Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017. Issue 4 (3rd May 2021)

• Record Type: Journal Article
• Authors: Dineen, Rebecca L.; Penno, Christophe; Kelleher, Philip; Bourin, Maxence J. B.; O'Connell‐Motherway, Mary; van Sinderen, Douwe
• Is Part Of: Microbial biotechnology. Volume 14:Issue 4(2021)
• Page Start: 1494
• Page End: 1511
• Publication Date: 2021-05-03
• Subjects: Microbial biotechnology -- Periodicals; Biotechnology; Microbiology; 660.62
• DOI: 10.1111/1751-7915.13810
• Languages: English
• ISSNs: 1751-7915

Abstract

Summary: pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum . RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an i teron‐ r egulated p lasmid (IRP) under strict auto‐regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low‐copy Bifidobacterium ‐ E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809. Abstract : In the current work, RepB was identified as the replicative initiator of the 190 kb conjugative megaplasmid pMP7017 from gut commensal Bifidobacterium breve JCM7017. RepB was shown to bind to repeat sequences downstream of its coding sequence and this region was determined to be essential for efficient replication. In addition, a stable low‐copy Bifidobacterium‐E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes.