<p>The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (&gt / 1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.</p>

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.

<p>The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity (&gt / 1%) was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.</p>

Magister Scientiae - MSc / The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study. / South Africa

Orientador: Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T14:04:11Z (GMT). No. of bitstreams: 1 Silva_CynthiaCanedoda_D.pdf: 9736339 bytes, checksum: 506af9b64177978ae4c3c5c0588e1b28 (MD5) Previous issue date: 2010 / Resumo: Os efluentes de refinarias contêm vários compostos tóxicos, que podem provocar sérios danos aos seres vivos se não tratados previamente. Os fenóis são poluentes encontrados em efluentes de diversas indústrias, incluindo as refinarias de petróleo, e são conhecidos como um dos compostos mais tóxicos encontrados no meio ambiente. Portanto, há necessidade constante de buscar formas de remover ou reduzir a presença destas substâncias nos efluentes. Este trabalho teve como objetivos analisar e explorar a diversidade microbiana presente em lodo de sistemas de tratamento de efluentes de refinarias petróleo, através da construção de bibliotecas de genes RNAr 16S e bibliotecas metagenômicas em vetores do tipo fosmídeo. Análises filogenéticas das bibliotecas de genes RNAr 16S e metagenômicas mostraram uma comunidade bacteriana bastante diversa, com predominância do Filo Proteobacteria, seguido de Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi e Bacteroidetes. Dentre os gêneros mais abundantes, se destacaram Thauera, Comamonas, Diaphorobacter e Thiobacillus. Espécies pertencentes a estes gêneros realizam o processo de desnitrificação e espécies de Thauera e Comamonas estão relacionadas com a degradação de compostos aromáticos. A grande diversidade bacteriana refletiu em uma ampla diversidade metabólica, sendo obtido um grande número de resultados para genes relacionados com processos biológicos importantes para o tratamento de efluentes, como o metabolismo de nitrogênio, enxofre, fósforo, além de genes relacionados com o catabolismo de compostos aromáticos, tais como benzoato, bifenil e fenol. Adicionalmente, foram observadas possíveis novas sequências para os genes que codificam enzimas-chaves responsáveis pela meta-clivagem de fenol e outros aromáticos, como fenol hidroxilase e catecol 2,3-dioxigenase. No presente estudo foram também realizadas triagens funcionais dos 13.200 clones para enzimas hidrolíticas, com detecção de dois hits para esterase e um para lipase. Este trabalho mostrou que a bioprospecção genômica microbiana é uma estratégia inovadora na área de tratamento de efluentes, que pode contribuir para o conhecimento da microbiota responsável pela degradação dos poluentes. / Abstract: Refinery wastewater has several toxic compounds that may cause serious damage to life. Phenolic compounds are found in several industrial effluents, including petroleum refinery, and represent a significant environmental toxicity hazard. Therefore, there is a constant need to search for new technologies able to remove and reduce the presence of these substances in wastewater. This study aimed to analyze and explore the microbial diversity present in sludge from refinery wastewater treatment systems through the construction of 16S rRNA genes libraries and metagenomic libraries in fosmid vectors. Sequencing and phylogenetic analyses of libraries showed a highly diverse bacterial community, with predominance of the Proteobacteria phylum, followed by Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi and Bacteroidetes. The most abundant genera were Thauera, Comamonas, Diaphorobacter and Thiobacillus. Species belonging to these genera are facultative anaerobic and may be involved with the denitrification process, and Thauera and Comamonas species are related with degradation of aromatic compounds. This bacterial diversity reflected in a broad metabolic diversity. Analysis of the metagenomic data derived from pyrosequencing revealed a lot of hits related with biological processes of great relevance for wastewater treatment, such as genes for nitrogen, sulfur and phosphorus metabolism, in addition to genes related to the catabolism of aromatic compounds, such as benzoate, biphenyl and phenol. Additionally, sequences representing possible new genes encoding for key-enzymes responsible for the meta-cleavage of phenol and other aromatic compounds, such as phenol hydroxylase and cathecol 2,3-dioxygenase, were found in the fosmid libraries. Finally, high-throughput functional screening of 13,200 clones for hydrolytic enzymes yielded two positive hits for