taxonômico, quanto em relação ao potencial biotecnológico que poderiam apresentar. Portanto, o presente estudo foi desenhado para caracterizar e identificar taxonomicamente leveduras depositadas no CRM - UNESP, bem como selecionar estirpes que produzem enzimas extracelulares degradadoras de polissacarídeos como amilase, celulase, xilanase, pectinase e ligninase. Usando uma abordagem polifásica, um total de 340 isolados de leveduras foi identificado, sendo que 71,2% compreendem 43 taxa de ascomicetos e os restantes 28,8% foram classificados em 27 taxa de basidiomicetos. O estudo também levou à descoberta de 8 prováveis novas espécies. Baseado nesta constatação, a classificação taxonômica e análise filogenética foi realizada para duas espécies anamórficas de ascomicetos e uma espécie teleomórfica de basidiomiceto. A descrição destas três espécies é apresentada neste estudo. Os resultados demonstraram que Wickerhamiella kiyanii FB1-1DASPT e W. pindamonhangabaensis H10YT pertencem à clade Wickerhamiella da ordem Saccharomycetales... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In recent time, there has been an increasing interest in exploring diverse ecological habitats in order to reveal the yeast biodiversity. The increased awareness in the biotechnological potentials of yeasts has also spurred attempts to search for new species with novel biotechnological capabilities. Aiming to explore and conserve the fungal diversity from various ecosystems, the UNESP - Central for Microbial Resources (UNESP - CMR) harbors various strains of ecologically diverse yeasts isolates, some of which were yet to be identified. Therefore, this study was designed to identify and characterize some yeasts from the UNESP - MRC and to select strains possessing extracellular plant polysaccharide degrading enzymes namely amylase, cellulase, xylanase, pectinase and ligninase. Using a polyphasic approach, a total of 340 strains were identified. Taxonomic classification grouped 71.2% of these isolates into 43 ascomycetous taxa while the remaining 28.8% were classified in 27 basidiomycetous taxa. The study also led to the discovery of 8 putative new species. As a result, we classified two anamorphic species in the Ascomycota and one teleomorphic species in the Basidiomycota. In this study we provide the description of both species. Our results demonstrated that the two ascomycetous species proposed as Wickerhamiella kiyanii FB1-1DASPT and W. pindamonhangabaensis H10YT belong to the Wickerhamiella clade of the Saccharomycetales (Saccharomycetes) while the basidiomycetous species proposed as Bulleromyces texanaensis ATT064T belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Agaricomycotina). In order to show the significance of intraspecific diversity in yeasts, in one of our studies, we subjected 11 strains, (including the type strain CBS 8960T) of Hannaella kunmingensis... (Complete abstract click electronic access below)
Mestre

Microbial life represents the majority of the diversity of life on planet earth. Microbes are found in all ecosystems. The microbial community of an ecosystem can be an indicator of its health and the foundation of the ecosystem function. Thus, an understanding of the microbial community of an ecosystem is vital to understanding the ecosystem itself. To fully grasp the microbial community structure, it is essential to understand the factors that shape the community composition and diversity of the ecosystem. This works focusses on the primary drivers of microbial composition in three ecosystems: an estuary, the gut of Drosophila melanogaster, and walnut grove soil.

In the benthic estuarine environment of Trunk River, physical perturbations in the water column above decaying seagrass altered the composition of the microbial community causing a visible microbial bloom. To understand the microbial community progression in the bloom, we simulated perturbed sites in the river and studied four different depths in the water column for two weeks. We found the bloom was largely made up of Prosthecochloris vibrioformis, a phototrophic sulfur oxidizer. The bloom appears to be driven by pH, salinity, and sulfide gradients, forming at a depth of ≈ 25cm beneath the surface of the water.

For the third chapter, we explored the effect of host diet on its gut microbial community. We created a controlled experiment in Drosophila melanogaster, a model organism. A population of D. melanogaster preconditioned on a balanced lab diet was split into two treatment diets, a high-sugar diet and a high-yeast diet. The microbial communities in the fecal matter of the flies were sampled for 4 days to understand their compositional changes. We found that a shift in the diet of D. melanogaster changed the phylogenetic, taxonomic, and functional compositions of the microbial communities. Each dietary change led to a distinct taxonomic, phylogenetic, and functional composition by the end of the experiment. The functional diversity of both treatment groups decreased, indicating a shift away from a diverse set of metabolic capabilities when subjected to a more comprehensive nutrition to a more specific set of metabolic capabilities adapted for the main nutrient, either sucrose or yeast extract.

In the fourth chapter, Pesticide-treated orchard soils were used to understand the effects of deliberate intervention (external factors) on microbial ecosystems. We also studied the potential of pathogens to colonize soil that had been exposed to such external factors. In a controlled experiment, soil was subjected to different fumigation treatments with and without subsequent amendment. It was then inoculated and incubated with Agrobacterium tumefaciens. The results indicate a fumigant-specific shift in the phylogenetic, taxonomic, genic, and functional composition of the soil. The low diversity fumigated soil was also better colonized by the A. tumefaciens. However, post-fumigation amendment with vermicompost increased the diversity, shifting the compositions towards non-fumigated vermicompost and suppressing A. tumefaciens colonization.

