Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains.
An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified &phi;CTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the &phi;CTX attachment site. The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI(q) also contained on the plasmid. Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI.
Hoang
T T
TT
Department of Microbiology, Colorado State University, Fort Collins, Colorado, 80523-1677, USA.
Kutchma
A J
AJ
Becher
A
A
Schweizer
H P
HP
D019677:Artificial Gene Fusion; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D016680:Genome, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D001616:beta-Galactosidase