Os manguezais compõem um bioma de interface entre o continente e o oceano em regiões intertropicais, ambiente este caracterizado por condições únicas ambientais e uma elevada biodiversidade. Este projeto tem como objetivo estudar, utilizando abordagens de metagenômica e metatranscriptomica, as comunidades microbianas encontradas nos manguezais localizados nos municípios de Bertioga/SP e Cananeia/SP, com enfoque nos genes relacionados ao processo de redução do sulfato. Para tanto, uma biblioteca metagenômica contendo 12.960 clones em vetor fosmídeo foi triada por meio de PCR específico para o gene dsrB, ao mesmo passo que esta foi completamente sequenciada em plataforma Illumina HiSeq2000. Foram obtidos três insertos metagenomicos (23D5, MGV 10001431 e MGV 10016026, com 31, 31 e 34 kb, respectivamente). Estes foram então anotados e analisados mais detalhadamente. A inserção 23D5 foi a única a apresentar genes essenciais para a redução dissimilatória do sulfato (apr, hdr, dsr, sat). A diversidade taxonômica dos grupos relacionados ao ciclo do enxofre demonstrou a predominância dos filos Bacteroidetes e Proteobacteria enquanto a análise filogenética para gene dsrB apresentou diferenças entre os três insertos, afiliando os mesmos a sequências similares a Firmicutes e Deltaproteobacteria e revelando serem diferentes das sequências presentes em base de dados. A análise de metatrascriptomica dos quatro manguezais apresentou um padrão de expressão diferencial para o cluster dsr de acordo com o estado de conservação dos manguezais estudados. Estes resultados compõem o primeiro acesso a fragmentos genômicos e a funcionalidade dos mesmos em microrganismos redutores de sulfato em solos de manguezais. / Mangrove is a biome composed of the interface between the continent and the ocean in tropical areas, characterizing by unique environmental conditions and high biodiversity. Here, we aimed to study, using metagenomic and metatranscriptomic approaches, the microbial communities identified in the mangroves located in the cities of Bertioga/SP and Cananeia/SP, focusing on genes related to the sulfate reduction process. For this purpose, a metagenomic library containing 12.960 clones in fosmid vector was screened by PCR for the specific dsrB gene, and the whole library was also completely sequenced by the Illumina HiSeq2000 platform. Three metagenomic inserts were obtained (23D5, MGV 10016026 and MGV 10001431, with 31, 31 and 34 kb, respectively), which were recorded and detail analyzed. The insertion 23D5 was the only one that presents essential genes for dissimilatory sulfate reduction (apr, hdr, dsr, sat). The taxonomic diversity of groups related to the sulfur cycle demonstrated the predominance of Bacteroidetes and Proteobacteria phyla, while phylogenetic analysis to dsrB gene showed differences between the three inserts, affiliating them to similar sequences of Firmicutes and Deltaproteobacteria, and revealing differ from the sequences present in the data base. The metatranscriptomic analysis of the four mangroves showed a pattern of differential expression for the DSR cluster according to the conservation status of the studied mangroves. These results constitute the first access of genomic fragments and functionality of the sulfate reducing microorganisms in mangrove soils.

