Identification of novel biosynthetic gene clusters and bacterial taxa from a soil microbiome using metagenomic approaches

Abstract

The microbial world is home to incredible taxonomic and metabolic diversity. Microorganisms are prolific producers of secondary metabolites, which are an important source of bioactive compounds, namely antibiotics, which are encoded by biosynthetic gene clusters (BGCs). The majority of the microbial diversity cannot be cultured and thus their taxonomic and metabolic diversity need to be addressed via culture independent methods. Metagenomics provides a platform to explore microorganisms yet-to be-cultured. The study of metagenomic BGCs rely on cloning environmental DNA into metagenomic libraries and screening target genes or pathways of interest using DNA amplification. However, this approach is hypothesized to overlook the diversity of BGCs. In order to expand the current ability to recover BGCs from metagenomic libraries, a new platform for targeted screening was developed using next generation sequencing (NGS), in conjunction with bioinformatic algorithms, allowing the sequencing of an entire metagenomic library and assignment of assembled sequences to their clones of origin. Then the NGS approach was validated and contrasted to the traditional amplification approach, showing that the NGS approach increased the number and diversity of BGCs and taxonomic markers detected. Furthermore, the NGS strategy was applied to fully annotate pathways prior to selection offering an innovative platform to guide prospecting of metagenomic libraries. Taxonomic markers from the metagenomic library indicated the presence of bacteria affiliated with the Candidate Phyla Radiation (CPR), a newly described group believed to not colonize soils. In order to study these ultra-small members of the soil microbiome, soil samples for a crop rotation experiment were collected from plots under controlled nutrient deficiency. A protocol was developed to enrich for ultra-small cells from soil samples, which were shotgun sequenced. Genomes from members of the ultra-small fraction were reconstructed to draft quality, showing a predominance of CPR. The MAGs were used to infer their complex carbohydrate degradation potential and reconstruct the community profiles across all soil samples to evaluate their responses to nutrient levels and pH in soils. Soil dwelling CPR were differentiated taxonomically and showed enrichment of genes associated with plant biomass degradation. No correlation between nutrient deficiency and community composition was found, but instead the community responded to changes in pH and metal levels in soil. Hence, they might play relevant roles in the soil microbiome while stressing the potential for metabolic and taxonomic diversity yet-to-be discovered in soil environments. Another relevant source of antibiotics are synthetic agents such as carbon. However, their ability to induce bacterial death is unclear, mainly due to the lack of appropriate experimental conditions to assess bacterial viability, namely the use of biologically incompatible dispersing agents and extraneous incubation conditions. To elucidate their toxicity, TSB growth medium, was employed as a dispersing agent for the first time and used to evaluate carbon nanotube toxicity against gram-positive and gram-negative bacteria, alongside other commonly used dispersing agents, such as surfactants. Bacterial cells are able to resist nanotube stress in biologically compatible media, however death is observed in the presence of sensitizing agents such as surfactants. Thus, carbon nanotubes, although unable to drive antimicrobial activity on their own are synergistic agents that can be used in conjunction with other compounds to increase antibiotic activity.