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.
Subject(s)
Bacillus/genetics , Cloning, Molecular/methods , Conjugation, Genetic , Genetic Vectors , Plasmids , Sequence Deletion , Bacillus/enzymology , Bacillus subtilis/genetics , Escherichia coli/genetics , Genes, Bacterial , Transformation, BacterialABSTRACT
The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis.
