Publication: METAGENÓMICA COMO UNA HERRAMIENTA PARA LA EVALUACIÓN DE LA PRESENCIA DE FAGOS EN AMBIENTES RICOS EN BACTERIAS SULFORREDUCTORAS Y SUS IMPLICANCIAS ECOLÓGICAS
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Date
2018
Authors
VALENCIA ALBORNOZ, RICARDO GABRIEL
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Abstract
En las últimas décadas, la metagenómica, el estudio de las secuencias de ADN ambiental, ha surgido como una alternativa para identificar la presencia de especies microbianas y comprender las interacciones entre éstas en comunidades complejas. En el desarrollo de esta técnica, las secuencias de ADN identificadas desde una muestra medioambiental pueden provenir de diferentes tipos de organismos, tales como eucariontes, procariontes y virus. La integración de la información obtenida en estos estudios permite generar una amplia gama de insumos con el fin de incrementar el conocimiento sobre las comunidades microbianas estudiadas que van desde la ponderación de abundancias relativas de especies microbianas, hasta la reconstrucción de novo de genomas de microorganismos no cultivables.Estudios basados en herramientas metagenómicas han reconocido que los virus son los organismos más abundantes y más diversos en el planeta. Su importancia no solo radica en su cantidad, sino también en que éstos son capaces de afectar de forma significativa procesos biogeoquímicos a escala global. En la actualidad, la comunidad científica ha puesto un particular énfasis en el rol ecológico de bacteriófagos o fagos, virus que afectan a bacterias, los cuales pueden modular la composición de las comunidades microbianas presentes en un determinado hábitat. Adicionalmente, los fagos forman un reservorio de nuevos genes, que pueden ayudar, tanto al fago como a su hospedero, a garantizar su permanencia y adaptación en el ambiente.La presente investigación tuvo como objetivo desarrollar una metodología capaz de identificar aquellos fagos más predominantes encontrados en medioambientes acuáticos naturales no intervenidos y comprobar si estos fagos presentan características genéticas similares. La integración de esta información entrega elementos valiosos para evaluar el rol ecológico de los fagos a nivel de comunidad microbiológica compleja, y cuáles son sus efectos a nivel de ecosistema.Durante el desarrollo de la memoria, se estimó la abundancia relativa y prevalencia (presencia) de fagos en 420 metagenomas acuáticos, compuestos por 140 metagenomas de origen marino, 140 genomas de origen estuarino y 140 genomas correspondientes a muestras de agua dulce. Esta estimación se realizó utilizando dos bases de datos de genes exclusivos de fagos, Phantome y Uniprot.En los metagenomas seleccionados, la abundancia relativa de los fagos permitió discriminar una fracción de fagos que posee un alto índice de abundancia, a los cuales se le atribuye una mayor tasa de replicación (fracción “activa”), de un grupo que posee abundancias relativas menores, conocidos como fracción “latente”. Esta fracción está asociada principalmente a fagos con tendencia a lisogenia o con una alta especificidad para infectar bacterias poco abundantes.Además, se confirmó que hay una estrecha relación entre abundancia relativa de un fago en un bioma y su prevalencia, es decir, estar presente en una mayor cantidad de muestras. Esto indica que los mecanismos que permiten a los fagos propagarse, también le proveen a éstos adaptación a diferentes ambientes. Respecto a la diversidad de bacteriófagos encontrada en los metagenomas, , Caudavirales, correspondientes a los virus que formados por una cápside, es el orden que domina en los fagos más abundantes, siguiendo la secuencia de mayor a menor, Siphoviridae, Myoviridae y Podoviridae.Durante este estudio se identificó que los bacteriófagos más abundantes en los tres biomas acuáticos está dominado por una gran cantidad de bacteriófagos incultivables, los cuales han sido previamente descritos solo por herramientas metagenómicas. Estos fagos fueron extraídos de muestras marinas y son un grupo poco explorado que, según el análisis, tiene una preponderancia única en estos sistemas. De los fagos descritos como cepas preciamente aisladas, cabe destacar la presencia de cianofagos como Planktothrix phage PaV-LD, Synechococcus phage S-SM2 y Prochlorococcus phage P-SSM2, explicado por la presencia de sus huéspedes, las cianobacterias, en estos ambientes y virófagos, como el virófago del Lago Orgánico, cuyas consecuencias en la dinámica fago-bacteria aún no son totalmente comprendidas.Con respecto a genes de bacteriófagos, se encontró un comportamiento similar al de abundancia, prevalencia y diversidad de fagos, sin la aparición del sesgo entre ambas bases de datos. Adicionalmente, estos resultados permiten inferir que aquellos