lipase and one for esterase. The data gathered together in this work showed that microbial genomic prospection is an innovative strategy for the wastewater treatment system that may undoubtedly contribute to the knowledge of microorganisms responsible by pollutant degradation. / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Biofilme sind eine der widerstandsfähigsten Formen mikrobiellen Lebens. Ihr frühzeitiges Auftreten in der Erdgeschichte konnte durch Stromatolithfunde bewiesen werden. Heutige Biofilme und mikrobielle Matten bieten somit eine Möglichkeit wichtige Einblicke und Erkenntnisse über das erste Leben auf unserem Planeten zu geben. In dieser Arbeit wurden die prokaryotischen Lebensgemeinschaften von verschiedenen Ökosystemen mittels metagenomischer und metatranskriptomischer Methoden analysiert. Mithilfe von „Next-Generation Sequencing“ wurden 16S rRNA Genanalysen, metatranskriptomische Analysen und funktionsbasierte Durchmusterungen von Fosmid-Metagenombanken durchgeführt. Die bakterielle Zusammensetzung und Diversität von kalzifizierenden Biofilmen und dem unterliegenden Kalktuff des Frischwasserbachs Westerhöfer Bach wurden analysiert. Es konnte gezeigt werden, dass der Biofilm hauptsächlich von filamentösen Cyanobacteria, aeroben Vertretern aus allen Klassen der Proteobacteria und Chloroflexi bevölkert wurde. Die bakterielle Diversität nahm flussabwärts zu, was auf Änderungen der physikochemischen Parameter zurückgeführt wird. Aufgrund geringerer UV-Einstrahlung waren im Kalktuff mehr Proteobacteria als Cyanobacteria vorhanden. Des Weiteren gab es deutliche Unterschiede zwischen den relativen Abundanzen der gesamten und aktiven proteobakteriellen Klassen im Biofilm. Die aktiven Funktionen der Biofilm-Mikrobiota einer Westerhöfer Bach Probe wurden mittels metatranskriptomischer Methoden genauer analysiert. Die meisten Transkripte der mikrobiellen Biofilmgemeinschaft umfassten Gene der Photosynthese, des Proteinmetabolismus, des Kohlenstoffmetabolismus und der Zellatmung. Um das metagenomische Potential des Westerhöfer Bach Biofilms zu erschließen, wurden vier „large-insert“ Metagenombanken konstruiert. Funktionsbasierende Durchmusterungsverfahren führten zur Identifikation von fünf bisher unbekannten Genen, die für proteolytische Enzyme kodieren und einem Gen-Cluster, welches für cellulolytische Enzyme kodiert. Bei dem zweiten untersuchten Habitat handelt es sich um eine mikrobielle Matte des hypersalinen Lake 21 auf Kiritimati. Die Mikrobialith-bildende Matte besteht aus neun klar abgegrenzten, unterschiedlich gefärbten Lagen, welche separat auf ihre bakterielle und archaelle Zusammensetzung analysiert wurden. Anhand der prokaryotischen Zusammensetzung und dem Sauerstoff- und Lichtgradienten ergab sich eine Einteilung der mikrobiellen Matte in drei Zonen. Im Allgemeinen erhöhte sich die prokaryotische Diversität mit Tiefe der Matte, wohingegen das Redoxpotential und der pH-Wert sanken. Passend zu den hydrochemischen Daten änderte sich die prokaryotische Zusammensetzung von der photisch-oxischen Zone, welche aus halophilen, oxygenen und anoxygenen Phototrophen und aeroben Heterotrophen bestand, zu Sulfat-reduzierenden Bakterien (SRB), Fermentierern und potentiell Sulfat-reduzierenden Archaeen in der Übergangszone. In der anoxischen Zone konnten hauptsächlich SRB, Fermentierer, Ammonium-oxidierende Archaea und geringe Mengen methanogene Archaeen detektiert werden. Von den kenianischen Natronseen Bogoria, Sonachi, Elementeita und Magadi wurde die prokaryotische Zusammensetzung und Diversität von Boden-, Sediment-, Wasser-, und mikrobiellen Mattenproben analysiert. Hier zeigte sich, dass Boden- sowie Sedimentproben hauptsächlich von Proteobacteria, Gemmatimonadetes, Firmicutes, Actinobacteria, Acidobacteria und Bacteroidetes bevölkert wurden, wohingegen in den Wasserproben Cyanobacteria vorherrschten. Die Archaeen wurden überwiegend von unterschiedlichen Vertretern der Halobacteria repräsentiert. In den humiden Proben wurden außerdem methanogene Archaeen und Thaumarchaeota nachgewiesen. Letztlich wurde in dieser Arbeit die bakterielle Zusammensetzung des Biofilms und des dazugehörigen Planktons von mikrobiellen Brennstoffzellen (MBZ) untersucht. Der erzeugte Datensatz demonstrierte, dass die aktive und gesamte bakterielle Lebensgemeinschaft in den einzelnen Replikaten minimal variierte. Generell zeigte sich, dass stromproduzierende MBZ eine niedrigere bakterielle Diversität aufwiesen als nicht stromproduzierende MBZ. Des Weiteren zeigte die Analyse, dass bisher unkultivierte Vertreter der Spezies Geobacter und Clostridium mit der Stromproduktion verbunden waren.

Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research. / Chemistry / M. Sc. (Life Sciences)