Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes.

Assembling a complete genome from a single bacterial cell, termed single-cell genomics, is challenging with current technologies. Recovery rates of complete genomes from fragmented assemblies of single-cell templates significantly vary. Although increasing the amount of genomic template material by standard cultivation improves recovery, most bacteria are unfortunately not amenable to traditional cultivation, possibly owing to the lack of unidentified, yet necessary, growth signals and/or specific symbiotic influences. To overcome this limitation, we adopted and modified the method of cocultivation of single-captured bacterial cells in gel microdroplets (GMDs) to improve full genomic sequence recovery. By completing multiple genomes of two novel species derived from single cells, we demonstrated its efficacy on diverse bacterial species using human oral and gut microbiome samples. Here we describe a detailed protocol for capturing single bacterial cells, cocultivating them in medium and isolating microcolonies in GMDs with flow cytometry. Beginning with preliminary studies, obtaining GMDs with single microcolonies for whole-genome amplification may take ∼4 weeks.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Artificial polyploidy improves bacterial single cell genome recovery.

BACKGROUND: Single cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell. METHODOLOGY/PRINCIPAL FINDINGS: We inhibited the bacterial cytoskeleton protein FtsZ in B.subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells. SIGNIFICANCE: The method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.

The importance of soil microorganisms for maintaining diverse plant communities in tallgrass prairie.

PREMISE OF THE STUDY: According to the "Janzen-Connell hypothesis," soil microorganisms have the potential to increase plant community diversity by mediating negative feedback on plant growth. Evidence for such microbe-driven negative feedback has been found in a variety of terrestrial systems. However, it is currently unknown how general this phenomenon is within most plant communities. Also unknown is the role of mutualists in generating such feedback: do they decrease the influence of soil-mediated negative feedback on plant fitness or do they increase its effect by proliferating with plant hosts to which they give the least benefit? • METHODS: We investigated soil-microbe-mediated feedback via a series of reciprocal transplant experiments in the greenhouse using soil from a restored tallgrass prairie and native tallgrass prairie plant species. • KEY RESULTS: We found that negative feedback was very common but that mutualists (arbuscular mycorrhizal fungi) influence plant growth in opposition to the overall negative feedback trend. • CONCLUSIONS: Widespread microbially mediated negative feedback indicates that plant community diversity and composition in tallgrass prairie are dependent on soil microorganisms. Native soil microorganisms should be considered in restoration efforts of tallgrass prairie and, potentially, other native plant communities.

Scale-dependent niche axes of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are mutualistic with most species of plants and are known to influence plant community diversity and composition. To better understand natural plant communities and the ecological processes they control it is important to understand what determines the distribution and diversity of AMF. We tested three putative niche axes: plant species composition, disturbance history, and soil chemistry against AMF species composition to determine which axis correlated most strongly with a changing AMF community. Due to a scale dependency we were not able to absolutely rank their importance, but we did find that each correlated significantly with AMF community change at our site. Among soil properties, pH and NO(3) were found to be especially good predictors of AMF community change. In a similar analysis of the plant community we found that time since disturbance had by far the largest impact on community composition.