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>

Philosophiae Doctor - PhD / Metagenomic sequencing of environmental samples provide direct access to genomic information of organisms within the respective environments. This sequence information represents a significant resource for the identification and subsequent characterization of potentially novel genes, or known genes with acquired novel characteristics. Within this context, the thermophilic environments are of particular interest due to its potential for deriving novel thermostable enzymes with biotechnological and industrial applications. In this work metagenomic library construction, random sequencing and sequence analysis strategies were employed to enhance identification and characterisation of potentially novel genes, from a thermophilic soil sample. High molecular weight metagenomic DNA was extracted from two Chinese hydrothermal soil samples. This was used as source material for the construction of four genomic DNA libraries. The combined libraries were estimated to contain in the order of 1.3 million genes, which provides a rich resource for gene identification. Approximately 70 kbp of sequence data was generated from one of the libraries as a resource for sequence-based analysis. Initial BLAST analysis predicted the presence of 53 ORFs/partial ORFs. The BLAST similarity scores for the investigated ORFs were sufficiently high (>40%) to infer homology with database proteins while also being indicative of novel sequence variants of these database matches. In an attempt to enhance the potential for deriving more full length ORFs a novel strategy, based on WGA technology, was employed. This resulted in the recovery of the near complete sequence of partial ORF5, directly from the WGA DNA of the environmental sample. While the full length ORF5 could not be recovered, the feasibility of this novel approach, for enhanced metagenomic sequence recovery was proved in principle. The implementation of multiple insilico strategies resulted in the identification of two ORFs, classified as homologs of the DUF29 and Usp protein families respectively. The functional inference obtained from the integrated in-silico predictions was furthermore highly suggestive of a putative nucleotide binding/interaction role for both ORFs. A putative novel DNA polymerase gene (denoted TC11pol) was identified from the sequence data. Expression and characterization of the full length TC11pol did however not result in detectable polymerase activity. The implementation of a homology modeling approach proved succesfull for deriving a structural model of the polymerase that was used for: (i) deriving functional inferences of the potential activities of the polymerase and (ii) deriving a 5’ exonuclease deletion mutant for functional analysis. Expression and subsequent functional characterization of the putative 5’exo- TC11pol mutant resulted in detectable polymerase and 3’-5’ exonuclease activity at 37 and 45 oC, following a heat denaturation step at 55 oC for 1 hour. It was, therefore concluded that the putative 5’exo- TC11pol mutant was functionally equivalent to the Klenow fragment of E. coli, while exhibiting increased thermostability. / South Africa

