Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Abstract

Bacillus licheniformis penicillinase genes, penP and penl, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penl(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48°C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB6OE24), the coding region was concluded to be the pen! gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penl promoter. The regulation of penP and pen! by the repressor is discussed.

Interaction of BlaI, the repressor for the β‐lactamase gene of Bacillus licheniformis, with the blaP and blaI promoters

“…DNase I footprinting analysis of BlaI binding to a DNA fragment containing both promoters confirmed that site-specific DNA binding is localized to the regions of dyad symmetry contained within the promoter regions of blaI and blaP ( fig.4), as previously predicted [5]. Footprinting of the complementary strand and DNA containing the isolated promoters revealed similar protected regions (not shown).…”

“…The gene encoding the repressor (blaI) has been cloned from Bacillus licheniformis 749 and from a closely related strain 9945A and has been shown to lie 5' to blaP [4,5]. Transcriptional analysis [6] demonstrated that blaI mRNA is transcribed divergently from blaP mRNA and that it is coinduced with blaP mRNA, suggesting that blair expression is autoregulated.…”

“…These regions contain highly homologous 23 bp regions of dyad symmetry believed to be the specific sites to which BlaI binds [5]. The blaP promoter contains two 23 bp regions of dyad symmetry whereas the blaI promoter contains only one.…”

Evolution of an inducible penicillin‐target protein in methicillin‐resistant Staphylococcus aureus by gene fusion

“…The same areas also contained promoter-like and Shine-Dalgarno (SD)-like sequences in opposite directions followed by a start codon for another open reading frame. This open reading frame extended for at least 250 bp in MRSA PBP (only 32 bp being shown in fig.l Bacillus licheniformis [12] and is supposed to be involved in regulation of these inducible ,&-lactamase genes.…”

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.…”