genes que posean alta abundancia y prevalencia, corresponderían a genes cuya función confiere alguna ventaja a la comunidad viral.Junto al análisis anterior, se construyó redes de similitud de secuencias de genes utilizando como base los 100 genotipos más abundantes en el ambiente marino. Estas redes permiten visualizar las distintas familias génicas presentes y así atribuir que familias son las que confieren a los fagos mayor abundancia (genes en la fracción activa). Los resultados de este análisis produjo un set de clústeres génicos ligados a proteínas estructurales de cola, helicasa, que está involucrada en la replicación del ADN y una enzima oxigenasa (2-Fe-oxoglutarato oxigenasa) que participa en reacciones redox dentro de la célula del huésped y que puede tener un papel en la reparación de ADN viral. Sin embargo, al tratar de contrastar estas familias génicas con la abundancia relativa de cada uno de representantes y con la familia viral de donde provienen, no se pudo determinar una correlación positiva entre estas características. Es decir, no hay vínculo aparente que enlace la abundancia de cierto gen con la abundancia de un taxón en particular o la abundancia de un gen filogenéticamente cercano.Para conocer si los profagos de una cierta comunidad bacteriana, en este caso bacterias sulforreductoras, están representados en muestras ambientales, se les calculó la abundancia relativa en los metagenomas provenientes de los tres biomas. Dentro de la lista de profagos más abundantes en ambientes marinos resaltan aquellos pertenecientes a una bacteria del género Desulfobacula, que tiene 15 representantes de 46 profagos en la lista. Su ubicuidad y cantidad representantes hace que estos profagos sean candidatos para aplicaciones posteriores, como también obtener un mayor entendimiento de la ecología de esta comunidad y su modificación en este grupo de biomas.En conjunto, los resultados indican que un cierto grupo de fagos es altamente abundante y prevalente en el ambiente acuático. Sin embargo, es difícil especificar características genéticas que expliquen por qué son abundantes a este nivel, según lo indicado por las redes de similitud y árboles aumentados. Sin embargo, se reconoce que estos fagos, además de ser poco explorados, juegan un rol importante tanto en la modulación de comunidades complejas, como también la regulación de ciclos biogeoquímicos globales en ambientes acuáticos.
In the last decades, metagenomics, the study of environmental DNA sequences, has emerged as an alternative for the understanding of interaction in complex microbial communities. Sequences in a sample come from different types of organisms, like eukaryotes, prokaryotes and virus. It possible to extract a variety of information from a metagenomic sample. Examples are estimations of abundance relative to specie or genera, and de novo reconstruction of genomes belonging to uncultivable microorganisms.Through metagenomics, it has been recognized that viruses are the most abundant organisms on Earth. Its importance lies not only in quantity but also in the fact that they are capable of significantly affecting biogeochemical processes on a global scale. Currently, the scientific community has placed particular emphasis on the ecological role of bacteriophages or phages, viruses that affect bacteria, which can modulate the composition of microbial communities present in a given habitat. Additionally, the phages form a reservoir of new genes, which can help both the phage and its host to ensure its permanence and adaptation in the environment.The bacteriophages are grouped mainly in three families: Myoviridae, Siphoviridae and Podovodirae, all belonging to the order Caudavirales, corresponding to the viruses formed by a capsid, where the genetic material is found, and a tail made of proteins. The genetic composition of these viruses has been studied, revealing difficulties in linking taxonomic classification with special genomic characteristics. Because viruses don’t possess a set of marker genes which are conserved throughout evolution, viral metagenomics becomes an alternative to explore in depth the gene content of phages in diverse environments.Our research aimed to develop a methodology capable of identifying those most predominant phages found in natural aquatic environments (not intervened) and to verify if these phages present similar genetic characteristics. The integration of this information provides valuable elements to make conclusions at an ecological level, related to the influence of the viral community on the ecosystem.During the development of the project, the relative abundance and prevalence (presence) of phage were estimated in 420 aquatic metagenomes, composed of 140 metagenomes of marine origin, 140 genomes of estuarine