APC Microbiome Ireland, University College Cork, Western Road, Cork, Ireland.
School of Microbiology, University College Cork, Western Road, Cork, Ireland.
Microb Biotechnol. 2021 Jul;14(4):1494-1511. doi: 10.1111/1751-7915.13810. Epub 2021 May 3.
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 iteron-regulated plasmid (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.
pMP7017 是从肠道共生双歧杆菌短双歧杆菌 JCM7017 中分离出来的一种可接合的大质粒，被证明编码两个假定的复制酶，这里分别命名为 RepA 和 RepB。在本研究中，RepB 被鉴定为 pMP7017 的复制起始子，因为 repB 基因及其周围区域足以允许在两种双歧杆菌物种，即短双歧杆菌和长双歧杆菌亚种长双歧杆菌中自主复制。RepB 被证明与编码序列下游的重复序列结合，该区域对有效复制至关重要。根据我们的结果，我们假设 pMP7017 是严格自动调节控制下的可重复调节质粒（IRP）。重组生产和纯化的 RepB 被确定在溶液中以二聚体形式存在，与其他 IRP 的复制酶不同，后者以二聚体和单体的混合物形式存在。此外，创建了稳定的低拷贝双歧杆菌-大肠杆菌穿梭载体 pRD1.3，可用于克隆和表达大片段基因，这在将编码来自短双歧杆菌 UCC2003 的胞外淀粉葡聚糖酶的 5.1kb apuB 基因克隆到长双歧杆菌亚种长双歧杆菌 NCIMB8809 中并进行异源表达时得到了证明。
