Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters

Abstract

Transcriptional lacZ fusions to the Pu and Pm promoters of the TOL (toluene degradation) plasmid inserted in monocopy in the chromosome of Pseudomonas putida showed a very different responsiveness to their respective aromatic effectors regarding growth phase. While a substantial XylS-dependent activation of Pm-lacZ was detected nearly instantly after m-toluate addition, XylR- and xylene-mediated induction of the sigma 54 promoter Pu became significant only after cells slowed down exponential growth and entered stationary phase. When Pu and Pm were fused to lacZ-tet reporters (i.e., promoterless lacZ genes coupled to a tet gene which confers resistance to tetracycline when cotranscribed with the leading gene) instead of lacZ alone, the resulting colonies displayed a distinct phenotype consisting of hyperfluorescence on agar plates after being sprayed with 4-methylumbelliferyl-beta-D-galactoside, simultaneously with being either sensitive (Pu) or resistant (Pm) to tetracycline. To examine whether the same phenotype could be scored in strains carrying transcriptional fusions of the lacZ-tet cassettes to other genes or promoters whose expression is silenced during growth and activated in stationary phase, we constructed mini-Tn5 lacZ-tet transposons for random genetic probing of promoters preferentially active at advanced stages of growth. Chromosomal insertions of this mobile element were selected by means of the constitutive resistance to kanamycin which is also specified by the transposon. A number of kanamycin-resistant colonies which are hyperfluorescent with 4-methylumbelliferyl-beta-D-galactosidase but sensitive to tetracycline and which reached full induction only at postexponential growth stages were obtained.