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

Tamminen MV, Virta MP - Front Microbiol (2015)

Bottom Line: The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry.The method provides a novel tool for enumerating functional cell populations in complex microbial communities.We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
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 nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete 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 labeled 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 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

No MeSH data available.


Related in: MedlinePlus

A logarithmic plot of the proportion of false positive background events to positive events in gate P1 versus the percentage of E. coli XL1 to E. coli MC1061 in the initial cell suspension.
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Figure 7: A logarithmic plot of the proportion of false positive background events to positive events in gate P1 versus the percentage of E. coli XL1 to E. coli MC1061 in the initial cell suspension.

Mentions: Six different suspensions of E. coli XL1 and E. coli MC1061 cells were prepared in which the percentage of E. coli XL1 ranged from 0 to 100% of the total. The genome of E. coli XL1 contains a tetracycline resistance gene, whereas the genome of E. coli MC1061 does not. The cells were processed as described above to amplify the genomes and label them based on the presence of the tetracycline resistance gene. When analyzed using a flow cytometer, picoreactors carrying the E. coli XL1-genome appeared as bright fluorescent events (Figure 6). The less-fluorescent events correspond to empty picoreactors and picoreactors containing the E. coli MC1061 genome. Altogether, 100,000 events were collected for each suspension. The proportion of E. coli XL1 to MC1061 cells remained similar between the initial cell suspension and the labeled picoreactors. Based on the scatterplots, as little as 0.1% E. coli XL1 cells in the initial cell suspension (relative to MC1061 cells) could be differentiated using the method. The number of fluorescent events correspond to the percentage of XL1 cells (power equation R2-value 0.97; Figure 7).


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

Tamminen MV, Virta MP - Front Microbiol (2015)

A logarithmic plot of the proportion of false positive background events to positive events in gate P1 versus the percentage of E. coli XL1 to E. coli MC1061 in the initial cell suspension.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356102&req=5

Figure 7: A logarithmic plot of the proportion of false positive background events to positive events in gate P1 versus the percentage of E. coli XL1 to E. coli MC1061 in the initial cell suspension.
Mentions: Six different suspensions of E. coli XL1 and E. coli MC1061 cells were prepared in which the percentage of E. coli XL1 ranged from 0 to 100% of the total. The genome of E. coli XL1 contains a tetracycline resistance gene, whereas the genome of E. coli MC1061 does not. The cells were processed as described above to amplify the genomes and label them based on the presence of the tetracycline resistance gene. When analyzed using a flow cytometer, picoreactors carrying the E. coli XL1-genome appeared as bright fluorescent events (Figure 6). The less-fluorescent events correspond to empty picoreactors and picoreactors containing the E. coli MC1061 genome. Altogether, 100,000 events were collected for each suspension. The proportion of E. coli XL1 to MC1061 cells remained similar between the initial cell suspension and the labeled picoreactors. Based on the scatterplots, as little as 0.1% E. coli XL1 cells in the initial cell suspension (relative to MC1061 cells) could be differentiated using the method. The number of fluorescent events correspond to the percentage of XL1 cells (power equation R2-value 0.97; Figure 7).

Bottom Line: The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry.The method provides a novel tool for enumerating functional cell populations in complex microbial communities.We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
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 nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete 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 labeled 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 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

No MeSH data available.


Related in: MedlinePlus