Analysis of a novel 8.9 kb cryptic plasmid from Bacteroides uniformis, its long-term stability and spread within human microbiota

The analysis of plasmid content in dominant Bacteroidales order intestinal strains isolated from the same child at a 5 year interval identified a 8.9 kb plasmid in Bacteroides uniformis BUN24 strain isolated at age 6 and indistinguishably sized plasmids in the isolates of B. uniformis, B. vulgatus, B. intesinalis, and Parabacteroides distasonis at age 11. We sequenced a B. uniformis BUN24 plasmid, designated pBUN24, and using molecular surveys of diverse species we established that this 8944 bp molecule (G + C content 43.5%) represents a novel family of small cryptic Bacteroidales plasmids. The replication region of pBUN24 was experimentally localized to a 1707-bp fragment that includes a putative repA gene, coding for a protein of Rep_3 superfamily of replication proteins of theta-type plasmids preceded by a putative iteron-containing origin of replication. The other open reading frames (ORFs) identified in pBUN24 sequence include a putative tad–ata-type toxin–antitoxin and mobA–mobB mobilization modules, as well as seven additional cryptic ORFs. The interaction of Tad and Ada components demonstrated by a pull-down assay and the toxicity of Tad in Escherichia coli host suggests the functionality of the plasmid addiction module. Re-sequencing of plasmids in two Bacteroides strains isolated at the age of 11 showed 100% nucleotide identity to pBUN24. This data supports the notion that this plasmid is transmissible to other Bacteroidales strains in the natural ecosystem. The possible roles of toxin–antitoxin system and other proteins encoded by pBUN24 in providing an apparent ecological advantage to the plasmid-harbouring strains of a bacterial symbiont in the human gut deserve further investigation.

► We identified a novel cryptic plasmid in a human intestinal strain of B. uniformis, pBUN24.
► We characterized the replication, plasmid addiction, and mobilization genes on pBUN24.
► Plasmid pBUN24 seems to be of high prevalence within normal intestinal populations of Bacteroidales order bacteria.
► We showed that pBUN24 is stably maintained within the intestinal microbiota of a healthy child for over 5 years.