ArticlePloS one2014
The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- ISAntibiotics (Basel, Switzerland) · 2025Article
- Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.Nucleic acids research · 2023Article
- Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator inFrontiers in microbiology · 2022Article
- Rationally designedAnimal diseases · 2021Article
- Generation and Evaluation of a Glaesserella (Haemophilus) parasuis Capsular Mutant.Infection and immunity · 2020Article
- Comparative Genomics ofInternational journal of genomics · 2020Article
- Identification and characterization of serovar-independent immunogens in Actinobacillus pleuropneumoniae.Veterinary research · 2017Article
- p518, a small floR plasmid from a South American isolate of Actinobacillus pleuropneumoniae.Veterinary microbiology · 2017Article
- The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.Open biology · 2017Article
- Complete Genome Sequence of MIDG2331, a Genetically Tractable Serovar 8 Clinical Isolate of Actinobacillus pleuropneumoniae.Genome announcements · 2016Article
- Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.Veterinary microbiology · 2015Article
- Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.The Journal of antimicrobial chemotherapy · 2015Article
- Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation.PloS one · 2015Article
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Authors and funding
9 authors.
Funding
Abstract
We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.
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