Implementación de una plataforma bioinformática para el análisis del plasmidoma microbiano en ambientes acuáticos contaminados

Abstract

The environmental plasmidome, encompassing the collective plasmids within an ecosystem, plays a key role in microbial adaptation and evolution. Plasmids, as mobile DNA molecules, enable the transfer of beneficial traits among bacteria, especially in response to environmental stresses. This study examines the plasmidome of Costa Rica’s heavily polluted Virilla River, impacted by urbanization and industrial activities. We analyzed water and sediment samples across various sites to understand how pollution affects plasmid diversity and characteristics. By employing protocols for plasmidic DNA extraction, high-throughput sequencing, and ad hoc bioinformatics pipelines, we discovered that contaminated sites exhibit increased plasmid diversity, with the highest species richness observed at the most polluted site during the rainy season. Different bioinformatics methods were applied at each step, enhancing the accuracy, reliability, and robustness of the results, which in turn provided more reliable biological insights. The most effective circular detection method identified 28,043 circular contigs, predominating plasmid hosts associated with Gammaproteobacteria. Our findings suggest that polluted urban environments may promote the formation of longer plasmidic contigs. The study also revealed a significant increase in antibiotic resistance genes (ARGs) at highly polluted sites, particularly in freshwater samples and during the rainy season. This heightened diversity in contaminated environments has implications for the dissemination of antibiotic and metal-resistance genes, as well as other adaptive traits within microbial communities. This research underscores the need to study plasmids in natural environments to better comprehend their ecological roles and potential risks in polluted ecosystems.El plasmidoma ambiental, que abarca todos los plásmidos presentes en un ecosistema, desempeña un papel fundamental en la adaptación y evolución de los microorganismos. Los plásmidos, como elementos móviles de ADN, facilitan la transferencia de rasgos beneficiosos entre bacterias, especialmente en respuesta a presiones ambientales. Para estudiar el plasmidoma, se emplearon diversos métodos bioinformáticos que mejoran la precisión, fiabilidad y solidez de los resultados, proporcionando conocimientos biológicos más confiables. La implementación de este protocolo bioinformático en la supercomputadora Kabré, junto con su disponibilidad en repositorios como GitLab, facilita el acceso de este recurso a la comunidad científica en Costa Rica. Aunque el protocolo fue diseñado inicialmente para estudiar el plasmidoma en muestras del río Virilla, también hizo posible analizar el plasmidoma en muestras de aguas profundas a nivel global, así como el viroma (totalidad de virus) en estas muestras. Es relevante destacar que el análisis del plasmidoma en el río Virilla, gravemente contaminado por la urbanización y las actividades industriales, permitió comprender cómo la contaminación influye en la diversidad y las características de los plásmidos en el agua y sedimento de diferentes sitios. Mediante protocolos de extracción de ADN adaptados y secuenciación de alto rendimiento, descubrimos que los sitios contaminados actúan como puntos críticos de diversidad plasmídica, lo cual tiene importantes implicaciones para la propagación de resistencia a antibióticos, metales y otros rasgos adaptativos en las comunidades microbianas. Este estudio subraya la importancia de investigar los plásmidos en entornos naturales para comprender mejor sus roles ecológicos y los riesgos potenciales en ecosistemas contaminados.Plasmids play a crucial role in facilitating genetic exchange and enhancing the adaptability of microbial communities. Despite their importance, environmental plasmids remain understudied, particularly those in fragile and underexplored ecosystems such as the deep-sea. In this paper we implemented a bioinformatics pipeline to study the composition, diversity, and functional attributes of plasmid communities (plasmidome) in 81 deep-sea metagenomes from the Tara and Malaspina expeditions, sampled from the Pacific, Atlantic, and Indian Oceans at depths ranging from 270 to 4005 m. We observed an association between depth and plasmid traits, with the 270-1000 m range (mesopelagic samples) exhibiting the highest number of plasmids and the largest plasmid sizes. Plasmids of Alphaproteobacteria and Gammaproteobacteria were predominant across the oceans, particularly in this depth range, which also showed the highest species diversity and abundance of metabolic pathways, including aromatic compound degradation. Surprisingly, relatively few antibiotic resistance genes were found in the deep-sea ecosystem, with most being found in the mesopelagic layer. These included classes such as beta-lactamase, biocide resistance, and aminoglycosides. Our study also identified the MOBP and MOBQ relaxase families as prevalent across various taxonomic classes. This research underscores the importance of studying the plasmidome independently from the chromosomal context. Our limited understanding of the deep-sea’s microbial ecology, especially its plasmidome, necessitates caution in human activities like mining. Such activities could have unforeseen impacts on this largely unexplored ecosystem.Deep-sea ecosystems remain poorly understood due to exploration challenges. Despite the advancements metagenomics has brought to our understanding of the ocean microbiome, the diversity and variability of marine viruses, particularly in the deep sea, are still not well characterized. For this study, raw reads from deep-sea shotgun sequencing samples were retrieved from Tara and Malaspina expeditions, encompassing depths from 270 to 4005 meters. A total of 64 samples containing viral reads were identified and analyzed through a comprehensive bioinformatics pipeline, including quality assessment, taxonomic classification, and metabolic annotation. The analysis reveals that microbial viral diversity significantly decreases with depth, with shallower waters exhibiting higher species richness and evenness. Furthermore, deep-sea viruses are predominantly novel, with a notable proportion of unclassified viral operational taxonomic units (vOTUs) with increasing presence of previously uncharacterized viral species at greater depths, emphasizing the underexplored reservoir of microbial viral diversity in these enviroment. Additionally, a higher abundance of auxiliary metabolic genes (AMGs) was observed at shallower depths, indicating potential roles in host metabolism and adaptation. Our findings underscore the impact of depth on microbial viral diversity and community composition in deep-sea and highlight the need for further exploration to fully understand their complexity and ecological roles.The environmental plasmidome, encompassing all plasmids present in an ecosystem, plays a crucial role in the adaptation and evolution of microorganisms. Plasmids, as mobile DNA elements, facilitate the horizontal transfer of beneficial traits among bacteria, particularly in response to environmental pressures. To study the plasmidome, various bioinformatics methods were employed to enhance the precision, reliability, and robustness of results, thereby providing more accurate biological insights. The implementation of this bioinformatics protocol on the Kabré supercomputer, along with its availability in repositories such as GitLab, ensures accessibility to the scientific community in Costa Rica. Although initially designed to examine the plasmidome in samples from the Virilla River, this protocol also enabled the investigation of plasmidomes in global deep-sea samples and facilitated the analysis of the virome (totality of viruses) in these samples. The analysis of the Virilla River plasmidome, a system heavily impacted by urbanization and industrial activities, revealed how pollution affects the diversity and characteristics of plasmids in water and sediment across various sites. Using optimized DNA extraction protocols and high-throughput sequencing, the study identified polluted sites as hotspots of plasmid diversity. This finding has profound implications for understanding the spread of antibiotic resistance, metal tolerance, and other adaptive traits within microbial communities. This study highlights the importance of investigating plasmids in natural environments to better understand their ecological roles and to assess the potential risks they pose in contaminated ecosystems.