C. Szpirer

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

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.

Retrotransfer or gene capture: a feature of conjugative plasmids, with ecological and evolutionary significance.

1 Laboratoire de Ge! ne! tique des Prokaryotes, Universite! Libre de Bruxelles, IBMM, B-6041-Gosselies, Belgium 2 Laboratory for Microbial Ecology and Technology, University of Gent, B-9000 Gent, Belgium 3 Environmental Technology, Flemish Institute for Technological Research, VITO, B-2400 Mol, Belgium 4 Laboratory of Microbiology, Radioactive Waste & Clean-up Division, Center of Studies for Nuclear Energy, SCK/CEN, B-2400 Mol, Belgium

Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob

It 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.

Separate-component-stabilization system for protein and DNA production without the use of antibiotics.

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.

The art of selective killing: Plasmid toxin/antitoxin systems and their technological applications

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

Assignment1 of SND1, the gene encoding coactivator p100, to human chromosome 7q31.3 and rat chromosome 4q23 by in situ hybridization

Originally identified in Epstein-Barr virus transformed B cells where it binds to EBNA-2 (a transcription factor encoded by EBV), the coactivator p100 also binds to the RNA polymerase II initiation factor TFIIE and also interacts with the oncoproteins c-myb and v-myb (Tong et al., 1995; Dash et al., 1996; Leverson et al., 1998). These and other results suggest that p100 and its rat homologue p105 play a role in the regulation of cell proliferation and in cellular transformation. In this study we assigned the gene encoding the coactivator p100 (symbol: SND1, staphylococcal nuclease domain containing 1) to human chromosome band 7q31.3 and its rat homolog (Snd1) to rat chromosome band 4q23.

Assignment of AR1, transcription factor 20 (TCF20), to human chromosome 22q13.3 with somatic cell hybrids and in situ hybridization

The mechanism of activation of genes encoding stromelysins is under intensive investigation since these enzymes are thought to contribute to tumor metastasis (reviewed by McDonnell et al., 1994). Stromelysins are metalloproteinases with relatively broad substrate specificity and can invade tumors causing release and movement of cells. The released cells can circulate, lodge into a capillary, permeate the walls of blood vessels, and proliferate to form a new tumor mass (McDonnell et al., 1994). A previous study has identified a mouse transcription factor (SPBP) that activates expression of the stromelysin-1 gene, possibly in response to mitogens and growth factors (Sanz et al., 1995; Kirstein et al., 1996). Here we report isolation and chromosomal localization of a human cDNA encoding a transcription factor (AR1) that shows extensive sequence identity to the mouse SPBP.

Assignment1 of the rat pleiomorphic adenoma genes (Plag1, Plagl1, Plagl2) by in situ hybridization and radiation hybrid mapping

PLAG1 (pleiomorphic adenoma gene 1), PLAGL1 and PLAGL2 (PLAG-like 1 and PLAG-like 2) constitute a subfamily of C2H2 zinc finger transcriptional regulators (Kas et al., 1998). Activation of PLAG1 on chromosome 8q12 is the most frequent gain of function mutation found in pleomorphic adenomas of the salivary glands (Kas et al., 1997b). PLAGL1 was independently isolated as ZAC, a gene that shares with the tumour suppressor p53 the ability to regulate apoptosis and cell cycle progression and also as LOT1, a gene with lost or decreased expression in malignantly transformed ovarian epithelial cells and breast tumour cells (Abdollahi et al., 1999; Bilanges et al., 1999). PLAGL1/ZAC/LOT1 is a strong candidate gene for transient neonatal diabetes (Kamiya et al., 2000). In the human, PLAG1 maps to chromosome 8q12, PLAGL1 to 6q24→q25 and PLAGL2 to 20q11 (Kas et al., 1997b, 1998 and unpublished data). We determined the regional location of the three rat homologous genes. Materials and methods

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR.

Chromosome evolution of MMU16 and RNO11: conserved synteny associated with gene order rearrangements explicable by intrachromosomal recombinations and neocentromere emergence

Comparative mapping between the rat and mouse genomes has shown that some chromosomes are entirely or almost entirely conserved with respect to gene content. Such is the case of rat chromosome 11 (RNO11) and mouse chromosome 16 (MMU16). We determined to what extent such an extensive conservation of synteny is associated with a conserved gene order. Therefore, we regionally localized several genes on RNO11. The comparison of the gene map of RNO11 and MMU16 unambiguously shows that the gene order has not been conserved in the Murinae lineage, thereby implying the occurrence of intrachromosomal evolutionary rearrangements. The transition from one chromosome configuration to the other one can be explained either by two intrachromosomal recombinations or by a single intrachromosomal recombination accompanied by neocentromere emergence.