Influence of soil nutrients on the presence and distribution of CPR bacteria in a long-term crop rotation experiment.

Bacteria affiliated with the Candidate Phyla Radiation (CPR) are a hyper-diverse group of ultra-small bacteria with versatile yet sparse metabolisms. However, most insights into this group come from a surprisingly small number of environments, and recovery of CPR bacteria from soils has been hindered due to their extremely low abundance within complex microbial assemblages. In this study we enriched soil samples from 14 different soil fertility treatments for ultra-small (<0.45 µm) bacteria in order to study rare soil CPR. 42 samples were sequenced, enabling the reconstruction of 27 quality CPR metagenome-assembled genomes (MAGs) further classified as Parcubacteria/Paceibacteria, Saccharibacteria/Saccharimonadia and ABY1, in addition to representative genomes from Gemmatimonadetes, Dependentiae and Chlamydae phyla. These genomes were fully annotated and used to reconstruct the CPR community across all 14 plots. Additionally, for five of these plots, the entire microbiota was reconstructed using 16S amplification, showing that specific soil CPR may form symbiotic relationships with a varied and circumstantial range of hosts. Cullars CPR had a prevalence of enzymes predicted to degrade plant-derived carbohydrates, which suggests they have a role in plant biomass degradation. Parcubacteria appear to be more apt at microfauna necromass degradation. Cullars Saccharibacteria and a Parcubacteria group were shown to carry a possible aerotolerance mechanism coupled with potential for aerobic respiration, which appear to be a unique adaptation to the oxic soil environment. Reconstruction of CPR communities across treatment plots showed that they were not impacted by changes in nutrient levels or microbiota composition, being only impacted by extreme conditions, causing some CPR to dominate the community. These findings corroborate the understanding that soil-dwelling CPR bacteria have a very broad symbiont range and have metabolic capabilities associated to soil environments which allows them to scavenge resources and form resilient communities. The contributions of these microbial dark matter species to soil ecology and plant interactions will be of significant interest in future studies.

Identification of PKS Gene Clusters from Metagenomic Libraries Using a Next-Generation Sequencing Approach.

Microbial secondary metabolites have been an important source of bioactive compounds with diverse applications from medicine to agriculture, noticeably those encoded by polyketide synthase (PKS) clusters due to their astounding chemical diversity. While most discovered compounds originate from culturable microorganisms, yet-to-be cultured microbes represent a reservoir of previously inaccessible compounds. The advent and development of metagenomics have allowed not only the characterization of these microorganisms but also their metabolic potential, making viable the prospection of environmental PKS for natural product discovery.Study of environmental PKSs often relies on the construction of metagenomic libraries and their mining, with clones containing PKS clusters identified via amplification of conserved domains and then screened for an activity of interest. Compounds produced by clones exhibiting the desired bioactivity can be isolated and characterized. However, these approaches can be less sensitive and biased against more divergent clusters, in addition to precluding the use of bioinformatics for cluster characterization prior to expression. While direct shotgun sequencing of metagenomes has identified and profiled a great number of PKSs from different environments and yet-to-be cultured microorganisms, it does not lend itself well to heterologous expression, the cruxes of natural product discovery.Here, we describe a strategy for sequencing entire metagenomic libraries while maintaining correspondence between sequence and clone, allowing the full characterization and annotation of all clusters present in a library using bioinformatic tools and then seamlessly passing clones of interest for activity screening through heterologous expression. Once a library is sequenced, the methods herein can be adapted for the mining of any biosynthetic gene cluster of interest within a metagenomic library.

Dispersant Effects on Single-Walled Carbon Nanotube Antibacterial Activity.

There is significant interest in understanding whether nanomaterials with outstanding mechanical or electrical properties also possess antibacterial properties. However, assessment of antibacterial activity is a complex problem at the interface of chemistry and microbiology. Results can be affected by many factors including nanomaterial size, surface chemistry, concentration, and the dispersion media. The difficulty of dispersing nanomaterials such as single-walled carbon nanotubes (SWNTs) has resulted in many studies being conducted in the presence of dispersion aides which may themselves contribute to bacterial stress. The recent discovery that a standard microbial growth media, tryptic soy broth (TSB), is an effective SWNT dispersant provides a new opportunity to investigate the potential antibacterial activity of SWNTs using dispersants that range from antibacterial to growth-supporting. The five dispersants chosen for this work were Sodium dodecyl sulfate (SDS), pluronic, lysozyme, DNA, and tryptic soy broth. Staphylococcus aureus and Salmonella enterica were used as the model Gram-positive and Gram-negative bacteria. Activity was measured in terms of colony forming unit (CFU) and optical density measurements. None of the systems exhibited activity against Salmonella. SDS was fatal to Staph. aureus regardless of the presence of SWNTs. The activity of pluronic and lysozyme against Staph. aureus was enhanced by the presence of SWNTs. In contrast, the DNA and TSB dispersions did not have any activity regardless of the presence of SWNTs. These results highlight that the purported antibacterial activity of SWNTs may only be effective against bacteria that are sensitized by the dispersant and suggests the need for additional research on the mechanisms by which SWNT-dispersant interactions can result in antibacterial activity.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.

Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity.

Single-Walled Carbon Nanotube Dispersion in Tryptic Soy Broth.

There has been little research on the dispersion of carbon nanotubes in dispersions of standard microbiological media. We report that tryptic soy broth (TSB) containing casein digest disperses single-walled carbon nanotubes (SWNT) at concentrations similar to those achieved in lysozyme (LSZ), one of the best known biomolecular SWNT dispersants. Similar to LSZ, the proposed mechanism for SWNT dispersion in TSB is favorable π-π stacking interactions with l-tryptophan. This is supported by similar SWNT concentrations in both LSZ and TSB supernatants, and the absence of appreciable dispersion in TSB that does not contain a source of l-tryptophan. Since l-tryptophan alone is insufficient to enable dispersion, it was previously hypothesized that LSZ's macromolecular structure created steric hindrance that was critical for SWNT dispersion. These new results show that intermediately sized l-tryptophan containing species can also enable dispersion. In addition, since TSB is a commonly used growth medium for microbiological research, its dispersive ability presents new research avenues for studying the effect of SWNT on prokaryotic cells without the need to oxidize SWNT or add dispersants that may induce microbial stress.