Impacts of sample handling and storage conditions on archiving physiologically active soil microbial communities.

Soil microbial communities are fundamental to ecosystem processes and plant growth, yet community composition is seasonally and successionally dynamic, which interferes with long-term iterative experimentation of plant-microbe interactions. We explore how soil sample handling (e.g. filtering) and sample storage conditions impact the ability to revive the original, physiologically active, soil microbial community. We obtained soil from agricultural fields in Montana and Oklahoma, USA and samples were sieved to 2 mm or filtered to 45 µm. Sieved and filtered soil samples were archived at -20°C or -80°C for 50 days and revived for 2 or 7 days. We extracted DNA and the more transient RNA pools from control and treatment samples and characterized microbial communities using 16S amplicon sequencing. Filtration and storage treatments significantly altered soil microbial communities, impacting both species richness and community composition. Storing sieved soil at -20°C did not alter species richness and resulted in the least disruption to the microbial community composition in comparison to nonarchived controls as characterized by RNA pools from soils of both sites. Filtration significantly altered composition but not species richness. Archiving sieved soil at -20°C could allow for long-term and repeated experimentation on preserved physiologically active microbial communities.

A novel conjugative transposon carrying an autonomously amplified plasmid.

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.