title: Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis.
abstract: Conjugative shuttle vectors of the pKVM series, based on an IncP transfer origin and the pMAD vector with a temperature sensitive replication were constructed to establish a markerless gene deletion protocol for Bacilli without natural competence such as the exoenzyme producer Bacillus licheniformis. The pKVM plasmids can be conjugated to strains of B. licheniformis and B. subtilis. For chromosomal gene deletion, regions flanking the target gene are fused and cloned in a pKVM vector prior to conjugative transfer from Escherichia coli to B. licheniformis. Appropriate markers on the vector backbone allow for the identification of the integration at the target locus and thereafter the vector excision, both events taking place via homologous recombination. The functionality of the deletion system was demonstrated with B. licheniformis by a markerless 939 bp in-frame deletion of the yqfD gene and the deletion of a 31 kbp genomic segment carrying a PBSX-like prophage.
authors.affiliation: Lehrstuhl für Mikrobiologie, TU-München, Freising, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rachinger
authors.forename: Melanie
authors.initials: M
authors.lastname: Bauch
authors.forename: Axel
authors.initials: A
authors.lastname: Strittmatter
authors.forename: Johannes
authors.initials: J
authors.lastname: Bongaerts
authors.forename: Stefan
authors.initials: S
authors.lastname: Evers
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Rolf
authors.initials: R
authors.lastname: Daniel
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Liebl
authors.forename: Heiko
authors.initials: H
authors.lastname: Liesegang
authors.forename: Armin
authors.initials: A
authors.lastname: Ehrenreich
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D010957:Plasmids; D017384:Sequence Deletion; D014169:Transformation, Bacterial
keywords: B. licheniformis; Markerless deletion; PBSX prophage; Temperature sensitive replication; Transconjugation