Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

Reyrat, Jean‐Marc; Pelicic, Vladimir; Gicquel, Brigitte; Rappuoli, Rino

doi:10.1128/.66.9.4011-4017.1998

Cited by 89 publications

(122 citation statements)

References 47 publications

(66 reference statements)

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“…Other advantages of rpsL over sacB counter-selection system include the small size of the rpsL-neo cassette (1.4 kb) compared to sacB-neo cassette (3 kb), which makes PCR amplification easy. Apart from the advantages of the rpsL counter-selection system, the strains selected using this method are limited in use because of their streptomycin resistance (Reyrat et al, 1998).…”

Section: Resultsmentioning

confidence: 99%

“…Selection for the positive phenotype of the introduced mutation has been difficult to achieve, making the use of a counter-selection approach very useful for the mutagenesis (Reyrat et al, 1998). A powerful counter-selection system for the introduction of mutations based on the wild-type rpsL gene responsible for streptomycin sensitivity has been described (Zhang et al, 2003;Rivero-Müller et al, 2007;Heermann et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…The prerequisite for this system to be effective is streptomycin-resistant strain. Resistance because of chromosomal mutations within rpsL is recessive in a merodiploid strain (Reyrat et al, 1998;Gill & Amyes, 2004). When both wild-type and mutant alleles of rpsL are expressed in the same strain, the strain becomes sensitive to streptomycin (Reyrat et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

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Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Tuntufye

Goddeeris

2011

FEMS Microbiol Lett

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Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Tuntufye

Goddeeris

2011

FEMS Microbiol Lett

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“…The efficiency of these approaches varies considerably among the serotypes of A. actinomycetemcomitans. We recently reported the construction of pJT1, a new suicide vector based on the pUC plasmids, that contains the counter-selectable marker sacB for sucrose sensitivity (Reyrat et al, 1998) and facilitates the efficient generation of scarless-markerless deletion mutations in A. actinomycetemcomitans (Ju arez-Rodr ıguez et al, 2013a). We also constructed several cloning and lacZ reporter plasmids for use in A. actinomycetemcomitans based on plasmid pYGK (Ju arez-Rodr ıguez et al, 2013a).…”

Section: Introductionmentioning

confidence: 99%

Integration host factor is required for replication of pYGK-derived plasmids inAggregatibacter actinomycetemcomitans

Torres-Escobar

Juárez-Rodríguez

Demuth

2014

FEMS Microbiol Lett

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In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β-subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα–IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

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“…To date, there are no counterselectable markers available for use with this bacterium. Yet, attenuated and phenotypically distinct mutants without antibiotic markers are most desirable to be developed as candidate vaccines, and counterselection using the Bacillus (subtilis sacB gene has been employed successfully in a wide variety of Gram-negative bacteria [8]. Therefore, in the present communication we describe a simple and highly e¤cacious procedure for the construction of unmarked A. pleuropneumoniae serotype 7 mutants.…”

Section: Introductionmentioning

confidence: 99%

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

Oswald

1999

FEMS Microbiology Letters

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Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km r ) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km r -sacB cassette. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

Cited by 89 publications

References 47 publications

Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Integration host factor is required for replication of pYGK-derived plasmids inAggregatibacter actinomycetemcomitans

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker

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