With this work, we have been able to implicate some of the important factors at play in the determination of microbial composition in various ecosystems. Additionally, we showed that the influence of these factors on microbial community is measurable on different metrics of composition.

O presente trabalho teve por objetivo avaliar o efeito de três ecossistemas sobre, a ocorrência de fungos rizosféricos, endofíticos e epifíticos. As áreas de estudo estão localizadas em uma propriedade rural, situada na cidade de Venâncio Aires, RS. Os fungos endofíticos e epifíticos foram isolados de amostras de raízes de guajuvira, (Patagonula americana L.), coletadas na mata (área 1), de uva-do-japão (Hovenia dulcis Thunb.), na área intermediária (área 2) e de fumo ou de milho, na lavoura (área 3) e, os fungos rizosféricos isolados de solo coletado junto as raízes dos vegetais citados. As amostras foram coletadas nos meses de janeiro, maio, setembro e novembro de 2004 e 2005. Os fungos foram identificados segundo características morfológicas com auxílio de chaves de identificação. As áreas 2 e 3 foram mais similares com relação aos fungos rizosféricos e epifíticos, enquanto, que, as áreas 1 e 2 foram mais similares com relação aos fungos endofíticos. A diversidade dos fungos isolados das três áreas não diferiu ao longo dos dois anos de coleta. A presença de Fusarium spp., em vegetais sem sintomas de doença indica que, as mesmas podem ser raças avirulentas ou patógenos latentes em equilíbrio com o hospedeiro e o ambiente. A presença de fungos antagonistas, principalmente Trichoderma spp. também, são responsáveis por esse equilíbrio, o qual ocorre nas três áreas.
The present paper aimed at assessing the effect of three ecosystems, on the occurrence of rhizospheric, endophytic and epiphytic fungi. The study areas are located in a farm in Venâncio Aires, Rio Grande do Sul. Samples were collected in the months of January, May, September and November 2004 and 2005. The endophytic and epiphytic fungi were isolated from samples of guajayvi roots (Patagonula americana L.), picked in the forest (area 1), of Japanese raisin tree (Hovenia dulcis Thunb.), in the intermediate area (area 2), and tobacco or corn, in the cultivated area (area 3) and rhizospheric fungi isolated from soil. The fungi were identified according to morphological characteristics, with the help of identification keys. Areas 2 and 3 were more alike concerning rhizospheric and epiphytic fungi; Areas 1 and 2, in turn, were more alike concerning endophytic fungi. The diversity of the isolated fungi from these three areas did not change during the two years of collection. The abundance of fungi with phytopathogenic potential such as the Fusarium spp. and Macrophomina phaseolina, mostly as endophytic in area 3 shows the influence of the host on them. The presence of antagonistic fungi, principally Trichoderma spp. also, are responsable for this equilibrium, which occured in the three areas.

Cyanobacteria are a group of photo-oxygenic bacteria found in nearly every ecosystem, but much cyanobacterial diversity, in various habitats, has yet to be explored. Cyanobacteria are often conspicuous components of photosynthetic flora, providing significant carbon and nitrogen inputs to surrounding systems. As possible primary colonizers of stone substrates not native to this region, cyanobacteria isolated from headstones may provide biogeographically informative data. An exploratory study of lichen-dominated microbial consortia, growing on headstones, was conducted to isolate and identify novel microaerophytic cyanobacteria, and resulted in the establishment of four novel cyanobacterial taxa. Phylogenetic analyses of photobionts in one tripartite lichen revealed two novel taxa: Brasilonema lichenoidesand Chroococcidiopsis lichenoides. Using a total evidence approach, analyzing ecology, morphology, ITS structure, and molecular data two additional taxa were described: Brasilonema geniculosusand Calothrix dumas. Analysis of secondary structures of the Internal Transcribed Spacer (ITS) regions of the 16S-23S operon in cyanobacteria are commonly used in cyanobacterial taxonomy studies and were applied to the identification of the new taxa in this study. However, the relationship between ITS structures, hairpin loops (helices) in a region of non-coding DNA, has not been thoroughly evaluated. The 16S-23S operon is one of many in prokaryotes with multiple copies and there is evidence that operons may vary due to differential selective pressures or drift. A study was undertaken analyzing ITS operons from 224 previously published cyanobacterial taxa for domain inclusion and exclusion, intragenomic heterogeneity of ITS operons, and the possible relevance of variable selective pressures affecting individual domains. Analysis revealed highly variable ITS domain inclusion even in complete sequences, as well as high variation between domains containing two or no tRNA sequences. Recommendations were made to standardize ITS analysis in the future to account for this possible variation. Further study is required to statistically demonstrate to what extent ITS secondary structures correlate with taxonomy.

