Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators

Abstract

. Microbiol. 76:217-230, 1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168: 1128Bacteriol. 168: -1132Bacteriol. 168: , 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (pen1c) from strain 749/C that constitutively produces penicillinase. Gelfand, Cetus Corporation. E. coli MC1000 (6) was kindly provided by Malcolm J. Casadaban, University of Chicago. Bacteriophage M13 mplO and mpll and plasmids pUC8, pUC13 (31, 37), pACYC184 (7), and pFC54.t (38) have been described previously. Plasmid pDH5413, a derivative of pDH5060 (8), was obtained from Shing Chang, Cetus Corporation, and its restriction map is shown in Fig. la. Plasmid pTW75 was constructed from p1201-P156-RBS1 (40) by elimination of the EcoRI site proximal to the ribosomebinding site. The construction of plasmids pHCW-P1, pHCW-P2, pHCW-P3, and pHCW-P4 are described in Fig. lb. Double-stranded phage DNA and plasmid DNA were prepared by the method of Birnboim and Doly (3).Expression of repressor genes and purification of penI. The flanking sequences of the penI+ and penIc genes were modified by oligonucleotide-directed site-specific mutagenesis to simplify subcloning into an E. coli expression vector. A HindlIl restriction site was introduced before the ATG initiation codon, and a BamHI site was introduced after the TGA codon of each of the four penl gene alleles. The penI genes were then subcloned as HindIII-BamHI cassettes into pFC54.t, and the resulting plasmids were used to transform strain DG116. Induction of the PL promoter was performed as described elsewhere (13).To purify the penicillinase repressor, we simplified the protocol for purifying X repressor (21)

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

“…5 The blaP gene encodes the class A ␤-lactamase of Bacillus licheniformis 749/I. In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1)(2)(3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4,5).…”

Functional domains of the penicillinase repressor of Bacillus licheniformis

“…1. This plasmid is a derivative of pDH5413 (25). It carries penI under the control of the trp promoter (15), followed by a polylinker region and the crystal protein transcriptional terminator (26).…”

“…penI was expressed in Escherichia coli, and its gene product was purified. The purified PENI was used in vitro for gel retardation and DNase I footprinting experiments to characterize its interactions with the regulatory regions of the pen genes, and three penI operators, positioned in the region between the penP and penI coding sequences, were identified (25). Since these operator sequences overlapped with the promoter sequences of penP and penI, we propose that the PENI repressed the expression of penP by physically blocking the RNA polymerase-binding site and that pen!…”

Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae

“…1 [6,7]). The N-terminal half of CopY shows 30% sequence similarity to the bacterial repressors of ß-lactamases, PenI, of Bacillus licheniformis [8] and related proteins and probably corresponds to the domain that recognizes the DNA promoter sequences [9]. The C-terminal region of CopY harbors a CxCxxxxCxC metal-binding motif.…”