FIG. 1.

Construction of mutant strains. (A) Schematic presentation of the htrB::pMutin4 mutation. The htrB gene was disrupted with pMutin4 by a single-crossover event (Campbell-type integration). Simultaneously, the spoVG-lacZ reporter gene of pMutin4 was placed under the transcriptional control of the htrB promoter region (PhtrB). The chromosomal fragment from the htrB region, which was amplified by PCR and cloned into pMutin4, is indicated by black bars. Only the restriction sites relevant for the construction are shown (B, BamHI; H, HindIII). lacI, E. coli lacI gene; ori pBR322, replication functions of pBR322; Ap^r, ampicillin resistance marker; Em^r, erythromycin resistance marker; T₁T₂, transcriptional terminators on pMutin4; P_spac,isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent promoter;htrB′, 3′-truncated htrB gene; ′htrB, 5′-truncated htrB gene. (B) Schematic presentation of the amyE region of the chromosome of strains containing an amyE::PcssRS-bgaB mutation. By a double-crossover event, the amyE gene was disrupted with a pDN220-derived cassette containing the bgaB reporter gene placed under the transcriptional control of the cssRS promoter region (PcssRS); Cm^r, chloramphenicol resistance marker; amyE′, 3′ truncated amyE gene; ′amyE, 5′ truncated amyE gene. (C) Schematic presentation of the amyE region of the chromosome of strains containing the amyE::XcssS mutation. By a double-crossover event, the amyE gene was disrupted with a pXcssS-derived cassette (XcssS), which contains the cssS gene placed under the transcriptional control of a xylose-inducible promoter (P_xylA). Cm^r, chloramphenicol resistance marker; xylR, gene specifying the XylR repressor protein; amyE′, 3′ truncated amyE gene; ′amyE, 5′ truncated amyE gene. (D) Schematic presentation of the PcssRS::pDN221 reporter strain. The promoter region of the cssRS genes was duplicated by the insertion of pDN221 into the chromosome via a single-crossover event (Campbell-type integration). Consequently, both the cssRS operon and the spoVG-lacZ reporter gene of pDN221 are placed under the transcriptional control of a cssRS promoter region (PcssRS). Only the restriction sites relevant for the construction are shown (B, BamHI; E, EcoRI). lacI, E. coli lacI gene; ori pBR322, replication functions of pBR322; Ap^r, ampicillin resistance marker; Em^r, erythromycin resistance marker; T₁T₂, transcriptional terminators on pDN221; P_spac, IPTG-dependent promoter.