Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis

Michael Rachinger, Melanie Bauch, Axel Strittmatter, Johannes Bongaerts, Stefan Evers, Karl Heinz Maurer, Rolf Daniel, Wolfgang Liebl, Heiko Liesegang, Armin Ehrenreich

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

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.

Original languageEnglish
Pages (from-to)365-369
Number of pages5
JournalJournal of Biotechnology
Volume167
Issue number4
DOIs
StatePublished - Sep 2013
Externally publishedYes

Keywords

  • B. licheniformis
  • Markerless deletion
  • PBSX prophage
  • Temperature sensitive replication
  • Transconjugation

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