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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 Gene2.64
· 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. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
Cite
Abstract
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 Gm R 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 (1341)
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References27
Newest
Published on Jan 1, 1989
Joseph Sambrook29
Estimated H-index: 29
,
Edward F. Fritsch16
Estimated H-index: 16
,
Tom Maniatis126
Estimated H-index: 126
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...
Published on Feb 1, 1998in Antimicrobial Agents and Chemotherapy4.71
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
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...
Published on Sep 1, 1997in Journal of Bacteriology3.23
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
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...
Published on Mar 1, 1997in Microbiology1.03
Tung T. Hoang14
Estimated H-index: 14
,
Scott Williams1
Estimated H-index: 1
+ 1 AuthorsJoseph S. Lam48
Estimated H-index: 48
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...
Published on Oct 1, 1996in Nucleic Acids Research11.15
L. Alexander Lyznik7
Estimated H-index: 7
(Purdue University),
K. V. Rao21
Estimated H-index: 21
(Purdue University),
Thomas K. Hodges38
Estimated H-index: 38
(Purdue University)
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...
Published on Jun 1, 1996in Gene2.64
Susan M. Dymecki35
Estimated H-index: 35
(CIS: Carnegie Institution for Science)
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...
Published on Jan 1, 1996
Herbert P. Schweizer1
Estimated H-index: 1
,
Thomas R. Klassen1
Estimated H-index: 1
,
Tung T. Hoang9
Estimated H-index: 9
Published on Jan 1, 1996in Gene2.64
Jadwiga Wild8
Estimated H-index: 8
(UW: University of Wisconsin-Madison),
Zdenka Hradecna12
Estimated H-index: 12
(UW: University of Wisconsin-Madison)
+ 1 AuthorsWaclaw Szybalski55
Estimated H-index: 55
(UW: University of Wisconsin-Madison)
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]....
Published on Jan 1, 1996
晶子 中澤1
Estimated H-index: 1
I Global Prospects Discovery of oxygenases in Pseudomonas Pseudomonas: a century of biodiversity Pseudomonas genetics and taxonomy II Metabolism of Hydrocarbons and Chlorinated Hydrocarbons Effects of two-liquid phase systems and the expression of the alk genes on the physiology of alkane-oxidizing strains Alternative pathways for biodegradation of alkyl and alkenylbenzenes Genes and enzymes for hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 Biodegradation of chlorobenzenes ...
Cited By1341
Newest
Published on 2019in Scientific Reports4.01
Robeena Farzand (University of Leicester), Kumar Rajakumar22
Estimated H-index: 22
(University of Leicester)
+ 3 AuthorsHelen M. O'Hare11
Estimated H-index: 11
(University of Leicester)
Klebsiella pneumoniae is a human pathogen, prominent in antimicrobial-resistant and nosocomial infection. The integrative and conjugative element ICEKp1 is present in a third of clinical isolates and more prevalent in invasive disease; it provides genetic diversity and enables the spread of virulence-associated genes. We report a second integrative conjugative element that can co-occur with ICEKp1 in K. pneumoniae. This element, ICEKp2, is similar to the Pseudomonas aeruginosa pathogenicity isla...
Published on Dec 1, 2019in Scientific Reports4.01
Guanfang Xie (WHU: Wuhan University), Man Zeng (WHU: Wuhan University)+ 1 AuthorsZhixiong Xie (WHU: Wuhan University)
Pseudomonas donghuensis HYS is the type strain of a recently identified species, P. donghuensis, which has pathogenic potential with an unclear virulence mechanism. In this study, we used Caenorhabditis elegans as a host to explore the virulence mechanism of P. donghuensis HYS. Based on a correlation between P. donghuensis HYS virulence and its repellence property, we identified 68 potential virulence-related genes, among them the Cbr/Crc system, which regulates the virulence of prokaryotic micr...