Les systèmes hydrothermaux associés à la serpentinisation sont anoxiques et riches en $H_2$, $CH_4$ et molécules organiques. Ces composants alimentent des micro-organismes qui colonisent les systèmes serpentinisés, et ce en dépit d’un pH élevé et de faibles concentrations en accepteurs d'électrons et en carbone dissous. Dans ce travail, les communautés microbiennes ont été étudiées en se focalisant sur Prony, un écosystème serpentinisé côtier de Nouvelle-Calédonie, puis, en comparant différents écosystèmes serpentinisés, pour faire émerger des similarités taxonomiques et fonctionnelles. À Prony, nos analyses de métabarcoding ont mis en évidence l'importance d’une biosphère rare. L'analyse de métagénomes a permis de reconstruire 82 génomes procaryotes. Un de ces génomes est phylogénétiquement proche des espèces du genre Serpentinomonas, bactéries chimiolithotrophes isolées du site serpentinisé The Cedars, qui détiennent le record d’alcalophilie. Ces espèces et d'autres phylotypes, tels que les taxons affiliés aux Lost City Methanosarcinales, ont été trouvés dans plusieurs sites serpentinisés et pourraient contribuer à la définition d'une signature biologique des phénomènes de serpentinisation. En ciblant spécifiquement les métabolismes enrichis dans les milieux serpentinisés, nous avons pu mettre en évidence l'importance du métabolisme de l'hydrogène, des mécanismes cellulaires de réponse aux stress et d’une voie de dégradation des phosphonates, reposant sur l’activité d'une C-P lyase. Cette voie métabolique, qui a un rôle clé dans l'assimilation du phosphore et la libération de molécules organiques, vient enrichir les modèles écologiques des systèmes serpentinisés
Serpentinizing hydrothermal systems are anoxic and enriched in $H_2$, $CH_4$ and organic molecules. These compounds support microbes that colonize serpentinizing systems, despite high pH and low concentrations of electron acceptors and dissolved inorganic carbon. In this work, two axes were explored to study the microbial communities. On the one hand, we focused on Prony, a coastal serpentinizing site in New Caledonia, and on the other hand we compared different serpentinizing systems to reveal taxonomic and functional similarities. At Prony, our metabarcoding analyses highlighted the importance of the rare biosphere. Moreover, 82 prokaryotic genomes were successfully reconstructed using five metagenomes from Prony. One of these genomes was phylogenetically close to the species of the genus Serpentinomonas, chemolithotrophic bacteria isolated at the serpentinizing site The Cedars that are capable of growth up to pH 12.5. These species, and other phylotypes, such as taxa affiliated with Lost City Methanosarcinales were identified in several serpentinizing sites and could contribute to the definition of a biological signature associated with serpentinization. By specifically targeting enriched metabolisms in serpentinizing environments, we highlighted key functions associated with hydrogen metabolism and environmental stress response mechanisms. The comparison of serpentinizing metagenomes revealed the importance of a phosphonate degradative pathway, based on the activity of a C-P lyase. This metabolic pathway, which plays a key role in the uptake of phosphorus and the release of organic molecules, was integrated into the ecological models of serpentinizing systems

Les systèmes hydrothermaux associés à la serpentinisation sont anoxiques et riches en H₂, CH₄ et molécules organiques. Ces composants alimentent des micro-organismes qui colonisent les systèmes serpentinisés, et ce en dépit d’un pH élevé et de faibles concentrations en accepteurs d'électrons et en carbone dissous. Dans ce travail, les communautés microbiennes ont été étudiées en se focalisant sur Prony, un écosystème serpentinisé côtier de Nouvelle-Calédonie, puis, en comparant différents écosystèmes serpentinisés, pour faire émerger des similarités taxonomiques et fonctionnelles. À Prony, nos analyses de métabarcoding ont mis en évidence l'importance d’une biosphère rare. L'analyse de métagénomes a permis de reconstruire 82 génomes procaryotes. Un de ces génomes est phylogénétiquement proche des espèces du genre Serpentinomonas, bactéries chimiolithotrophes isolées du site serpentinisé The Cedars, qui détiennent le record d’alcalophilie. Ces espèces et d'autres phylotypes, tels que les taxons affiliés aux Lost City Methanosarcinales, ont été trouvés dans plusieurs sites serpentinisés et pourraient contribuer à la définition d'une signature biologique des phénomènes de serpentinisation. En ciblant spécifiquement les métabolismes enrichis dans les milieux serpentinisés, nous avons pu mettre en évidence l'importance du métabolisme de l'hydrogène, des mécanismes cellulaires de réponse aux stress et d’une voie de dégradation des phosphonates, reposant sur l’activité d'une C-P lyase. Cette voie métabolique, qui a un rôle clé dans l'assimilation du phosphore et la libération de molécules organiques, vient enrichir les modèles écologiques des systèmes serpentinisés
Serpentinizing hydrothermal systems are anoxic and enriched in H₂, CH₄ and organic molecules. These compounds support microbes that colonize serpentinizing systems, despite high pH and low concentrations of electron acceptors and dissolved inorganic carbon. In this work, two axes were explored to study the microbial communities. On the one hand, we focused on Prony, a coastal serpentinizing site in New Caledonia, and on the other hand we compared different serpentinizing systems to reveal taxonomic and functional similarities. At Prony, our metabarcoding analyses highlighted the importance of the rare biosphere. Moreover, 82 prokaryotic genomes were successfully reconstructed using five metagenomes from Prony. One of these genomes was phylogenetically close to the species of the genus Serpentinomonas, chemolithotrophic bacteria isolated at the serpentinizing site The Cedars that are capable of growth up to pH 12.5. These species, and other phylotypes, such as taxa affiliated with Lost City Methanosarcinales were identified in several serpentinizing sites and could contribute to the definition of a biological signature associated with serpentinization. By specifically targeting enriched metabolisms in serpentinizing environments, we highlighted key functions associated with hydrogen metabolism and environmental stress response mechanisms. The comparison of serpentinizing metagenomes revealed the importance of a phosphonate degradative pathway, based on the activity of a C-P lyase. This metabolic pathway, which plays a key role in the uptake of phosphorus and the release of organic molecules, was integrated into the ecological models of serpentinizing systems

