title: The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.
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.
authors.affiliation: Section of Paediatrics, Imperial College London, St Mary's Campus, London, United Kingdom.
authors.forename: Janine T
authors.initials: JT
authors.lastname: Bossé
authors.affiliation: Section of Paediatrics, Imperial College London, St Mary's Campus, London, United Kingdom; Laboratório de Genética Molecular de Micro-organismos, Departamento de Microbiologia - DMB - BIOAGRO, Universidade Federal de Viçosa - Viçosa, Brazil.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Soares-Bazzolli
authors.affiliation: Section of Paediatrics, Imperial College London, St Mary's Campus, London, United Kingdom.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, North Mymms, Hatfield, United Kingdom.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
authors.affiliation: Section of Paediatrics, Imperial College London, St Mary's Campus, London, United Kingdom.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics; D016254:Mutagenesis, Insertional; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins