English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Plus monthly

Global: Zendy Tools annual

Global: Zendy Tools monthly

Global: Zendy Free Plan

Background: Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some unpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. Methodology/Principal Findings: We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a support for genetic material and therefore allow degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labelled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1) or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.01% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to complex environmental microbial communities as a target gene-based screen for individual microbes. Conclusions/Significance: The genomic support for complete cell degradation allows an exposure of individual genomes of environmental bacteria. The genome exposure followed by genome amplification and labelling combines the benefits of in situ-PCR and FISH methods and permits the detection cells with single copy chromosomal targets in complex mixtures of microbial cells. The method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells