SummaryIt is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two-hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP-type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.

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.

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Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.