Background: The large-scale analysis of phenomic data (i.e., full phenotypic traits of an organism, such as shape, metabolic substrates, and growth conditions) in microbial bioinformatics has been hampered by the lack of tools to rapidly and accurately extract phenotypic data from existing legacy text in the field of microbiology. To quickly obtain knowledge on the distribution and evolution of microbial traits, an information extraction system needed to be developed to extract phenotypic characters from large numbers of taxonomic descriptions so they can be used as input to existing phylogenetic analysis software packages. Results: We report the development and evaluation of Microbial Phenomics Information Extractor (MicroPIE, version 0.1.0). MicroPIE is a natural language processing application that uses a robust supervised classification algorithm (Support Vector Machine) to identify characters from sentences in prokaryotic taxonomic descriptions, followed by a combination of algorithms applying linguistic rules with groups of known terms to extract characters as well as character states. The input to MicroPIE is a set of taxonomic descriptions (clean text). The output is a taxon-by-character matrix-with taxa in the rows and a set of 42 pre-defined characters (e.g., optimum growth temperature) in the columns. The performance of MicroPIE was evaluated against a gold standard matrix and another student-made matrix. Results show that, compared to the gold standard, MicroPIE extracted 21 characters (50%) with a Relaxed F1 score > 0.80 and 16 characters (38%) with Relaxed F1 scores ranging between 0.50 and 0.80. Inclusion of a character prediction component (SVM) improved the overall performance of MicroPIE, notably the precision. Evaluated against the same gold standard, MicroPIE performed significantly better than the undergraduate students. Conclusion: MicroPIE is a promising new tool for the rapid and efficient extraction of phenotypic character information from prokaryotic taxonomic descriptions. However, further development, including incorporation of ontologies, will be necessary to improve the performance of the extraction for some character types.

