Gene Replacement in Bordetella pertussis by Transformation with Linear DNA

Abstract

We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.

Harnessing CRISPR–Cas systems for bacterial genome editing

“…A few studies have employed linear DNA to elicit gene replacement by double-crossover recombination, with success in E. coli, Bordetella pertussis, S. thermophilus, and S. pneumoniae [22,[39][40][41]. Synthetic DNA molecules are easily designed and affordable for engineered mutagenesis and splicing by overlap extension PCR also allows the generation of editing templates [42].…”

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1

“…Although pPhAaroACmr was treated with the Klenow fragment and deoxynucleoside triphosphates prior to treatment with T4 ligase, examination showed that most of the sample remained linear rather than circular. The results, then, are not totally unexpected, since there have been few reports of the successful establishment of linear DNA into bacteria (10,16,24).…”

Selection and biochemical studies of pyrimidine‐requiring mutants of Lactobacillus plantarum

“…The relative activity is the ratio of the specific activity in the mutant strains and the specific activity in CCM 1904. Carbamylaspartate was detected with the colorimetric method of Prescott and Jones (1969) at 0°C only in AM1055 extract and not in the wild-type strain CCM 1904 or in the other DHOase-deficient mutants. 0 AM1223 is a double mutant deficient in DHOdehase and in OMPdecase.…”