Lydia C. Cameron (UMN: University of Minnesota), Benjamin M. Bonis1
Estimated H-index: 1
(UMN: University of Minnesota)
+ 3 AuthorsRyan C. Hunter18
Estimated H-index: 18
(UMN: University of Minnesota)
Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxida...
Published on Jun 24, 2019in Scientific Reports4.01
Elizabet Monteagudo-Cascales (CSIC: Spanish National Research Council), Sofía M. García-Mauriño4
Estimated H-index: 4
(CSIC: Spanish National Research Council)
+ 1 AuthorsInés Canosa14
Estimated H-index: 14
(CSIC: Spanish National Research Council)
The histidine kinase CbrA of the CbrAB two-component system of Pseudomonas putida is a key element to recognise the activating signal and mediate auto- and trans-phosphorylation of the response element CbrB. CbrA is encoded by the gene cbrA which is located downstream of a putative open reading frame we have named cbrX. We describe the role of the CbrX product in the expression of CbrA and show there is translational coupling of the genes. We also explore the role of the transmembrane (TM) and P...
Published on Dec 1, 2019in Scientific Reports4.01
Xiaoyin Feng (SCAU: South China Agricultural University), Maxim Kostylev12
Estimated H-index: 12
(UW: University of Washington)
+ 1 AuthorsE. P. Greenberg83
Estimated H-index: 83
(SCAU: South China Agricultural University)
Pseudomonas aeruginosa quorum sensing (QS) regulates expression of dozens of genes in a cell density-dependent manner. Many QS-regulated genes code for production of extracellular factors, “public goods” that can benefit the entire population. This cooperation encourages individuals to cheat by using but not producing public goods. QS also controls expression of a limited number of genes encoding “private” cellular enzymes like Nuh, an enzyme involved in adenosine catabolism. Growth of P. aerugi...
Published on Mar 20, 2019in Scientific Reports4.01
Paula Blanco9
Estimated H-index: 9
(CSIC: Spanish National Research Council),
Fernando Corona7
Estimated H-index: 7
(CSIC: Spanish National Research Council),
José Manuel Lavié Martínez55
Estimated H-index: 55
(CSIC: Spanish National Research Council)
The emergence of antibiotic resistant Gram-negative bacteria has become a serious global health issue. In this study, we have employed the intrinsically resistant opportunistic pathogen Stenotrophomonas maltophilia as a model to study the mechanisms involved in the acquisition of mutation-driven resistance to antibiotics. To this aim, laboratory experimental evolution studies, followed by whole-genome sequencing, were performed in the presence of the third-generation cephalosporin ceftazidime. U...
Published on Dec 1, 2019in Nature Communications11.88
Hao Huang (CityU: City University of Hong Kong), Xiaolong Shao1
Estimated H-index: 1
(NKU: Nankai University)
+ 4 AuthorsXin Deng18
Estimated H-index: 18
(CityU: City University of Hong Kong)
The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed ...
Published on Jun 22, 2019in Molecular Biology and Evolution14.80
Sara Hernando-Amado7
Estimated H-index: 7
(CSIC: Spanish National Research Council),
Fernando Sanz-García2
Estimated H-index: 2
(CSIC: Spanish National Research Council),
José Manuel Lavié Martínez55
Estimated H-index: 55
(CSIC: Spanish National Research Council)
Published on 2019in Molecular Plant Pathology4.38
Alexander Kutschera1
Estimated H-index: 1
(TUM: Technische Universität München),
Ursula Schombel4
Estimated H-index: 4
+ 2 AuthorsStefanie Ranf11
Estimated H-index: 11
(TUM: Technische Universität München)
Published on Sep 19, 2019in bioRxiv
Geoffrey D. Vrla (Princeton University), Mark Esposito5
Estimated H-index: 5
(Princeton University)
+ 3 AuthorsZemer Gitai25
Estimated H-index: 25
(Princeton University)
Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the signaling pathways and downstream toxins specifically induced by surface association to stimulate P. aeruginosa virulence are not fully understood. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and represent a major class of surface-dependent cytotoxins. AQ cytotoxicity is...
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