Le sol représente le support de la production agricole. A l’interface avec les autres compartiments de la biosphère, il remplit de nombreuses fonctions essentielles à la fourniture de services écosystémiques nécessaires au bien-être de nos sociétés. C’est aussi une ressource non renouvelable dont les propriétés physicochimiques et biologiques ont été altérées par le développement de l’agriculture intensive. La prise de conscience actuelle de cet état de fait a révélé la nécessité de définir de nouveaux modes de gestion adaptés à la préservation et à l’utilisation durable des sols. Elle a ainsi marqué l’entrée dans l’ère de l’agroécologie qui prône un modèle de production optimisant notamment les services rendus par la biodiversité afin de réduire le recours aux intrants et à l’utilisation d’énergie. Pour atteindre cet objectif, le développement d’une gamme d’indicateurs permettant d’évaluer les pratiques/systèmes agricoles en rendant compte de la qualité biologique du sol est donc indispensable. Cette thèse, dont l’objectif est de contribuer au développement de bioindicateurs microbiens de la qualité du sol, s’inscrit dans ce contexte agroécologique. Le choix de travailler sur les communautés microbiennes se justifie pleinement dans cette problématique car elles sont (i) présentes avec une forte densité et diversité dans tous les environnements, (ii) fortement impliquées dans le fonctionnement biologique et les services rendus par le sol, et (iii) elles répondent de façon très sensible aux changements des conditions environnementales en termes de modification de biomasse, de structure/diversité et d’activité. Elles offrent donc un potentiel important en termes de développement de bioindicateurs. Ce travail a porté plus précisément sur l’évaluation de deux indicateurs complémentaires : (i) la biomasse moléculaire microbienne et (ii) la diversité taxonomique microbienne. Dans une première partie nous avons éprouvé la robustesse de ces deux indicateurs en évaluant les biais associés à chacune des étapes techniques des procédures mises en œuvre pour leur mesure. Nous avons ensuite utilisé ces deux indicateurs dans différents contextes agronomiques pour évaluer leur pertinence. Un premier travail a ainsi consisté à suivre la réhabilitation du patrimoine microbien, par l’implantation d’une culture à vocation énergétique, d’un sol pollué irrigué pendant une centaine d’années par des eaux usées. Une seconde application a porté sur l’étude de l’impact de différentes pratiques agricoles sur les communautés microbiennes selon l’intensité du travail du sol (labour vs. travail réduit), la gestion des résidus de culture (export vs. restitution), et le type de culture (annuelle vs. pérenne).Les résultats montrent que la biomasse moléculaire microbienne et la diversité taxonomique obtenue par séquençage massif sont deux bioindicateurs robustes et sensibles pour décrire la qualité microbiologique des sols agricoles dans des contextes très variés. Ces deux indicateurs permettent de mettre en évidence aussi bien des perturbations des sols que l’impact positif de pratiques innovantes. Ils peuvent donc représenter des outils performants pour l’évaluation des systèmes agricoles, aidant à une amélioration de leur mode de gestion et, à long terme, permettant une utilisation durable des ressources fournies par ces sols
Soil is the support of agricultural production. It performs many functions essential to the provision of ecosystem services necessary for the well-being of our societies. Soil physicochemical and biological properties have been altered by the development of intensive agriculture while it is a non-renewable resource, revealing the need to develop new management practices suitable for the sustainability of soil quality. This also marked the entry into the “Agroecology” era, which promotes the development of new agricultural systems optimizing services provided by biodiversity to reduce the use of inputs and energy use. To achieve this aim, the development of a range of indicators to assess the impact of agricultural practices on the biological quality of the soil is essential. This thesis, which aims to contribute to the development of microbial bio-indicators of soil quality, is a part of this agroecological context. The choice to work on microbial communities is fully justified because they are (i) present with a high abundance and diversity in all environments, (ii) heavily involved in biological functioning and the soil ecosystem services, (iii) they respond very sensitive to changes in environmental conditions in terms of biomass, diversity and activity. They therefore have significant potential in terms of bio-indicators of development. This work has focused specifically on the evaluation of two complementary bioindicators: (i) the microbial molecular biomass and (ii) the microbial taxonomic diversity. In a first part we tested the robustness of these two bioindicators by assessing the biases associated with each of the procedure technical steps used for their measurement. We then used these bioindicators in different agricultural contexts to assess their sensitivity. A first work has followed the rehabilitation of microbial patrimony of a polluted soil irrigated for a hundred years by sewage, by implanting a bioenergy crop. A second application has focused on the impact of different agricultural practices on microbial communities depending on the intensity of tillage (tillage vs. reduced tillage), management of crop residues (export vs. restitution), and the crop type (annual vs. perennial). Results highlighted that microbial molecular biomass and microbial taxonomic diversity achieved by high throughput sequencing are both robust and sensitive bioindicators to describe the microbiological quality of agricultural soils in very different contexts. Both bioindicators allow evidencing soil disturbances but also the positive impact of innovative practices. They may therefore represent powerful tools for the assessment of agricultural systems, helping to improve their long term management, allowing a sustainable use of resources provided by soils

In 1992, a marine Synechococcus was discovered in a meromictic lake in the Vestfold Hills, Antarctica. This thesis describes the ecology and taxonomy of this organism. Ace Lake is a saltwater, meromictic that was isolated from the marine environment approximately 6000 years ago. In 1992, The lake was 25 m deep, the top 12 m was oxygenated and the lake had a salinity range of 16 to 40 g kg-1 salt. The recent discovery of Synechococcus in Ace Lake was aided by flow cytometric methods. In Ace Lake, Synechococcus occured in the highest densities below the pycnocline with maximum numbers occurring just above the oxic/anoxic interface. Synechococcus bloomed in spring with numbers declining again in early January. At the peak of the bloom in 1992, a density of 8 x 106 cells mr1 was recorded at 11 m in the lake. No diel periodicity in the growth of Synechococcus was detected. Synechococcus was also present in two of ten other meromictic lakes and basins. The organism occured throughout the aerobic zone in Pendant Lake, in densities of approximately 10-7 cells m1-1, and below the pycnocline in Lake Abraxas in densities of 1.4 x 10 7 cells m1-1. It is possible that salinity restricts the distribution of Synechococcus in the meromictic lakes of the Vestfold Hills. Synechococcus strains were isolated from Ace Lake, Pendant Lake and Lake Abraxas for further characterisation. The three strains were similar in size and had the same lipid soluble pigment signature, with two unknown carotenoid pigments present in addition to the chlorophyll a, zeaxanthin and bb carotene. The three strains had phycoerythrin as their principle accessory light harvesting pigment. They were genetically similar (99. 7 % similarity in the 16S rRNA sequence) and had a G + C content of between 57 and 58 mol %. They were also genetically similar (95. 7 % similarity in the 16S rRNA sequence) to another marine picocyano bacteria, Prochlorococcus marinus. Based on the square root temperature dependence model, the minimum and maximum theoretical growth temperatures of the Ace Lake Synechococcus strain was -8° C, and 29.8° C. The optimal theoretical growth temperature was 19.7° C. ln-situ growth rates of the Ace Lake Synechococcus strain at 6 m, 8 m and 10 m in Ace Lake were determined. These rates were -0.118 d-1, 0.072 d-1 and 0.341 d-1 respectively. An increase in water temperature and a re~~on in light intensity increased the in-situ growth rate of the Ace Lake Synechococcus population. The grazing pressure on Synechococcus in Ace Lake was not determined. It is probable, however, that the distribution and abundance of Synechococcus in Ace Lake, Pendant Lake and Lake Abraxas is controlled by grazing. Chapter 1 summarises and reviews the current ecological and taxonomic research that has been undertaken on Ace Lake. Chapter 2 describes the flow cytometric techniques that were developed to study Synechococcus in Antarctic Lakes. Chapter 3 discusses the ecology of Synechococcus in Ace Lake and chapter 4 the distribution of Synechococcus in meromictic lakes in the Vestfold Hills. Chapter 5 describes the taxonomic characteristics of three Antarctic Synechococcus strains and chapter 6 discusses controls of Synechococcus growth in Ace Lake.

