Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1

Abstract

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.

Conjugative Plasmid Transfer in Gram-Positive Bacteria

“…5B). In spite of these similarities, mutation of each of the seven Tyr residues of the pBBR1-Mob protein did not affect the frequency of conjugation of the plasmid, whereas changes at two conserved residues, Asp120 and Glu121, totally abolished plasmid transfer (205). These results leave open the question whether this family of Mob proteins cleaves its target DNA through a Tyr-mediated transesterification reaction or by a water-mediated nucleophilic attack performed by a Glu residue, as proposed for the closing reaction carried out by the replication initiator protein of plasmid pC194 (150).…”

“…Based on the conservation of the N-terminal but not of the C-terminal moiety, it was proposed that the former region of MobM could be involved in the nicking reaction, a hypothesis supported by the conservation of a Tyr residue among the Mob proteins of RCR plasmids (91). For the Mob protein of plasmid pBBR1 (a broad-host-range theta replicating plasmid from the gram-negative host Bordetella bronchiseptica), it was shown that the protein is related to pMV158 MobM (205) and the pBBR1 oriT region has homologies to the pMV158 oriT (Fig. 5B).…”

“…DNase I footprinting assays showed that purified MobM protein protected the IR-2 sequence (90). Since the Ϫ10 extended region of the promoter that directs synthesis of the mobM mRNA (at least in lactococcal cells) is also included within the IR-2 (66), it would appear that the protein regulates its own synthesis, similarly to the Mob protein of plasmid pBBR1 (205), a hypothesis that is currently under investigation (C. de Antonio and M. Espinosa, personal communication). Consequently, this pMV158-DNA region was defined as the oriT plasmid in vitro (90,91) and was later shown to promote transfer of pSC101 when MobM was provided in trans (65).…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Correspondingly, in the pIP501 system, relaxase binding to oriT autoregulates the expression of the relaxase and downstream transfer genes (163). Gram-negative plasmid pBBR1 has a nic region identical to that of pMV158, and reminiscent of pIP501, the binding of pBBR1 relaxase at oriT autoregulates downstream tra gene expression (255). It is postulated that this mode of regulation evolved for the feedback control of plasmid transfer rates to minimize the metabolic burden associated with conjugative DNA transfer (163).…”

The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501

“…In TraA, no putative Leu-zipper motif was found. For Mob, the mobilization protein encoded by the mobilizable broad-host-range plasmid pBBR1, the nic site of which is identical with that of pMV158, autoregulation by the binding of Mob to its promoter region overlapping with oriT has been demonstrated (Szpirer et al, 2001). …”