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GENERAL RESEARCH GUIDELINES
Community genetic diversity as a public good for evolution of innovative functions
Comparative genomics suggests that a large fraction of the metabolic genes present in most bacterial genomes have been horizontally exchanged. The higher number of HGT cases among physiological and overlapping niche-dependent bacteria suggests that ecological interactions are a key factor favoring gene exchange in bacteria. However, most HGT studies have been conducted by analyzing organisms that did not coexist in the exact same habitat, confounding the identification of the putative donor, the time scale of the event, and the functional relevance of the exchange. I believed that a better understanding of the role of ecology in HGT requires a more detailed examination of microbial interactions on a temporal and spatial scale. My previous work suggests that cooperative adaptations (e.g., syntrophism) may be facilitated by HGT, however, how genomes change and adjust to these conditions is unknown. To address how novel functions evolve, I will study the cooperative degradation of newly synthesized chemical compounds (e.g. chlorinated compounds, artificial sweeteners) in different systems (e.g. bioreactor, human microbiomes). Using non-disruptive sampling technologies (e.g. sucrose gradients), stable isotope probing, physicochemical quantification, metagenomics and transcriptomics, my goal is to elucidate the effect of genomic changes in genome diversity that facilitate cooperative interactions. This work will have a direct impact on our understanding of how microbial communities assemble and how interaction within an environment leads to the evolution of functions (metabolic pathways and services).
The genomic basis for cooperation in bacteria
Degradation of most compounds requires the cooperation of phylogenetically unrelated bacteria. While these processes are relevant to understanding how microbial communities assemble and evolve, how recognition and complementation mechanisms arise is unknown. My previous work suggests that cooperative adaptations (e.g., syntrophism) may be facilitated by HGT, however, how genomes change and adjust to these conditions is unknown. To address how these mechanisms evolve, I will study the cooperative degradation of newly synthesized chemical compounds (e.g. chlorinated compounds, artificial sweeteners) in different systems (e.g. bioreactor, human microbiomes). Using non-disruptive sampling technologies, stable isotope probing, physicochemical quantification, metagenomics and transcriptomics, my goal is to elucidate genomic changes that facilitate cooperative interactions. This work will have a direct impact on our understanding of how microbial communities assemble and how interaction within an environment leads to the evolution of functions (metabolic pathways and services).
Tools for exploring fine-level diversity in microbial communities
Sequencing of universal genetic markers, such as the 16S rRNA gene, is the de facto molecular tool for the identification of bacterial composition in studies of microbial communities. However, due to their high conservation, these markers are not suitable for accurately differentiating between more recently diverged lineages and, therefore, the contribution of this diversity remains unknown. Assessment of such fine-level diversity has become ecologically and evolutionarily important, as lineages within the same species may have differences in evolutionary history, gene content, function, and ecology. Exploration of this fine-level diversity has been scarce due to the laborious search and extraction of gene orthologs present in specific taxonomic groups and the difficulty of finding low-redundancy primers that can efficiently amplify the homologous region. To overcome these limitations, we present a python-based application, called PROTÉGÉ (PROTEIN coding genes for high-resolution phylogenetic labeling), which integrates automatic search and extraction of orthologous genes with in-silico design, selection and evaluation of low-degeneracy primers. for high-throughput sequencing.
SPECIFIC RESEARCH PROJECTS
Evolution of interactions between the endophytic microbiota of Theobroma cacao
Relationship of cocoa (Theobroma cacao L.) seed microbial endophyte diversity with germination and grafting of regional materials from Tumaco, Nariño.
FUNDED PROJECT (UNAL-AGROSAVIA)
The Pacific region of the department of Nariño has a high potential for the development of cocoa cultivation, due to optimal agroclimatic conditions and high genetic diversity of regional materials. However, currently the use of cocoa diversity in the region faces several obstacles, one of these is the lack of propagation studies that allow the multiplication of these materials, which is reflected as one of the highest priority demands of the sector to develop and adjust schemes for mass propagation of regional cocoa material (http://www.siembra.co/).
Regional materials are adapted to the environmental conditions of the region; however, their propagation is difficult due to their low percentage of grafting. Microbial endophytes can affect plant development and growth; therefore, the analysis of the diversity of endophytes in cocoa tissues and their use as inoculants is an alternative to modulate seedling activity and improve the propagation of regional materials. Since the cocoa produced in Tumaco is considered of high quality, the development of propagation technologies for regional materials will strengthen the production of fine and aromatic cocoa, which offers a better price for the farmer.
This research proposal seeks, first, to explore the differences and similarities of the endophytic communities (fungi and bacteria) of regional materials and universal cocoa rootstocks, in order to determine if there is a relationship between these endophytic communities and grafting success. Second, it seeks to isolate endophytic microorganisms with growth-promoting capacity and evaluate their effect on the grafting success of regional cocoa materials. This knowledge will allow the development of technologies to improve the propagation schemes of materials from the department of Nariño.
Diversity of the microbiome associated with wild plants of Theobroma cacao L. and sister species in the Amazon and Choco regions.
