Bifidobacterium longum DJO10A is a dominant human isolate with broad-spectrum antimicrobial capabilities (A. Islam, D. D. O’Sullivan, and D. J. O’Sullivan, unpublished data). As this strain has potentially very interesting characteristics, it was chosen for an in-depth analysis. Functional analysis of its characteristics requires molecular tools, and these are not plentiful for bifidobacteria. It was therefore the objective of this study to analyze the two native plasmids of this strain and construct a shuttle cloning vector for bifidobacteria.
The mode of replication of the large plasmid in B. longum
DJO10A, pDOJH10L, was predicted to be rolling circular replication. This plasmid contains DNA iterons, which have been shown to be involved in plasmid replication and copy number (22
). It also contains a DnaA box that is involved in the initiation of replication in rolling circular plasmids (26
). To replicate in bifidobacteria, it is likely that the host DnaA protein binds to the DnaA box region, causing an open complex formation of duplex DNA at the AT-rich region, thus enhancing the unwinding of the origin for initiation of replication. Replication initiation also involves a Rep protein encoded by pDOJH10L. Interestingly, this plasmid contains three rep
genes, but repB
became truncated during the cointegration event, leading to the formation of pDOJH10L, and the iteron structures corresponding to RepA are incomplete. RepC, which originated from a gene fusion event during the formation of pDOJH10L, is likely the functional Rep gene of this plasmid. This Rep protein likely functions with the complete replication region (iteron structure, DnaA box, and AT-rich region) located upstream from the truncated repB
gene during the replication of this plasmid. Mechanistic studies have shown that Rep proteins function as initiators of replication when in a monomer formation and as repressors when in a dimer formation (17
). The RepC protein of pDOJH10L would therefore likely have a central role in initiating its replication and controlling its copy number.
All sequenced plasmids from bifidobacteria to date have been proposed to replicate by rolling circular replication. Interestingly, the small plasmid from B. longum
DJO10A replicates via a different mechanism, having all the characteristics of a theta replicating plasmid. In addition, the DNA sequence of pDOJH10S did not show any significant homology to previously sequenced bifidobacterial plasmids, suggesting it may be a novel plasmid type for bifidobacteria. The Rep protein ofpDOJH10S has high homology to several known Rep proteins, with the highest homology to the theta replicase from Rhodococcus rhodochrous
. Interestingly, the G+C content of pDOJH10S (66.2%) is significantly higher than that of the genome of B. longum
DJO10A (60.1%), further substantiating a recent acquisition of this plasmid in bifidobacteria by horizontal gene transfer. As its G+C content is similar to that of Rhodococcus rhodochrous
, which is listed as 67 to 70% (11
), it is likely that Rhodococcus
is the original origin of the plasmid. Both Rhodococcus
are closely related, being members of the Actinomycetes
. The opportunity for horizontal gene transfer from Rhodococcus
also exists as members of this genus are frequently found in the feces of animals (40
A probable mechanism for the transfer of this plasmid from Rhodococcus into Bifidobacterium is via conjugation. Plasmid pDOJH10S contains a Mob and a putative oriT region that contain all the components necessary for plasmid mobilization. While neither pDOJH10S nor pDOJH10L contain tra genes, Tra functions can be supplied in trans to facilitate plasmid mobilization. Plasmid pDOJH10L has two Mob proteins, but it has only one putative oriT upstream of MobB. The lack of a putative oriT upstream of MobA is most likely the result of a cointegration event during the formation of pDOJH10L from at least two other bifidobacterial plasmids.
The evidence for a cointegration event during the formation of pDOJH10L is very convincing, given the more than 96 and 98% identity with two other plasmids from another B. longum strain (Fig. ). To date, this is the first cointegration event reported for a plasmid in bifidobacteria, suggesting it may not be a very frequent event. Analysis of the cointegration event indicates that it involved at least one other plasmid during pDOJH10L formation, as an extra rep gene is also present. The selective pressure for obtaining this third rep gene appears to be the disruption of both the RepA and RepB replication regions during the formation of pDOJH10L. Thus, RepC is likely involved in replicating the plasmid via the complete replication region upstream of the truncated repB gene.
Several Bifidobacterium-E. coli
shuttle vectors have been constructed, and all are based on RCR plasmids from bifidobacteria (19
). However, plasmids that utilize RCR display both structural and segregational instabilities in gram-positive bacteria, while theta replicating plasmids display much greater stabilities (10
). In this study, the theta replicating pDOJH10S plasmid was used as the basis for a shuttle cloning vector, as this would likely lead to a more stable cloning vector. It was successfully electroporated into B. longum
and E. coli
but could not be introduced into Lactobacillus bulgaricus
. It also does not replicate in Lactococcus lactis
(J. Yang and D. J. O’Sullivan, unpublished data), illustrating the limited hosts capable of replicating this vector. This is consistent with other studies on plasmid replication origins from bifidobacteria, as they have not been able to replicate in lactic acid bacteria (19
). The availability of a theta-replicating vector for stable gene cloning in bifidobacteria will facilitate functional genomic analysis of this gut-friendly inhabitant.