Dataset Information

FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

ABSTRACT: Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

SUBMITTER: Stringer AM

PROVIDER: S-EPMC3459970 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

Stringer Anne M AM Singh Navjot N Yermakova Anastasiya A Petrone Brianna L BL Amarasinghe Jayaleka J JJ Reyes-Diaz Lucia L Mantis Nicholas J NJ Wade Joseph T JT

PloS one 20120927 9

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletio ...[more]

PMID: 23028641

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Project description:BACKGROUND:Salmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana. METHODS:This was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR. RESULTS:Of 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550?bp with high ciprofloxacin MIC of 0.5??g/ml. CONCLUSION:This study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.

Dataset Information

FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

Publications

FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

Similar Datasets

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