origin and 140 genomes corresponding to freshwater samples. For the estimation, two exclusive phage gene databases were used.In the selected metagenomes, the relative abundance of the phages allows the identification of a fraction of phage with a high abundance index, related to higher rates of replication ("active" fraction) and a group with lower relative abundances are attributed, known as the "latent" fraction. This fraction is mainly associated with phage with a tendency to lysogenia or with a high specificity to infect low abundant bacteria.It was also confirmed that there is a close relationship between relative abundance of a phage in a biome and its prevalence, that is, to be present in a greater number of samples. This indicates that the mechanisms that allow the phage to spread, also provide adaptation to different media. Regarding the diversity of bacteriophages found in metagenomes, this index is very similar when comparing the three groups. It should be noted that there are differences when using a database on the other for the calculation of diversity, due to biases related to the content of the database itself, which does not occur in the estimation of abundance.With this information, tables can be constructed with the names of the most abundant bacteriophages in the three aquatic biomes. At the top of the table, a large number of uncultivable bacteriophages obtained from metagenomic samples appear. These phage were extracted from marine samples and are a little explored group that, according to the analysis, has a unique preponderance in these systems. Of the described phage, the presence in the list of cyanophages such as Planktothrix phage PaV-LD, Synechococcus phage S-SM2 and Prochlorococcus phage P-SSM2 is noteworthy, explained by the presence of their hosts, cyanobacteria, in these environments and in the case of the virophages, like Organic Lake virus, whose consequences in the dynamic phage-bacterium are not yet fully understood.With regard to bacteriophage genes, the same behavior was found with respect to abundance, prevalence and diversity of genes, without the appearance of bias between both databases. This confirms the nature of the gene to register a certain level of abundance in an environment, depending on whether it confers adaptation to a population. Thus, a classification of genes similar to the "active" and "latent" fraction in phages can be performed.Together with the previous analysis, networks of similarity of gene sequences were constructed using as base the 100 most abundant genotypes in the marine environment. These networks allow us to visualize the different gene families present and thus attribute which families are those that confer phage greater abundance (genes in the active fraction). We observed gene clusters linked to structural tail proteins, helicase that is involved in DNA replication and an oxygenase enzyme (2-Fe-oxoglutarate oxygenase) that participates in redox reactions within the host cell and that may play a role in the repair of viral DNA.However, when trying to contrast these gene families with the relative abundance of each of the representatives and the viral family from which they originate, it wasn’t possible to determine a positive correlation between these characteristics, that is, there is no apparent link between the abundance of a certain gene with the abundance of a particular taxon or the abundance of a phylogenetically close gene.To know if the prophages of a certain bacterial community, in this case sulphate reducing bacteria, are represented in environmental samples, the relative abundance in the metagenomes coming from the three biomes was calculated. Within the list of most abundant prophages in marine environments those belonging to a bacterium of the Desulfobacula genus stand out, which has 15 representatives of 46 prophets in the list. Its ubiquity and number of representatives makes these prophages candidates for later applications.Overall, the results indicate the presence of a certain group of highly abundant phages in the aquatic environment, however, it is difficult to specify genetic characteristics that explain why they are abundant at this level, as indicated by similarity networks and augmented trees. However, it is recognized that these phage, besides being little explored, play an important role in aquatic environments. Taking only the community of sulphate reducing bacteria, there are prophages that also stand out in terms of abundance and that may be promising to understand the ecology of this community and its modification in this group of biomes.This reinforces previous studies that show the ubiquity of this pattern in the viral world and the need to continue looking for characteristics of the dominant phages on Earth.
