The Bacillus licheniformis BlaP beta-lactamase as a model protein scaffold to study the insertion of protein fragments

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.     

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

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.     

Functional domains of the penicillinase repressor of Bacillus licheniformis.

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.     

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

The location of the repressor gene, blaI, for the beta-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 blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, 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 BlaI 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 BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP.     

Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.

Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.     

Phosphate enrichment and fed-batch operation for prolonged beta-lactamase production by Bacillus licheniformis

AIMS: Investigation of the phosphate effect and feeding strategy, i.e. linear and exponential feeding, to improve beta-lactamase production by Bacillus licheniformis considering the viability of the cells. METHODS AND RESULTS: Effect of phosphate enrichment on beta-lactamase production was investigated and resulted in 1.2-fold increase in beta-lactamase activity. Thereafter, exponential and linear feed profiles were established, after an initial batch phase for t = 0-7.5 h. The highest beta-lactamase activity was obtained at fed-batch operation with exponential feeding (FBO1) condition as A = 106 U cm(-3), which is c. 1.7-fold higher than that of the phosphate-enriched batch operation (PE-BO). CONCLUSIONS: Biphasic variations in beta-lactamase production was enhanced to monophasic variation with the exponential feeding strategy where the activity was obtained as A = 106 U cm(-3) at t = 16 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphate enrichment decreases the intracellular ammonium concentration and organic acid excretion, but increrases beta-lactamase production. When batch operation (BO) and PE-BO are compared, it is seen that succinic acid formation decreased with the phosphate enrichment as a result of smooth operation of the tricarboxylic acid cycle. At FBO1 despite the increased lactic and acetic acid formation, beta-lactamase production increased 1.7-fold, and 92% of the cells were alive at the end of the fermentation.     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Crystallization and preliminary X-ray data for the exocellular beta-lactamase of Bacillus licheniformis 749/C

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is P21, with unit cell dimensions a = 66.77 A, b = 93.77 A, c = 43.57 A and beta = 104.5 degrees. The asymmetric unit consists of two molecules of 28,500 Mr each. The crystals are suitable for structure analysis to at least 2 A resolution.     

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

Bacillus licheniformis penicillinase genes, penP and penI, 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 penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees 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 pPTB60E24), the coding region was concluded to be the penI 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 penI promoter. The regulation of penP and penI by the repressor is discussed.     

A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis.

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.     

Viability of Bacillus licheniformis and Bacillus thuringiensis spores as a model for predicting the fate of bacillus anthracis spores during composting of dead livestock

Safe disposal of dead livestock and contaminated manure is essential for the effective control of infectious disease outbreaks. Composting has been shown to be an effective method of disposal, but no information exists on its ability to contain diseases caused by spore-forming bacteria, such as Bacillus anthracis. Duplicate composters (east and west), each containing 16 dead cattle, were constructed (final capacity, 85,000 kg). Spores (10(7) CFU/g manure) of Bacillus licheniformis and Bacillus thuringiensis were mixed with autoclaved feedlot manure and placed in either sterile vials or porous nylon bags. Compost temperatures in the west composter were slightly higher than in the east composter. Viable B. thuringiensis spores were reduced to ≤10(2) CFU in all samples after 112 days but were isolated from bags (west composter) at ≤10(2) and at 10(5) CFU (east composter) after 230 days. In contrast, B. licheniformis was at ≤10(2) CFU in vials (west composter) after 112 days but remained at 10(6) CFU after 230 days (east composter). Similarly, B. licheniformis in bags was not detected after 230 days in the west composter but remained at 10(7) CFU in the east composter. Our study suggests that spore viability was reduced in the west composter by exposure to compost and elevated temperatures over time. Different temperature profiles may explain why spores remained viable in the east structure but were largely rendered nonviable in the west structure. Under practical conditions, variation in composting microclimates may preclude the complete inactivation of Bacillus spores, including those of B. anthracis, during composting. However, composting may still have merit as a method of biocontainment, reducing and diluting the transfer of infectious spores into the environment.     

Use of the beta-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis.

The isolation of auxotrophic mutants of Bacillus licheniformis, a microbe containing constitutive beta-lactamase activity, was found to be facilitated by the addition of clavulanic acid and cefotaxime during enrichment.     

Molecular cloning of the gene for the beta-lactamase of Bacillus licheniformis and its expression in Escherichia coli

Summary The structural gene, pen, for the -lactamase of B. licheniformis has been cloned into a vector and shown to be expressed at a low rate in E. coli. The cloned pen gene appears to be expressed from a promoter within the fragment of B. licheniformis DNA, since its rate of expression is not affected by the presence of the phage repressor, the absence of the phage's positive-control functions, or the position or orientation of the gene within the phage genome.