A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants

Published on May 1, 1998in Gene2.638
· DOI :10.1016/S0378-1119(98)00130-9
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
RoxAnn R. Karkhoff-Schweizer10
Estimated H-index: 10
(CSU: Colorado State University)
+ 1 AuthorsHerbert P. Schweizer46
Estimated H-index: 46
(CSU: Colorado State University)
Abstract An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZα-allele for blue–white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa ΔpabC strain.
  • References (27)
  • Citations (1412)
📖 Papers frequently viewed together
1 Author (Jeffrey H Miller)
19.6k Citations
175 Citations
1,930 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1Joseph SambrookH-Index: 31
#2Edward F. FritschH-Index: 16
Last. Tom ManiatisH-Index: 127
view all 3 authors...
Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefe...
148k Citations
Many strains of Pseudomonas aeruginosa are resistant to the antibiotics cerulenin and thiolactomycin, potent inhibitors of bacterial fatty acid biosynthesis. A novel yeast Flp recombinase-based technique was used to isolate an unmarked mexAB-oprM deletion encoding an efflux system mediating resistance to multiple antibiotics in P. aeruginosa. The experiments showed that the MexAB-OprM system is responsible for the intrinsic resistance of this bacterium to cerulenin and thiolactomycin. Whereas th...
104 CitationsSource
#1Tung T. Hoang (CSU: Colorado State University)H-Index: 9
#2Herbert P. Schweizer (CSU: Colorado State University)H-Index: 46
The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophil...
73 CitationsSource
#1Tung T. HoangH-Index: 14
#2Scott WilliamsH-Index: 1
Last. Joseph S. LamH-Index: 49
view all 4 authors...
asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, t...
32 CitationsSource
#1L. Alexander Lyznik (Purdue University)H-Index: 7
#2Khareedu Venkateswara Rao (Purdue University)H-Index: 21
Last. Thomas K. Hodges (Purdue University)H-Index: 40
view all 3 authors...
Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment. Genomic sequencing in the region of the FRT site (following the r...
174 CitationsSource
#1Susan M. Dymecki (CIS: Carnegie Institution for Science)H-Index: 36
Abstract Site-specific recombinases can serve as powerful tools to target genetic manipulations to specific cell populations in culture and in the organism. A series of vectors for engineering gene activation, deletion and integration in mammalian cells using Flp recombinase is described here. The vectors are modular in design so that specific cassettes can be linked depending on the application. Using these vectors, efficient Flp-mediated lacZ activation and β-galactosidase (βGal) detection has...
70 CitationsSource
#1Jadwiga Wild (UW: University of Wisconsin-Madison)H-Index: 8
#2Zdenka Hradecna (UW: University of Wisconsin-Madison)H-Index: 12
Last. Waclaw Szybalski (UW: University of Wisconsin-Madison)H-Index: 54
view all 4 authors...
Abstract A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRTelements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Posfai et al. (1994) Nucleic Acids Res. 22, 2392–2398]....
44 CitationsSource
#1C S Kristensen (DTU: Technical University of Denmark)H-Index: 6
#2Leo Eberl (DTU: Technical University of Denmark)H-Index: 73
Last. V De Lorenzo (DTU: Technical University of Denmark)H-Index: 1
view all 6 authors...
The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA fla...
115 CitationsSource
Abstract Two cassettes with tetracycline-resistance (Tc R ) and kanamycin-resistance (Km R ) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli . In both cassettes, the resistance determinants are flanked by the short direct repeats ( FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzy...
1,332 CitationsSource
#1Herbert P. Schweizer (U of C: University of Calgary)H-Index: 11
#2Tung T. Hoang (U of C: University of Calgary)H-Index: 3
Abstract A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZα allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restri...
315 CitationsSource
Cited By1412
#1Eisha R Mhatre (University of Pittsburgh)
#2Daniel Snyder (University of Pittsburgh)H-Index: 4
Last. Vaughn S. Cooper (University of Pittsburgh)H-Index: 23
view all 9 authors...
Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia c...
#1Alecia T. Dent (UMB: University of Maryland, Baltimore)
#2Angela Wilks (UMD: University of Maryland, College Park)H-Index: 2
Last. A. Wilks
view all 2 authors...
Pseudomonas aeruginosa exhibits a high requirement for iron which it can acquire via several mechanisms including the acquisition and utilization of heme. P. aeruginosa encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system that encodes an extra cytoplasmic function (ECF) factor system. Previous studies have shown release of heme from the extracellular hemophore HasAp to the outer memb...
#1Xiaoyang Li (NUS: National University of Singapore)H-Index: 2
#2Tingting Zhu (NUS: National University of Singapore)
Last. Shen Q. Pan (NUS: National University of Singapore)H-Index: 13
view all 4 authors...
Agrobacterium tumefaciens is the causal agent of crown gall disease in nature; in the laboratory the bacterium is widely used for plant genetic modification. The bacterium delivers a single-stranded transferred DNA (T-DNA) and a group of crucial virulence proteins into host cells. A putative T-complex is formed inside host cells that is composed of T-DNA and virulence proteins VirD2 and VirE2, which protect the foreign DNA from degradation and guide its way into the host nucleus. However, little...
#1Anne D. Villela (McMaster University)
#2Hanjeong Harvey (McMaster University)H-Index: 11
Last. Lori L. Burrows (McMaster University)H-Index: 42
view all 4 authors...
TfpW is an oligosaccharyltransferase that modifies the subunits of type IV pili from group IV strains of Pseudomonas aeruginosa with oligomers of -1,5-linked-D-arabinofuranose (D-Araf). Besides its oligosaccharyltransferase activity, TfpW may be responsible for periplasmic translocation and polymerization of D-Araf. Here we investigated these potential roles of TfpW in Pa5196 pilin glycosylation. Topology studies confirmed the periplasmic location of loop 1 and the large C-terminus domain, howev...
#1Georgia Doing (Dartmouth College)H-Index: 2
#2Katja Koeppen (Dartmouth College)H-Index: 10
Last. Deborah A. Hogan (Dartmouth College)H-Index: 38
view all 4 authors...
Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here we describe the focal role of ethanol, a common microbial metabolite, in mixed-species co-cultures by dual RNA-seq transcriptomic analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase,...
1 CitationsSource
#1Randi L. Guest (Princeton University)
#2Daniel Samé Guerra (Princeton University)
Last. Thomas J. Silhavy (Princeton University)H-Index: 80
view all 5 authors...
Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential ...
#1Diogo de Abreu Meireles (USP: University of São Paulo)H-Index: 5
#2Cesar Henrique Yokomizo (USP: University of São Paulo)
Last. Luis Eduardo Soares Netto (USP: University of São Paulo)H-Index: 29
view all 3 authors...
YbbN/CnoX are proteins that display a Trx domain linked to a tetratricopeptide (TPR) domain, which are involved in protein-protein interactions and protein folding processes. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl (bleach). EcYbbN contains a SQHC motif within the Trx domain and displays no thiol-disulfide oxidoreductase activity. EcYbbN also presents a second Cys residue at Trx domain (Cys63) 24 residues away from SQHF motif that ca...
#1Tietao Wang (MOE: Chinese Ministry of Education)
#2Zhaoyu Hu (HUST: Huazhong University of Science and Technology)
Last. Haihua Liang (MOE: Chinese Ministry of Education)H-Index: 14
view all 10 authors...
The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell-wall-derived muropeptides. AmpD is cytoplasmic and plays a role in recycling of cell-wall muropeptides, with a link to antibiotic resistance. AmpDh2 is a periplasmic soluble enzyme with the former anchored to the inner leaflet of the outer membrane. We document herein that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa d...