Molecular characterisation of a conjugative Bifidobacterium megaplasmid

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Dineen, Rebecca L.
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University College Cork
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Bifidobacterium species are highly abundant autochthonous bacteria of the human gut microbiota, particularly during host infancy. Various members of the Bifidobacterium genus have been associated with a plethora of health-promoting attributes, among which maintaining gut homeostasis, limiting pathogenic bacterial invasion as well as their purported ability to modulate host immune responses are notable examples. Due to their positive association with human health, this genus has received substantial scientific attention and commercial interest. Plasmids were once considered an atypical feature of Bifidobacterium and those identified within this genus were primarily small cryptic plasmids which were presumed to replicate by a so-called rolling circle mechanism. However, the recent availability of long-read single-molecule sequencing technologies precipitated the resolution of the first reported bifidobacterial megaplasmid isolated from the common and abundant inhabitant of the human gut, Bifidobacterium breve JCM7017. The discovery of this >190 kb conjugative megaplasmid, denoted pMP7017, and the subsequent identification of pMP7017 homologs in several B. longum subsp. longum strains, highlights the prevalence of this megaplasmid family within this genus, representing an unexplored feature. Conjugative plasmids such as pMP7017 play a central role in bacterial evolution and have the potential to significantly influence the activity of the microbiome community and, by extension, impact human health and physiology. The research described in this thesis covers the replication functions of megaplasmid pMP7017 and exploits these functions for the development of important molecular tools that facilitate the genetic engineering of these genetically recalcitrant bacteria. As pMP7017 represents the first and, thus far, only experimentally validated conjugative plasmid of bifidobacterial origin, research performed within the context of this thesis also concerned examination of the conjugative functions of this megaplasmid. While the molecular characterisation of pMP7017 represents an opportunity for the development of much needed molecular tools and provides a starting point in the understanding of bifidobacterial DNA transfer systems, an integrated approach of comparative analyses and metagenomic data mining has generated highly relevant and insightful information concerning the biology and distribution of pMP7017 and related megaplasmids.
Bifidobacteria , Microbiota , Plasmid , Megaplasmid , Transcriptome , Host-impact
Dineen, R. L. 2023. Molecular characterisation of a conjugative Bifidobacterium megaplasmid. PhD Thesis, University College Cork.