J O Lampen’s research while affiliated with Rutgers, The State University of New Jersey and other places

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.

A cloning vector has been constructed which allows production and export by Escherichia coli of the Met346-Arg601 carboxy terminal domain of the 601 amino acid BLAR sensory-transducer involved in beta-lactamase inducibility in Bacillus licheniformis. The polypeptide, referred to as BLAR-CTD, accumulates in the periplasm of E. coli in the form of a water-soluble, Mr 26,000 penicillin-binding protein. These data and homology searches suggest that BLAR has a membrane topology similar to that of other sensory-transducers involved in chemotaxis.

The blaR gene of Bacillus licheniformis encodes the signal transducer for induction of the class A beta-lactamase. The protein product, BlaR, has a hydrophilic carboxy region that binds beta-lactams and shows high sequence homology to the class D beta-lactamases, particularly the OXA-2 beta-lactamase of Salmonella typhimurium. The BlaR-beta-lactam complex is stable and may provide the continuing stimulus needed for the prolonged production of the enzyme.

BlaI repressor for the beta-lactamase gene (blaP) of Bacillus licheniformis 749, was shown to repress expression of blaP and of the repressor gene (blaI), using the purified protein in a DNA-directed in vitro translation assay. Binding of BlaI to the promoter regions of blaP and blaI was examined by equilibrium and competitive binding assays. BlaI binds to the blaP promoter with an equal or only slightly higher affinity than to the blaI promoter. DNase I footprinting shows that BlaI binds directly to the blaP and blaI promoters, such that RNA polymerase binding and/or transcript elongation would be blocked.

Induction of beta-lactamase (blaP) in Bacillus licheniformis involves the regulatory genes blaI (repressor), blaR1 (coinducer) and R2 (function unknown). Transcription of the bla genes during induction was followed by Northern hybridization. In the first 30 min 2.3-kb transcripts encoding blaI and blaR1 were present. Subsequently, blaP mRNA and short transcripts encoding only blaI accumulated and reached a peak at 1 h. All bla transcripts turn over rapidly. Active repressor is not required for the burst of blaI-blaR1 mRNA. The production of blaI-blaR1 mRNA, and thus of BlaR1, is probably controlled both at initiation of transcription and at a later step in its synthesis and degradation.

A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes. Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B. licheniformis 749.

The repressor gene, blaI, for the beta-lactamase of Bacillus licheniformis 749 was functional when cloned in Escherichia coli, but addition of a beta-lactam did not lead to induction. One plasmid contained fragments from the inducible strain (source of repressor), the other carried fragments from the blaI- mutant 749/C (target). blaI lies just 5' to the promoter for the structural gene, blaP, and the target is the promoter region between the two genes. Interaction with both promoters seemed necessary for full repression. BlaI is a hydrophilic protein (Mr 15036) with the some structural similarities to repressors from Gram-negative bacteria.

The location of the repressor gene, blal, for the β-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blal open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blal, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074–1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of Blal as 5–10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of Blal to DNA was detected by the slower migration of protein DNA complexs during polyacrylamide gel electrophoresis. Blal was shown to selectively bind DNA fragments carrying the promoter regions of blal and blaP.