FIG. 4.

Schematic representations of the prophage 1 region and the pMutin mutagenesis system. (A) Genes in the B. subtilis prophage 1 region. (B) Disruption or deletion of chromosomal genes of B. subtilis with pMutin plasmids as described by Vagner et al. (49). To disrupt a certain gene (orfX), an internal fragment of orfX is amplified by PCR and cloned into pMutin. Transformation of B. subtilis with the pMutin plasmid containing the cloned fragment of orfX will result in the chromosomal integration of this plasmid by single-crossover recombination (SCO) into orfX. As a consequence of this integration, orfX is disrupted and the genes downstream of orfX (depicted as orfY) are placed under the control of the IPTG-inducible Pspac promoter of the integrated pMutin plasmid. To delete orfX, the flanking regions of orfX is amplified by PCR and cloned into pMutin in the same orientation but opposite order relative to that on the chromosome. Transformation of B. subtilis with the resulting pMutin plasmid results in the chromosomal integration of this plasmid by double-crossover recombination (DCO). Consequently, orfX is replaced by the integrated pMutin plasmid, while the genes downstream of orfX (depicted as orfY) are placed under the control of the IPTG-inducible Pspac promoter. PorfX, promoter of orfX; X′, 3′-truncated orfX; ′X, 5′-truncated orfX.