FUNDED PROJECT (COLCIENCIAS)
The study of the communities of microorganisms associated with different plant tissues has gained enormous interest in the agricultural area due to the growing evidence that these communities can affect the development, productivity and resilience of crops. These communities play in many cases important roles, for example in tissue protection against pathogens, nutrient fixation and translocation, drought resistance and resistance to herbivory. This is because the plant-associated habitat is a dynamic environment, which affects the structure and composition of microbial species colonizing roots, stems, branches, fruits and leaves. In the case of endophytic communities, they vary spatially in the plant, or may depend on the interaction with other endophytic or phytopathogenic microorganisms. These endophytic microbial populations can increase host plant health by enhancing tolerance to heavy metals and drought, reducing the establishment of herbivores or phytopathogens, and promoting plant growth.
In the case of Theobroma cacao, very little is known about the composition of the microbial communities associated with the plant under cultivated conditions and there are no reports on the composition under wild conditions. The lack of information on the presence of microorganisms under wild conditions is due to the fact that the parental plants that gave rise to the cultivated cocoa materials were extracted from their natural conditions and subsequently located in other areas of development and production. Therefore, determining the diversity of the cocoa microbiome under an evolutionary framework and under wild conditions offers a unique opportunity to explore, among other topics, the effect of ecosystem diversity on the composition of the cocoa microbiota, the effect of the evolutionary proximity of sister species and genera on the composition of the communities that colonize their tissues, and to determine patterns of coexistence within the microbial communities that can be associated with diseased and healthy tissues in wild and cultivated states.
Exploring the diversity and culturability of intestinal microbiomes of livestock animals.
Development of methodologies to separate and study rumen microorganisms in more depth.
The rumen microbial community is an important element in livestock health, nutrition, productivity and climate impact. Despite historical and current efforts to characterize this microbial diversity, many of its members remain unidentified, making it difficult to associate microbial groups with functions. In this project, we developed a low-cost methodology for the treatment of rumen samples that separates the microbial community on the basis of cell size, allowing the identification of subtle compositional changes. Briefly, the sample is centrifuged through a series of sucrose density gradients and the cells migrate to their corresponding density fraction. From each fraction, DNA is extracted and 16S rRNA gene amplicons are sequenced. We tested our methodology in four animals under two different conditions, fasting and post-feeding. Each fraction was examined by confocal microscopy showing that the same sucrose fraction consistently separated microorganisms of similar cell size regardless of animal or treatment. Analysis of microbial composition by metabarcoding revealed that our methodology detected bacterial families of low abundance and population changes between fasting and post-feeding treatments that could not be observed by bulk DNA analysis. In conclusion, the sucrose-based method is a powerful, low-cost approach to disentangle, enrich, and potentially isolate uncharacterized members of the rumen microbiome.
Development and evaluation of culture methodologies for the recovery of rumen microorganisms using metabarcoding.
The study of the rumen has been successful in advancing the understanding of how ruminal nutrition functions, however, part of this understanding must be linked to the ability to isolate and culture microbial populations in order to understand the structures and roles that occur in this ecosystem. The rumen microbiota is fundamental in cattle for the assimilation of plant material, however, the low cultivability hinders the understanding of its ecological function and biotechnological potential.
This project seeks to develop a cultivation strategy to improve the recovery of ruminal microorganisms. The strategy combines the evaluation of cultivation parameters with population analysis using metabarcoding. Among the objectives of this project was the evaluation of alternative culture methods for the isolation of rumen microorganisms, this objective was approached from the development of eight culture media, which have been described in the literature as suitable for the recovery of anaerobic microorganisms, in addition to the use of two variables; A dilution variable that has been described as a tool to favor poorly cultured populations and an incubation time variable, trying to represent and benefit the metabolism of microorganisms of slow growth or dependent on metabolites produced by others.
Characterization of the cecal bacterial microbiota of Cavia porcellus populations.
To characterize the cecal bacterial microbiota of Cavia porcellus populations in municipalities of the department of Nariño, in order to identify the factors that influence its structure and taxonomic composition.
It is clear that livestock systems have been the object of research due to their importance in food production; however, there are also less known species of small size (micro livestock) that are not conventional and have potential in protein production, such as Cavia porcellus, also known as guinea pig, guinea pig or guinea pig. This productive system has presented a limited progress in the study of its microbiome even though it is part of different regions of Latin America. However, in countries where this species is used as a pet or animal model, it has been reported that its intestinal microbiota was dominated by the Fila Bacteroidetes, Firmicutes, and an outstanding amount of Akkermansia with a function on mucin degradation.
In Colombia, the first and perhaps the only study of intestinal bacterial populations in guinea pigs was carried out by means of sowing and cultivation techniques, which recorded 86.4% of Gram-negative bacteria such as Shigella sp, Escherichia coli, Yersinia sp and 15.4% of Gram-negative bacteria such as Staphylococcus dorado and Staphylococcus viridans. Although these studies provide valuable information, they still lack relevant data for production systems, such as the presence of pathogenic bacteria circulating in the gastrointestinal system that can affect the health of the animal and the consumer.
Based on the above, and taking into account that C. porcellus consumes vegetable polysaccharides, used at the level of the caecum in fermentation processes producing organic acids by the action of cellulolytic enzymes, it is necessary to carry out studies to understand the microbiome of this region and the possible pathogenic agents that have not yet been fully identified. Finally, at the livestock level, the results of this study could contribute to the improvement of sanitary plans and thus increase the knowledge of the intestinal microbial composition of this species.
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