A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.
A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.
Kessler
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B
GBF-National Research Centre for Biotechnology, Braunschweig, FRG.
de Lorenzo
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V
Timmis
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KN
D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003062:Codon; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007763:Lac Operon; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics
D003062:Codon; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004274:DNA, Recombinant