In January 2010, a magnitude 7.0 earthquake devastated Haiti, one of the poorest countries in the Western Hemisphere. Haiti’s weak sanitation infrastructure and limitations in the public health system made the country susceptible to the spread of waterborne diseases. Following the earthquake, cholera rapidly spread through Haiti, killing 4,672 people in 5 months and leaving thousands hospitalized (MSNBC, 2010). Cholera is an infectious diarrheal disease caused by the pathogen, Vibrio cholerae, which results in severe dehydration with a high mortality risk. The source of the epidemic was traced to the Artibonite River, the island’s longest and most essential drinking water source (Encyclopaedia Britannica. n.d.). The origin of the contamination was later discovered to be unsanitary conditions left from United Nations peacekeepers from Nepal. Eight years later, cholera cases are still prevalent, although numbers have declined recently due to aid from private organizations (Dowell, S.F. et al 2011, Katz, J.M. 2013). However, with climate-related increases in ocean water temperatures, scientists expect hurricanes to intensify and increase damage to developing countries (Center for Climate and Energy Solutions. n.d.). Natural disasters promote the spread of waterborne illness by isolating people from safe drinking water and destroying public health infrastructure such as happened with the cholera outbreak in Haiti (Funari, E. et al 2013). To prevent future waterborne disease epidemics in such areas with limited resources, it would be beneficial to improve environmental surveillance through development of rapid, reliable, and portable detection methods for waterborne pathogens. The advent of high-throughput sequencing technologies has enabled the detection and characterization of microbial communities in their natural environments, an approach known as metagenomics. Metagenomic sequencing, unlike more traditional PCR methodologies, is capable of sequencing thousands of organisms in a sample. This metagenomic shotgun sequencing approach detects the abundance of microbes and bacterial diversity in the environment (Illumina, n.d.). The Oxford Nanopore MinION is a shotgun sequencing device that is optimal for portable, rapid detection of the microbial diversity in an environmental sample (Oxford MinION, n.d.). This handheld device has enormous potential for field use in emergency preparedness and disease response, particularly in developing countries where more advanced analytical equipment may be inaccessible due to lack of facilities or damaged infrastructure. Having access to quick, infield assessment technology for rapidly emerging outbreaks would be beneficial to a disease-specific public health response. Current protocols recommend that DNA is extracted from environmental samples as rapidly as possible after collection. If cooling is available with an insulated ice chest, samples may be transported/stored for periods ranging from 6 to 24 hours. The shorter timeframes minimize unwanted shifts in microbial structure (U.S. Geological Survey, 1997, WHO, n.d.). Access to cold storage in remote areas is unlikely, and the use of liquid preservation methods could assist in maintaining quality of DNA, and hence produce more accurate data in metagenomic analyses. In the absence of cold storage facilities, infield filtration coupled with preservation techniques are necessary to maintain samples integrity for transport to laboratory facilities. This thesis aimed to develop an infield filtration and sequencing protocol, coupled with the Oxford Nanopore MinION sequencing platform, to identify the potential bacteria, viruses, protozoa, fungi, antimicrobial resistance (AMR), pathogenic strains, and virulence associated genes for use in remote locations. Five locations across Lake Warner, Massachusetts were used for method development, coupled with Millipore Sterivex filters for field filtration to determine the most effective method for sample preparation in remote locations. Additionally, a chemical preservation method was assessed using dimethyl sulfoxide, disodium EDTA, and saturated NaCl (DESS). A study by Gray et al, found that liquid preservation methods (DNAgard, RNAlater, and DESS) outperformed the card-based preservatives (FTA cards and FTA Elute cards) in terms of bacterial recovery (Gray, M.A., et al 2013). DESS was selected for investigation in this thesis because of the low cost compared to the other liquid-based preservatives. Lake Warner in Hadley, Massachusetts, which is heavily used for fishing and boating activities, flows into the Connecticut River via the Mill River. Historically, the lake experienced high Escherichia coli (E. coli) levels due to pollution from primary effluent released in the 1950s from the Amherst Wastewater Treatment Plant (Johnson, J., 2015). Similar to Vibrio cholerae, E.coli spp is a waterborne bacteria caused by fecal contamination. Although most E.coli are natural inhabitants of the gastrointestinal tract, pathogenic serotypes can result in severe complications in vulnerable populations such as kidney failure in children and the elderly adults. (Todar, K., 2012). Lake Warner was chosen for the method development because of its history of E. coli pollution and recreational traffic as well as its general accessibility for study. Designing a methodology for rapid detection of pathogenic bacteria using a metagenomic approach could help improve surveillance for environmental pathogens that pose future epidemic risk. These tools are becoming increasingly important for prediction and response to waterborne diseases as climate impacts increase the frequency, intensity, and duration of extreme weather events that damage critical infrastructure for vulnerable populations (van Aalst, M.K. 2006).

This diploma thesis deals with taxonomy independent methods for classification of metagenomic signals, aquired by a MinION sequencer. It describes the formation and character of metagenomic data and already existing methods of metagenomic data classification and their development. This thesis also evaluates an impact of the third generation sequencing techniques in the world of metagenomics and further specialises on the function of the Oxford Nanopore MinION sequencing device. Lastly, a custom method for metagenomic data classification, based on data obtained from a MinION sequencer, is proposed and compared with an already existing method of classification.

