A general system to integratelacZ fusions into the chromosomes of gram-negative eubacteria: regulation of thePm promoter of theTOL plasmid studied with all controlling elements in monocopy

Abstract

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.

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

“…P. putida strains KT2442 hom.fg/xylRS and its derivative HFPu (Pu::lacZ, xylR ϩ ) have been described previously (36,37). KT2442PuXhoCla (PuXhoCla::lacZ, xylR ϩ ), KT2442PuScra1 (PuScra1::lacZ, xylR ϩ ), and KT2442PuScra2 (PuScra2::lacZ, xylR ϩ ) carrying mutant Pu::lacZ fusions in the same location of the chromosome as HF Pu were obtained as follows.…”

“…The replacement of the 47-bp XhoI-ClaI fragment of pUC-PuClaXho for synthetic XhoI-ClaI fragments harboring scrambled sequences from nucleotides Ϫ105 to Ϫ120 and from Ϫ95 to Ϫ120 gave rise to plasmids pUC-PuScra1 and pUC-PuScra2, respectively. The Pu versions present in pUC-PuClaXho, pUC-PuScra1, and pUCPuScra2, respectively, were rescued as 312-bp EcoRI-BamH fragments, fused to lacZ by cloning in the corresponding sites of pBK16 vector (36) and recombined with the homology fragment inserted in the chromosome of KT2442 hom.fg/xylRS as described previously (36). All cloned inserts and DNA fragments were verified before use by automated DNA sequencing.…”

“…From this procedure, we obtained two Pu derivatives, PuScra1 and PuScra2 (Fig. 1), that, along with their parental PuXhoCla, were fused to lacZ and recombined into the chromosome of P. putida KT2442 hom.fg/xylRS as described previously (36). As shown in Fig.…”

Investigation of the algT operon sequence in mucoid and non-mucoid Pseudomonas aeruginosa isolates from 115 Scandinavian patients with cystic fibrosis and in 88 in vitro non-mucoid revertants

“…Triparental mating was used to mobilize the algT-containing recombinant plasmid pCD100 from E. coli JM109 to non-mucoid P. aeruginosa isolates showing a mucA algT mutant genotype with the conjugation helper plasmid pRK600 (Kessler et al, 1992), as previously described (Hoffmann et al, 2005). Transconjugants of P. aeruginosa were selected on PIA agar containing 80 mg tetracycline ml 21 , and complementation of algT mutations was scored by the mucoid phenotype observed on PIA plates after incubation at 37 uC to confirm that the mutations found in algT were responsible for the non-mucoid phenotype.…”

Cross talk between catabolic pathways in Pseudomonas putida: XylS-dependent and -independent activation of the TOL meta operon requires the same cis-acting sequences within the Pm promoter

“…1). Recent observations from different laboratories (1,12,13) have shown that, in the absence of XylS, the Pm promoter is still responsive to benzoate (but not to 3-methylbenzoate), perhaps by recruiting the regulator of the chromosomal benABCD pathway, BenR (12). Since xylS and benR do not share a high degree of homology, at least as determined by DNA-DNA hybridization (12), Pm would be a type of promoter which can be activated by either of two different regulators.…”