by Yip Chi Wai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1992.
Includes bibliographical references (leaves 70-87).
ABSTRACT --- p.i
ACKNOWLEDGEMENT --- p.iv
TABLE OF CONTENTS --- p.v
LIST OF TABLES --- p.ix
LIST OF FIGURES --- p.xiii
INTRODUCTION --- p.1
LITERATURE REVIEW --- p.3
Chapter I. --- Mycobacterial Infections --- p.3
Chapter A. --- Mycobacterium tuberculosis --- p.3
Chapter B. --- Atypical mycobacteria --- p.3
Chapter II. --- Identification of Mycobacteria --- p.5
Chapter A. --- Conventional methods --- p.6
Chapter 1. --- Mycobacterium tuberculosis --- p.6
Chapter 2. --- Atypical mycobacteria --- p.7
Chapter B. --- Rapid identification methods --- p.8
Chapter 1. --- Identification by fatty acid analysis --- p.8
Chapter 2. --- Identification by mycolic acid analysis --- p.9
Chapter III. --- In vitro Susceptibility Testing of Mycobacteria --- p.11
Chapter A. --- Mycobacterium tuberculosis --- p.11
Chapter 1. --- Principle --- p.12
Chapter 2. --- Methods of susceptibility testing --- p.13
Chapter a. --- The absolute concentration method --- p.13
Chapter b. --- The resistance ratio method --- p.15
Chapter c. --- The 1% proportion method --- p.16
Chapter d. --- Radiometric method --- p.18
Chapter e. --- Other methods --- p.19
Chapter B. --- Atypical mycobacteria --- p.20
Chapter IV. --- Plasmid Analysis in Mycobacteria --- p.22
Chapter A. --- Discovery of plasmids in mycobacteria --- p.22
Chapter B. --- Methodologies in the studies of mycobacterial plasmids --- p.23
Chapter C. --- Possible roles of plasmid in epidemiology of mycobacteria --- p.24
MATERIALS AND METHODS --- p.26
Chapter I. --- Bacterial Strains and Strain Maintenance --- p.26
Chapter A. --- Strains collection --- p.26
Chapter B. --- Strains maintenance --- p.26
Chapter II. --- Culture Media and Culture Conditions --- p.26
Chapter III. --- Identification of Mycobacteria --- p.26
Chapter A. --- Conventional methods --- p.26
Chapter B. --- Fatty acid profile analysis --- p.27
Chapter 1. --- Bacterial isolates --- p.27
Chapter 2. --- Standards and reagents --- p.27
Chapter 3. --- Preparation of methyl ester for GC/GC-MS --- p.28
Chapter 4. --- Instrumentation --- p.28
Chapter a. --- Gas chromatography-mass spectrometry (GC-MS) --- p.28
Chapter b. --- Gas liquid chromatography (GLC) --- p.28
Chapter 5. --- Fatty acid profile analysis --- p.29
Chapter a. --- Calibration --- p.30
Chapter b. --- Identification of mycobacterial fatty acids --- p.30
Chapter c. --- Construction of mycobacterial fatty acid profiles --- p.30
Chapter 6. --- Discriminant analysis --- p.31
Chapter IV. --- In Vitro Drug Susceptibility Test --- p.31
Chapter A. --- Test strains --- p.31
Chapter B. --- Preparation of drug-containing media --- p.32
Chapter C. --- Minmum inhibition concentration (MIC) determination --- p.32
Chapter V. --- Heavy Metal Tolerance Test --- p.33
Chapter A. --- Bacterial strains --- p.33
Chapter B. --- Reagent and media preparation --- p.34
Chapter 1. --- Heavy metal stock solution preparation --- p.34
Chapter 2. --- Media preparation --- p.34
Chapter C. --- Minimum inhibition concentration (MIC) determination --- p.34
Chapter VI. --- Plasmid Analysis of Mycobacteria --- p.35
Chapter A. --- Bacterial strains --- p.35
Chapter B. --- Extraction procedures --- p.35
Chapter 1. --- Modified Kado & Liu method --- p.35
Chapter 2. --- French press procedure --- p.36
Chapter 3. --- Spheroplasts preparation procedure --- p.37
Chapter C. --- Electrophoresis procedure --- p.37
Chapter D. --- Statistical analysis for correlation between plasmid and drug resistance or heavy metal tolerance --- p.38
RESULTS --- p.39
Chapter I. --- Identification of Mycobacteria --- p.39
Chapter A. --- General characteristics of the chromatographic profile --- p.39
Chapter B. --- Discriminant analysis --- p.40
Chapter 1. --- Gas chromatography-mass spectrometry (GC-MS) --- p.40
Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.40
Chapter b. --- Rapidly growing mycobacter-ia --- p.41
Chapter c. --- Pigmented mycobacteria --- p.41
Chapter 2. --- Gas liquid chromatography (GLC) --- p.41
Chapter a. --- Slowly growing non-pigmented mycobacteria --- p.42
Chapter b. --- Rapidly growing mycobacteria --- p.42
Chapter c. --- Pigmented mycobacteria --- p.43
Chapter II. --- Vitro Drug Susceptibility Test --- p.43
Chapter A. --- Mycobacterium tuberculosis --- p.43
Chapter B. --- Atypical mycobacteria --- p.45
Chapter 1. --- General characteristics --- p.45
Chapter 2. --- Sensitivity pattern of different species --- p.46
Chapter a. --- Mycobacterium kansasii --- p.46
Chapter b. --- Mycobacterium avium- intracellulare complex --- p.46
Chapter c. --- Mycobacterium scrofulaceum --- p.47
Chapter d. --- Mycobacterium terrae complex --- p.47
Chapter e. --- Mycobacterium fortuitum --- p.47
Chapter f. --- Mycobacterium chelonae --- p.48
Chapter III. --- Heavy Metal Tolerance Test --- p.48
Chapter IV. --- Plasmid in Mycobacteria --- p.48
Chapter A. --- Mycobacterium tuberculosis --- p.48
Chapter B. --- Atypical mycobacteria --- p.49
Chapter 1. --- General characteristics --- p.49
Chapter 2. --- Correlation between drug resistance and plasmid --- p.50
Chapter 3. --- Correlation between heavy metal tolerance and plasmid --- p.50
DISCUSSION --- p.52
Chapter I. --- Identification of Mycobacteria --- p.52
Chapter II. --- In Vitro Drug Susceptibility Test --- p.56
Chapter A. --- Mycobacterium tuberculosis --- p.56
Chapter B. --- Atypical mycobacteria --- p.60
Chapter 1. --- Mycobacterium kansasii --- p.61
Chapter 2. --- Mycobacterium avium- intracellulare complex --- p.61
Chapter 3. --- Mycobacterium scrofulaceum --- p.62
Chapter 4. --- Mycobacterium terrae complex --- p.62
Chapter 5. --- Mycobacterium fortuitum --- p.63
Chapter 6. --- Mycobacterium chelonae --- p.64
Chapter III. --- Plasmid Analysis in Mycobacteria --- p.64
Chapter A. --- Mycobacterium tuberculosis --- p.64
Chapter B. --- Atypical mycobacteria --- p.66
SUMMARYS AND CONCLUSIONS --- p.68
LITERATURE CITED --- p.70
Chapter APPENDIX - --- Tables --- p.88
Figures --- p.144