Since the soils nutrient composition along with the associated biotic and abiotic factors direct the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies have been focused on sediment types with unique features characteristic of extreme environments. Red Sea lagoon ecosystems are environments with such unique features as they are highly saline. However, not much is known about the potential of their microbiomes to produce bioactive compounds. Here, we explored sediment types such as mangrove mud, microbial mat, and barren soil collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL) as sources for antibiotic bioprospecting. Our antibiotic bioprospecting process started with a metagenomic study that provides a more precise view of the microbial community inhabiting these sites and serves as a preliminary screen for potential antibiotics. Taking the outcomes of the metagenomic screening into account, the next step we established a library of culturable strains from the analyzed samples. We screened each strain from that library for antibiotic activity against four target strains (Staphylococcus aureus ATCC 25923, Escherichia coli dh5 α, Pseudomonas syringae pv. tomato dc3000 and Salmonella typhimurium dt2) and for the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes known to support synthesis of secondary metabolites that act like antimicrobial agents. The metagenomics study showed a shift in dominant phyla consistent with a historical exposure to hydrocarbon contamination and that AKL unexpectedly displayed more contamination than RHL. This may be due to dominant phyla in AKL being consistent with early hydrocarbon exposure (when contamination levels are still high) and the dominant phyla in RHL being consistent with late hydrocarbon exposure (when contamination levels are lower as a result of an extended period of hydrocarbon degradation). Additionally, RHL samples showed a higher percentage of enzymes associated with antibiotic synthesis, PKS and NRPS. When considering sediment type, mangrove mud samples showed a higher percentage of enzymes associated with antibiotic synthesis than microbial mat samples. Taken together, RHL was shown to be the better location with an increased probability of successful antibiotic bioprospecting, while the best sediment type in RHL for this purpose is microbial mat. Moreover, the phylum Actinobacteria tends to be the common target for research when it comes to antibiotic bioprospecting. However this culture-independent metagenomic study suggests the tremendous potential of Proteobacteria, Bacteroidetes, Cyanobacteria and Firmicutes for this purpose. Taking into account that the metagenomic screen suggests other phyla beyond Actinobacteria for antibiotic bioprospecting, the culture-dependent experiments were not designed to target actinobacteria alone. A total of 251 bacterial strains were isolated from the three collected sediments. Phylogenetic characterization of 251 bacterial isolates, based on 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity of ≤ 98 % to other strain sequences in the NCBI database. We demonstrate that 52 of the 251 isolates exhibit the potential to produce an antimicrobial effect. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 52 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards all target strains. In conclusion, this study demonstrated the significant microbial diversity associated with Red Sea harbor/lagoon systems and their potential to produce antimicrobial compounds and novel secondary metabolites. To the best of our knowledge, this is the first study that has analyzed the microbiomes in Red Sea lagoons for antibiotic bioprospecting.

No description available.

No description available.

<p>Microbial metagenomics is the exploration of taxonomical diversity of microbial communities in environmental habitats using large, exhaustive DNA sequence datasets. However, due to inherent limitations of sequencing technology and the complexity of environmental genomes, current analytical approaches do not reveal the existence of all microbes that may be present. In this study, a new classification approach is proposed based upon unique proteins that are specific for different clades of Alphaproteobacteria to predict the presence and absence of species from these groups of bacteria in published metagenomic datasets. In this work, 264 previously–identified, published conserved signature proteins (CSPs) characteristic of individual taxonomic clades of Alphaproteobacteria are used as probes to detect the presence of bacteria in metagenomic datasets. Although public genome sequence information has increased manifold since these CSPs were initially identified 6 years ago, results indicate that nearly all of these CSPs (259 of 265) are specific for their previously characterized clades. Furthermore, they are confirmed to be present in the newly–identified and sequenced members of these clades. In view of their specificity and predictive ability in different monophyletic clades of Alphaproteobacteria, the sequences of these CSPs provide reliable probes to determine the presence or absence of these Alphaproteobacteria in metagenomic datasets. In this work, CSPs are used to determine the presence of Alphaproteobacteria diversity in 10 published metagenomic datasets (bioreactor, compost, wastewater, activated sludge, groundwater, freshwater sediment, microbial mat, marine, hydrothermal vent and whale fall metagenomes), which cover diverse environment and ecosystems. It is indicated that the BLAST searches with these CSPs can be used to efficiently identify Alphaproteobacteria species in these metagenome dataset and substantial differences can be determined in the distribution and relative abundance of different Alphaproteobacteria species in the tested metagenome datasets. Thus the CSPs, which are specific for different microbial taxa, provide novel and powerful means for identification of microbes and for their taxonomic profiling in metagenomic datasets.</p> / Master of Science (MSc)