In the last decades, metagenomics, the study of environmental DNA sequences, has emerged as an alternative for the understanding of interaction in complex microbial communities. Sequences in a sample come from different types of organisms, like eukaryotes, prokaryotes and virus. It possible to extract a variety of information from a metagenomic sample. Examples are estimations of abundance relative to specie or genera, and de novo reconstruction of genomes belonging to uncultivable microorganisms.Through metagenomics, it has been recognized that viruses are the most abundant organisms on Earth. Its importance lies not only in quantity but also in the fact that they are capable of significantly affecting biogeochemical processes on a global scale. Currently, the scientific community has placed particular emphasis on the ecological role of bacteriophages or phages, viruses that affect bacteria, which can modulate the composition of microbial communities present in a given habitat. Additionally, the phages form a reservoir of new genes, which can help both the phage and its host to ensure its permanence and adaptation in the environment.The bacteriophages are grouped mainly in three families: Myoviridae, Siphoviridae and Podovodirae, all belonging to the order Caudavirales, corresponding to the viruses formed by a capsid, where the genetic material is found, and a tail made of proteins. The genetic composition of these viruses has been studied, revealing difficulties in linking taxonomic classification with special genomic characteristics. Because viruses don’t possess a set of marker genes which are conserved throughout evolution, viral metagenomics becomes an alternative to explore in depth the gene content of phages in diverse environments.Our research aimed to develop a methodology capable of identifying those most predominant phages found in natural aquatic environments (not intervened) and to verify if these phages present similar genetic characteristics. The integration of this information provides valuable elements to make conclusions at an ecological level, related to the influence of the viral community on the ecosystem.During the development of the project, the relative abundance and prevalence (presence) of phage were estimated in 420 aquatic metagenomes, composed of 140 metagenomes of marine origin, 140 genomes of estuarine origin and 140 genomes corresponding to freshwater samples. For the estimation, two exclusive phage gene databases were used.In the selected metagenomes, the relative abundance of the phages allows the identification of a fraction of phage with a high abundance index, related to higher rates of replication ("active" fraction) and a group with lower relative abundances are attributed, known as the "latent" fraction. This fraction is mainly associated with phage with a tendency to lysogenia or with a high specificity to infect low abundant bacteria.It was also confirmed that there is a close relationship between relative abundance of a phage in a biome and its prevalence, that is, to be present in a greater number of samples. This indicates that the mechanisms that allow the phage to spread, also provide adaptation to different media. Regarding the diversity of bacteriophages found in metagenomes, this index is very similar when comparing the three groups. It should be noted that there are differences when using a database on the other for the calculation of diversity, due to biases related to the content of the database itself, which does not occur in the estimation of abundance.With this information, tables can be constructed with the names of the most abundant bacteriophages in the three aquatic biomes. At the top of the table, a large number of uncultivable bacteriophages obtained from metagenomic samples appear. These phage were extracted from marine samples and are a little explored group that, according to the analysis, has a unique preponderance in these systems. Of the described phage, the presence in the list of cyanophages such as Planktothrix phage PaV-LD, Synechococcus phage S-SM2 and Prochlorococcus phage P-SSM2 is noteworthy, explained by the presence of their hosts, cyanobacteria, in these environments and in the case of the virophages, like Organic Lake virus, whose consequences in the dynamic phage-bacterium are not yet fully understood.With regard to bacteriophage genes, the same behavior was found with respect to abundance, prevalence and diversity of genes, without the appearance of bias between both databases. This confirms the nature of the gene to register a certain level of abundance in an environment, depending on whether it confers adaptation to a population. Thus, a classification of genes similar to the "active" and "latent" fraction in phages can be performed.Together with the previous analysis, networks of similarity of gene sequences were constructed using as base the 100 most abundant genotypes in the marine environment. These networks allow us to visualize the different gene families present and thus attribute which families are those that confer phage greater abundance (genes in the active fraction). We observed gene clusters linked to structural tail proteins, helicase that is involved in DNA replication and an oxygenase enzyme (2-Fe-oxoglutarate oxygenase) that participates in redox reactions within the host cell and that may play a role in the repair of viral DNA.However, when trying to contrast these gene families with the relative abundance of each of the representatives and the viral family from which they originate, it wasn’t possible to determine a positive correlation between these characteristics, that is, there is no apparent link between the abundance of a certain gene with the abundance of a particular taxon or the abundance of a phylogenetically close gene.To know if the prophages of a certain bacterial community, in this case sulphate reducing bacteria, are represented in environmental samples, the relative abundance in the metagenomes coming from the three biomes was calculated. Within the list of most abundant prophages in marine environments those belonging to a bacterium of the Desulfobacula genus stand out, which has 15 representatives of 46 prophets in the list. Its ubiquity and number of representatives makes these prophages candidates for later applications.Overall, the results indicate the presence of a certain group of highly abundant phages in the aquatic environment, however, it is difficult to specify genetic characteristics that explain why they are abundant at this level, as indicated by similarity networks and augmented trees. However, it is recognized that these phage, besides being little explored, play an important role in aquatic environments. Taking only the community of sulphate reducing bacteria, there are prophages that also stand out in terms of abundance and that may be promising to understand the ecology of this community and its modification in this group of biomes.This reinforces previous studies that show the ubiquity of this pattern in the viral world and the need to continue looking for characteristics of the dominant phages on Earth.
Description
Catalogado desde la version PDF de la tesis.
Keywords
BACTERIAS SULFORREDUCTORAS , BACTERIOFAGOS , MUESTRAS METAGENOMICAS ACUATICAS