Although there are many examples of microbial biogeography, few microbes have been studied at high taxonomic resolution over large spatial scales. As a result, the environmental and ecological processes that drive niche partitioning, diversity, composition, and activity of microbial taxa are often poorly understood. To address this gap, I examine the most abundant phytoplankton in the global ocean, Prochlorococcus sp., a marine cyanobacterium. Using amplicon libraries of the Prochlorococcus internal transcribed spacer (ITS) region and 23S rRNA gene as markers, I demonstrate several key differences between the two major high light (HL) clades of Prochlorococcus. First, by examining ITS amplicon libraries at high taxonomic resolution it is revealed that “sub-ecotype” clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Whereas unique combinations of environmental traits drive the distribution of the HL-I sub-ecotype clades, the HL-II sub-ecotype clades appear ecologically coherent. Second, using 23S rRNA and rDNA libraries I show that activity (rRNA) and abundance (rDNA) are highly correlated for Prochlorococcus across all sites and operational taxonomic units (OTUs) in the surface ocean, demonstrating a tight coupling between activity and abundance. Finally, I investigate the associations between Prochlorococcus and the rest of the microbial community in the North Pacific and find region-specific trends in both strength and sign. Associations with other microbes are strongest for HL-I in the temperate region and strongest for HL-II in the sub-tropical gyre. This dissertation clarifies the relative importance of the environment, geography, community, and taxonomy in terms of their role in creating complex assemblages of Prochlorococcus and helps improve our understanding of how marine microbial communities are assembled in situ.

