TY - JOUR
T1 - Integration-proficient plasmids for Pseudomonas aeruginosa
T2 - Site- specific integration and use for engineering of reporter and expression strains
AU - Hoang, Tung T.
AU - Kutchma, Alecks J.
AU - Becher, Anna
AU - Schweizer, Herbert P.
N1 - Funding Information:
This work was supported by Grant GM56685 from the National Institutes of Health and a grant from the Research Council of the College of Veterinary Medicine at Colorado State University to H.P.S. T.T.H. was supported by Student Traineeship HOANG99H0 from the American Cystic Fibrosis Foundation. We thank B. H. Iglewski and L. Passador for providing the lasB–lacZ fusion plasmid pLPBL, U. Ochsner for pUO58-24, and F. Lutz for p1000 and pIBH.
PY - 2000/1
Y1 - 2000/1
N2 - 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 φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Ω elements), and (6) the φCTX attachment site. The MCS and Ω 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.
AB - 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 φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Ω elements), and (6) the φCTX attachment site. The MCS and Ω 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.
KW - Flp recombinase
KW - GFP
KW - Integration
KW - P. aeruginosa
KW - Phage CTX
KW - β-galactosidase
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U2 - 10.1006/plas.1999.1441
DO - 10.1006/plas.1999.1441
M3 - Article
C2 - 10610820
AN - SCOPUS:0033968474
SN - 0147-619X
VL - 43
SP - 59
EP - 72
JO - Plasmid
JF - Plasmid
IS - 1
ER -