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Transposon-aided capture (TRACA) of plasmids resident in the human gut mobile metagenome


The bacterial community of the human gut is a complex ecosystem composed of >1,000 species, the majority of which are Gram positive and uncultured. To study plasmids resident within this community, we developed a culture-independent transposon aided capture method (TRACA), which does not rely on any plasmid-encoded traits. TRACA facilitated acquisition of plasmids from metagenomic DNA extracts, and subsequent maintenance and selection in an Escherichia coli host. We confirmed the presence of the transposon in captured plasmids and demonstrate that these plasmids are mainly of a Gram-positive origin. Sequencing of plasmids designated pTRACA10 (7 kb) and pTRACA17 (2.7 kb) revealed genes involved in plasmid mobilization and replication. From the homologies of these genes we conclude that pTRACA17 originates from a Gram-positive host belonging to the Firmicute division. pTRACA10 had two additional open reading frames with similarity to a conserved hypothetical protein and phosphoesterase or phosphohydrolase enzymes (Clusters of Orthologous Groups number 4186). Both plasmids lacked any conventional selectable markers.

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Figure 1: Overview of the TRACA method.
Figure 2: Maps of TRACA acquired plasmids pTRACA10 and pTRACA17 from complete sequence data.

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We thank M. Claesson for assistance with annotation of plasmid sequences, and E. Brodie and G. Andersen for the 16S rRNA microarray analysis. This work was supported by the Irish Government under the National Development Plan (2000–2006) and Science Foundation Ireland.

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B.V.J., TRACA concept and method development, all experimental work and manuscript preparation. J.R.M., TRACA method development and manuscript preparation.

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Correspondence to Julian R Marchesi.

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The authors declare no competing financial interests.

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Jones, B., Marchesi, J. Transposon-aided capture (TRACA) of plasmids resident in the human gut mobile metagenome. Nat Methods 4, 55–61 (2007).

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