Tipificación y caracterización genómica y morfológica de virus microbianos presentes en econichos acídicos industriales.
Conicyt InstrumentBecas Nacionales
AuthorCovarrubias Pizarro, Paulo Cesar
Viruses are the most abundant biological entities known and represent the largest reservoir of uncharacterized genetic diversity on Earth. Their interactions with microbial hosts represent one of the most frequent biological associations in ecology, and yet they have not been adequately addressed in most envronmentes, acidic industrial econiches linked to bioleaching (Chile ́s main biotechnology) included. While lytic phages acts as the primary a... Ver más
Viruses are the most abundant biological entities known and represent the largest reservoir of uncharacterized genetic diversity on Earth. Their interactions with microbial hosts represent one of the most frequent biological associations in ecology, and yet they have not been adequately addressed in most envronmentes, acidic industrial econiches linked to bioleaching (Chile ́s main biotechnology) included. While lytic phages acts as the primary agents of microbial mortality, and thus as drivers of nutrient and energy cycling, lysogenic phages confer morphological or metabolic properties to their hosts, that frequently increase host fitness. Due to their ability to modulate microbial communities (by selective lysis) and for their role as agents of horizontal gene transfer (transduction), viruses have an important applied potential. In this thesis the study of viruses that could affect the survival and biological fitness of bioleaching microorganisms was addressed, aiming to gain insights into the role of viruses in the dynamics of microbial acidophiles and their effect on the stability of the biotechnological process directed by the host microbes. For this purpose, the presence, diversity and nature of free viruses in industrial bioleaching liquors and of temperate viruses or proviruses in metagenomic and genomic datasets derived from these environments was assessed. Also, functional aspects of the biology of a virus (designated AcaML1) that infects a relevant bioleaching model microorganism (Acidithiobacillus caldus ATCC 51756T) were investigated. Using filtration and concentration strategies, the subcellular fraction in acidic solutions obtained from various industrial bioleaching environments in Chile was recovered (heap, dump and reactor). In all tested cases, DNA-containing particles, ranging in size between 50 and 200 nm were obtained at concentrations of 106 to 108 particles/L, depending on the system. The estimated particle concentration was found to be 10 to 100 times higher than that of the microorganisms present. However, only a minor fraction of the particles recovered had viral morphotypes according to transmission electron microscopy studies, and a significant proportion of accompanying vesicular structures were detected in all samples. Given the low yield of free viruses, an exhaustive search of proviruses associated with acidophilic microbial communities and isolates was carried out, using public and private metagenomic and genomic data recovered from industrial acidic environments. In both datasets, viral sequences (Siphoviridae, Podoviridae, Myoviridae, Inoviridae) were identified, although in low proportions. Viral segments recovered were generally small (between 5 and 31 Kb), lacked viral morphogenetic signature genes and contained few genes typically associated with chimeric MGEs (e.g. ICEAfe1 of Acidithiobacillus ferrooxidans ATCC 23270T). Even if no complete virus could be reconstructed from the metagenomes analysed, potentially complete Caudoviralea proviruses were recovered from the genomes of Thiomonas sp. 3As and Acidithiobacillus caldus ATCC 51756T. Within the group of acidophilic bacteria analysed these few proviruses found were rather an exception than a norm. The 59 Kb-long provirus identified in the genome of strain ATCC 51756T, designated Acidithiobacillus caldus Myovirus Like 1 or AcaML1, encoding 74 open reading frames and organized modularly, was found to occur in other strains of the species, conserved in shape and sequence. DNA damage proved to induce AcaML1 out of the genome, giving rise to an episomal replicative intermediary and to icosahedral VLPs. Invariably, the recovered particles showed absence of the tail and tail fibers characteristic of the Myoviridae, and proved unable to lyse its host or an alternative (provirus negative) "sensitive" strain. The acidophiles investigated in this thesis carried CRISPR loci present in their genomes in proportions comparable to those of bacteria and archaea from other ecological niches. The proportion of spacers with sequence identity to protospacers of viral origin (all directed against viruses of the Caudovirales order) turned out to be low, in agreement with the other results obtained in this thesis. Taken together, these results point to the almost null presence of free viruses and integrated proviruses in industrial acidic econiches. Despite this, the identification of AcaML1 provirus in the genome of A. caldus ATCC 51756T (and other strains of the species) provides the basis for future fundamental and applied studies of significance to the area of mining biotechnology. Ver menos
InstitutionUNIVERSIDAD ANDRES BELLO
Date de publicación2019
QUATRINI NYQVIST, Raquel