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Most bacterial strains harbor plasmids that are maintained with remarkable stability. A large variety of plasmids encode systems that act when other control mechanisms have failed, i.e., when plasmid-free progeny is generated during replication. The mechanisms that control plasmid maintenance by T/A loci are well known: the antagonistic regulators that neutralize the toxins are metabolically unstable. Rapid depletion of these unstable regulators occurs in newborn, plasmid-free cells. As the same cells have inherited stable toxin molecules from the mother cell, the toxin will no longer be neutralized by the antitoxin, leading to the killing of the plasmid-free cells. This mechanism effectively reduces the proliferation of plasmid-free cells in growing bacterial populations (1). The most widely studied T/A system so far is the ccd system located on the F plasmid (2). The ccd system is composed of two genes, ccdA and ccdB, encoding small proteins: the CcdA antidote (8.7 kDa) and the CcdB toxin (11.7 kDa). The CcdB protein acts as a poison because it selectively targets the Escherichia coli DNA gyrase, a bacterial topoisomerase II. Early studies of this T/A system were performed at the Universite Libre de Bruxelles (ULB). Today new applications are commercialized by Delphi Genetics SA, a spin off company of the ULB founded by the researchers who developed the use of T/A systems as selectable markers. POSITIVE SELECTION VECTORS

RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin ( ori ) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.

The use of PCR-amplified DNA-fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent Aspel sites and a unique HindII site. Cleavage of these vectors with Aspel produce linearized molecules with a single thymidine nucleotide at the 3' ends allowing TA cloning of Taq-amplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZ alpha-CcdB fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15. This bifunctionality allowed us to show that small PCR products (< 1000 bp) that do not disrupt lacZ alpha efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.

RepHI1B is one of the replicons that is specific to IncHI1 multireplicon plasmids. Its general organization resembles that of several replicons that control their copy number by an iteron mechanism. The RepHI1B replicon (2.4 kb) contains: (i) an 882 bp repA gene coding for a 32 kDa replication protein (RepA), sharing significant similarity with the initiator proteins of other replicons belonging to various incompatibility (Inc) groups, including P1 (IncY), Rts1 (IncT), RepFIB (IncFI), and RepHI1A (IncHI1); (ii) two sets of 17 bp DNA repeats (iterons), one upstream and one downstream from repA. By complementation testing, we identified the replication origin (ori) of RepHI1B in a 223 bp locus upstream from repA. By primer extension we mapped two promoters of repA (Pr1 and Pr2) in the ori sequence. We used repA::lacZ transcriptional fusions to study regulation of the repA gene. This analysis showed that repA is transcriptionally autoregulated. Gel mobility shift assays demonstrated that RepA binds specifically to the origin and to iterons overlapping the Pr1 and Pr2 promoters. A G to A transition at nucleotide position 13 of the iteron located in Pr2 (repeat 5) drastically decreases autoregulation of repA by inhibiting binding of RepA.

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

The IncHI1 plasmids are multireplicon plasmids. They contain at least three autoreplicative regions, one of which is closely related to the RepFIA replicon of F. Two other IncHI1-specific replicons, RepHI1A and RepHI1B, have been recently isolated and mapped on the R27 (IncHI1) genome (P. Gabant, P. Newnham, D. Taylor, and M. Couturier, J. Bacteriol. 175, 76977701, 1993). In the present work, the DNA sequence of RepHI1B was determined. It reveals DNA repeats of 17 base pairs located upstream and downstream from a gene coding for a 32 kilodalton protein (RepA) required for replication. Interestingly, RepA presents significant homology with other Rep proteins encoded by plasmids belonging to different incompatibility groups: P1 (IncY), Rts1 (IncT), RepFIB (IncFI) and RepHI1A (IncHI1). All these results provide strong evidence that the RepHI1B replicon of the IncHI1 subgroup belongs to the group of plasmids which control their copy number by an iteron mechanism.