Functional domains of the penicillinase repressor of Bacillus licheniformis

Abstract

The penicillinase repressor (PENI) negatively regulates expression of the penicillinase gene (penP) in Bacillus licheniformis by binding to its operators located within the promoter region of penP. penI codes for a protein with 128 amino acids. Filter-binding analyses suggest that the active form of the repressor is a dimer. Genetic analyses of PENI derivatives showed that the repressor carrying either a 6-amino-acid deletion near the N terminus or a 14-amino-acid deletion at the C terminus was functionally inactive in vivo. A repressor derivative carrying a 6-amino-acid deletion within its N-terminal region was extensively purified and used in DNA footprinting and subunit cross-linking analyses. The results of these studies showed that the repressor derivative had lost its ability to bind operator specifically even though it could dimerize effectively. In similar studies, we demonstrated that an N-terminal portion of PENI with a molecular mass of 10 kDa derived by digestion with papain was able to bind operator specifically but with reduced affinity and had completely lost its ability to dimerize. These data suggest that the repressor has two functional and separable domains. The amino-terminal domain of the repressor is responsible for operator recognition, and the carboxyl-terminal domain is involved in subunit dimerization.The inducible enzyme penicillinase produced by Bacillus licheniformis is encoded by the chromosomal gene penP (3). In the absence of penicillin, the expression of the penP gene is negatively regulated at the level of transcription by a repressor. Genetic analysis (3,19) indicates that this penicillinase repressor (PENI) is encoded by the closely linked gene penl.The mechanism involved in the induction of the penP gene by exogenous penicillin is unknown, but two additional regulatory genes, penRl and penR2, are believed to play a role in the signal transfer process. penRi codes for the penicillin receptor protein, which is located in the bacterial membrane (7,11). penP and penI are transcribed divergently from within a 364-nucleotide region separating the coding regions. The translation initiation codon of penR1 is located 5 bases downstream of the coding region ofpenI. Therefore, penI and penRi are likely transcribed as a polycistronic mRNA from the penI promoter. 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

Characterization of a copper-transport operon, copYAZ, from Streptococcus mutans The GenBank accession number for the cop operon sequence reported in this paper is AF296446.

“…hirae), and the hypothetical protein encoded by orfY of L. sake. These regulatory proteins have been shown to repress transcription when the effector molecules (β-lactam antibiotics) or ions (Cu# + ) are absent or at physiological levels by binding to inverted repeat sequences overlapping the promoter (Hiramatsu et al, 1992 ;Smith & Murray, 1992 ;Strausak & Solioz, 1997 ;Wittman et al, 1993 ;Wunderli-Ye & Solioz, 1999b). Our studies on the CAT reporter gene suggest that Strep.…”

Quantitation of mecA Transcription in Oxacillin-Resistant Staphylococcus aureus Clinical Isolates

“…First, nonsense and frameshift mutations were predicted to truncate the protein at positions that would have removed the carboxyl terminus. The cleavage site that inactivates the repressor following induction, presumably by preventing dimerization, is 22 amino acids from the carboxyl terminus in BlaI (19) and in the same relative position in MecI (G. Archer and M. Bosilevac, unpublished observations) (Fig. 2).…”

“…The repressors bind to specific palindromic sequences that overlap divergent promoters for mecA and mecR1 (8,18). A helix-turn-helix DNA binding motif has been assigned to the amino terminus of the repressor protein based on structure predictions (11), and the carboxyl terminus has been assumed to be involved in repressor dimerization by analogy to other repressors and by the location of the repressor cleavage site (19,20). However, the structure of neither MecI nor BlaI has been solved.…”

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

“…After deletion of the C-terminal domain, the repressor becomes unable to dimerize and to form a stable complex with its operators. DNase footprinting experiments and filter binding reactions revealed the presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and show the deduced 5Ј-AAAGTATTACATATGTAACNTTT-3Ј consensus sequence (Fig.…”

“…The blaI gene encodes a 128-residue protein characterized by two functional and separate domains (11). The DNA-binding domain, a helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the Cterminal region.…”