Discovery of a conjugative megaplasmid in Bifidobacterium breve

Abstract

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.

FIG 1

pMP7017 general features. Genome atlas representing the circular sequence of the magaplasmid pMP7017 with the relative ORFs organization is shown. Highlighted are the four main functional regions (REG1 to -4) with relative genetic features. A green line also divides pMP7017 into two putative cointegrates.

FIG 2

Amino acid usage comparison and tRNA genes. (a) Heatmap and hierarchical clustering showing the amino acid frequencies computed for the predicted proteome of pMP7017 and the host. For representative reasons, a sample of equal numbers of 227 randomly selected ORFs of B. breve JCM 7017 and pMP7017 are displayed in this heatmap. (b) Bidimensional barplot representing the cumulative amino acid frequency in pMP7017 and the host with locus map indicating the 14 consecutive tRNA genes encountered in pMP7017 sequence. A table with the total number of tRNA in the plasmid and chromosome is also included.

FIG 3

CRISPR/Cas systems. (a) Locus map showing the organization of the CRISPR/cas locus in B. breve JCM 7017 chromosome and in the megaplasmid. (b) Heatmap showing the BLASTP analysis of the 74 spacer regions found in the CRISPR/cas systems of the B. breve JCM 7017 chromosome and relative megaplasmid, aligned against NCBI nonredundant (nr) database. A green line also indicates matches within representatives of B. breve species.

FIG 4

Presence of pMP7017 in B. breve and B. longum subsp. longum. (a) Dot plot alignment showing the comparison of pMP7017 megaplasmid with the contigs of B. longum subsp. longum of which ORFs displayed significant match in BLASTP alignment (e.g., B. longum subsp. longum 1-6B, B. longum subsp. longum 2-2B, and B. longum subsp. longum 44B). Delimited by green bars are the regions displaying the most of synteny. (b) BLAST-based genome atlas showing the comparative analysis conducted on pMP7017 against the draft sequence of B. longum subsp longum 1-6B, B. longum subsp. longum 2-2B, and B. longum subsp. longum 44B. From the outer to the inner circle and shaded in gradient from dark red to orange: B. breve JCM 7017 pMP7017, B. longum subsp. longum 44B, B. longum subsp. longum 2-2B, and B. longum subsp. longum 1-6B. (c) PFGE experiment showing the presence of a megaplasmid of a size comparable to that of pMP7017 (about 200 kb) also in B. longum subsp. longum 1-6B, B. longum subsp. longum 2-2B, and B. longum subsp. longum 44B, but absent in B. longum subsp. longum 35B and B. breve UCC2003 as a negative control.

FIG 5

Conjugal transfer of pMP7017. PFGE experiments for B. breve UCC2003 or B. longum NCIMB 8809 transconjugants conducted with AscI (a) and SpeI (b) enzymes, respectively. The presence of megaplasmid in the parent strain, B. breve JCM 7017, and the donor strain, B. breve JCM 7017_199, is depicted in each panel. The presence of pMP7017 in B. breve UCC2003 trasconjugants is evidenced by a 111-kb fragment in total DNA digests with AscI, while the megaplasmid presence in B. longum NCIMB 8809 transconjugants is verified by the presence of a 196-kb fragment.