Jadwiga Wild

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing

A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.

Copy-Control pBAC/oriV Vectors for Genomic Cloning

The use of the improved BAC system for cloning genomic DNA and library constructions is described. This system retains all the advantages of the original BACs but, in addition, permits, on command, amplification of the BAC plasmids and cloned DNA. This system consists of (1) plasmid pBAC/oriV containing an additional replication origin, oriV, and (2) a host carrying the up-mutants of the trfA replicator gene expressed from the l-arabinose-inducible Para promoter. The pBAC/oriV clones are always maintained in the single-copy state, but if more DNA is required, they could be amplified up to 100-fold, depending on the size of the cloned insert.

Conjugal Transfer of Plasmid R6K γ ori Minireplicon Derivatives from Escherichia coli to Various Genera of Pathogenic Bacteria

Three R6K-derived γ ori minireplicons were successfully transferred by conjugation from Escherichia coli to several species of pathogenic bacteria. The pFL129 replicon encodes the wild-type initiation replication protein π, while plasmids pFL130 and pAG101 encode mutant forms of the π protein conferring the plasmid copy-up phenotype. Plasmids could be transferred to all recipient species tested, although high efficiency conjugal transfer was only obtained with genera of the Enterobacteriaceae. The efficiency of plasmid transfer to all recipients was lower for the copy-up derivatives, pFL130 and pAG101, than for pFL129. The three γ ori replicons were stably maintained in all transconjugants except pFL129 in Listeria monocytogenes. The two mutant plasmids retained their copy-up phenotype in the new bacterial hosts.