Dissertation

Microbial ecology seeks to describe the diversity and distribution of microorganisms in various habitats within the context of environmental variables. High throughput sequencing has greatly boosted the number and scope of projects aiming to study and analyse these organisms, with ever-increasing amounts of data being generated. Amplicon based taxonomic analysis, which determines the presence of microbial taxa in different environments on the basis of marker gene annotations, often uses percentage identity as the main metric to determine sequence similarity against databases. This data is then used to study the distribution of biodiversity as well as the response of microbial communities to stressors. However, the 16S rRNA gene displays varying degrees of sequence conservation along its length and is therefore prone to provide different results depending on the part of 16S rRNA gene used for sequencing and analysis. Furthermore, sequence alignment is primarily performed using the popular BLAST sequence alignment tool, which incurs a great computational performance penalty although newer, more efficient tools are being developed. A new approach that is fast and more accurate is critically needed to process the avalanche of data. Additionally, repositories of environmental metadata can provide contextual information to sequence annotations, potentially enhancing analysis if they can be incorporated into bioinformatics pipelines. The overarching aim of this work was to enhance the taxonomic annotation of bacterial sequences by developing a weighted scheme that utilizes inherent evolutionary conservation in the bacterial 16S rRNA gene sequences and by adding contextual, environmental information pertaining to these sequences in a systematic fashion.

Sargassum é uma macroalga frondosa com uma distribuição global ampla em recifes temperados e tropicais. Esta alga providencia habitat e refúgio a diversas espécies marinhas, mas também afecta outras negativamente. Apesar da sua importância ecológica, muito pouco se sabe sobre a vida microbiana nas superfícies de Sargassum. Através do estudo das comunidades microbianas presentes no biofilme da alga Sargassum sp., em paralelo com a água e o sedimento, ao longo de uma série temporal, pretende-se com este estudo entender as dinâmicas de sucessão microbianas do biofilme de Sargassum sp. e de que forma a comunidade microbiana se altera com as alterações ambientais. A amostragem de Sargassum sp., sedimento e água foi feita em Magnetic Island, na Grande Barreira de Coral na Australia, ao longo de um periodo de 13 meses. A sequenciação do gene 16S do rRNA de 30 amostras de Sargassum sp., usando a tecnologia Illumina, permitiu uma análise aprofundada da diversidade taxonomica do biofilme, ao mesmo tempo que o possível perfil funcional de taxa bacterianos chave, foi aferido usando o programa FAPROTAX. A implementação de análise estatística multivariada de ordenação, análises de correlação e estatísticas de abundância diferencial, permitiram investigar a resposta das comunidades bacterianas a parâmetros ambientais abióticos tais como: temperatura e nutrientes orgânicos e inorgânicos. Os biofilmes de Sargassum sp. revelaram estar dominados por bactérias dos Filos Firmicutes (23-45%), Proteobacteria (35-38%) e Bacteroidetes (13-30%) com diferenças observadas entre o Inverno e o Verão. As comunidades bacterianas da água e do sedimento mantiveram-se estáveis ao longo de toda a série temporal equanto que as comunidades associadas com Sargassum sp. revelaram flutuações ao nível da composição bacteriana. Os parâmetros ambientais explicam: 56% da variação da comunidade bacteriana da água, 46% da variação das comunidades associadas com o sedimento e 29% da variação do biofilme de Sargassum sp. Estes resultados sugerem que os mecanismos que medeiam as alterações na composição da comunidade microbiana de Sargassum sp. podem estar relacionados com factores bióticos tais como interação bactéria-bactéria ou interacção entre o hospedeiro e as bactérias associadas (endo e epifíticas), ou ainda interações com outros macroorganismos e seus microbiomas. Este estudo destaca a importância de examinar simultaneamente factores bióticos e ambientais, como forças condutoras de alteração dos microbiomas associados a espécies estruturantes dos recifes de coral.
The canopy-forming macroalgae Sargassum is widely distributed throughout temperate and tropical reefs globally, playing an important role in habitat/refugia provision for diverse marine species, but also affecting others negatively. Despite its ecological importance, little is known about the microbes living on the alga’s surface. This study aimed to understand microbial successional dynamics within the Sargassum sp. biofilm and determine how environmental variations affect the microbial community, by investigating the microbial communities of Sargassum sp. biofilms, seawater and sediment in tandem over a time series. Sargassum sp., sediment and seawater samples were collected over a 13-month period from Magnetic Island on the Great Barrier Reef. Illumina 16S rRNA gene sequencing of 30 Sargassum sp. samples provided an in-depth analysis of the taxonomic diversity existing on the biofilm while putative functional roles of key bacterial taxa were assigned with FAPROTAX. Unconstrained ordination, correlation analyses and differential abundance statistics were used to investigate the response of bacterial communities to measured environmental parameters such as temperature, organic and inorganic nutrients. Sargassum sp. biofilms were dominated by bacteria belonging to Firmicutes (23-45%), Proteobacteria (35-38%) and Bacteroidetes (13-30%) phyla, with differences in community structure observed between winter and summer. Microbial communities of seawater and sediment were stable throughout the time series, while the communities associated with Sargassum sp. fluctuated in composition. Environmental parameters explained 56% of the variation in microbial community composition for seawater, 46% for sediment-associated communities and 29% for epiphytic Sargassum sp. biofilm communities. These results suggest that the Sargassum microbiome could be primarily mediated by biotic factors such as bacteriabacteria interactions or interactions between the host and its associated bacteria (endophytes and epiphytes) and interactions with other species and their microbiomes. This study highlights the importance of simultaneously examining biotic and environmental drivers to determine factors structuring the microbiome of key reef species.